Production of closed linear dna
Abstract
An in vitro process for the production of closed linear deoxyribonucleic acid (DNA) comprises: (a) contacting a DNA template comprising at least one protelomerase target sequence with at least one DNA polymerase in the presence of one or more primers under conditions promoting amplification of said template; and (b) contacting amplified DNA produced in (a) with at least one protelomerase under conditions promoting production of closed linear DNA. A kit provides components necessary in the process.

Term
No projected expiry on record.
- Priority
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- Today
11 claims: 9 independent, 2 dependent
- 1ФОРМУЛА ИЗОБРЕТЕНИЯ 1. Бесклеточный способ получения закрытой линейной дезоксирибонуклеиновой кислоты (ДНК) ίη νίίτο, включающий:(a) приведение ДНК-матрицы, содержащей по меньшей мере одну протеломеразную последовательность-мишень, в контакт по меньшей мере с одной ДНК-полимеразой, в присутствии одного или нескольких праймеров в условиях, способствующих амплификации указанной матрицы;и (b) приведение амплифицированной ДНК, полученной в (а), в контакт по меньшей мере с одной протеломеразой в условиях, способствующих получению закрытой линейной ДНК.
- 2Способ по п.1, в котором указанную ДНК-матрицу инкубируют в условиях, способствующих амплификации указанной матрицы путем вытеснения реплицированных цепей посредством репликации другой цепи по типу вытеснения цепи, необязательно, в котором амплификацию указанной матрицы проводят с помощью амплификации по типу катящегося кольца (КСА).
- 3Способ по п.1 или 2, в котором:(a) указанные праймеры являются праймерами со случайной последовательностью;и/или (b) указанная ДНК-полимераза представляет собой рЫ29 с последовательностью 8ЕЦ ГО N0: 2 или ее вариант и/или указанная протеломераза представляет собой ТеГО бактериофага N15 с последовательностью 8ЕЦ ГО N0: 15 или ее вариант;и/или (c) амплифицированная ДНК, полученная в (а), содержит конкатемеры, включающие тандемные единицы последовательности ДНК, амплифицированные с указанной ДНК-матрицы, необязательно, в котором указанные конкатемеры разделяют на одиночные единицы амплифицированной последовательности ДНК с помощью указанной протеломеразы.
- 4Способ по любому из предшествующих пунктов, в котором указанная протеломеразная последовательность-мишень содержит ДНК-последовательность совершенного инвертированного повтора.
- 5Способ по любому из предшествующих пунктов, в котором:(a) указанная ДНК-матрица представляет собой закрытую кольцевую ДНК или (b) указанная ДНК-матрица представляет собой закрытую линейную ДНК, предпочтительно в котором указанную ДНК-матрицу инкубируют при денатурирующих условиях для образования закрытой кольцевой ДНК.
- 6Способ по любому из предшествующих пунктов, в котором указанная ДНК-матрица содержит экспрессионную кассету, содержащую эукариотический промотор, функционально соединенный с представляющей интерес кодирующей последовательностью, и, необязательно, эукариотической последовательностью терминации транскрипции;в котором указанная представляющая интерес кодирующая последовательность, необязательно, представляет собой кодирующую последовательность человека или кодирующую последовательность из патогена, инфицирующего человека, и, необязательно, в котором указанная экспрессионная кассета фланкирована с каждой стороны последовательностью-мишенью протеломеразы, причем предпочтительно указанный способ служит для получения закрытой линейной экспрессионной ДНК-кассеты.
- 7Способ по любому из предшествующих пунктов, в котором:(I) указанный способ дополнительно включает очистку закрытой линейной ДНК, полученной в (Ь);и/или (II) указанный способ включает: (a) приведение одноцепочечной указанной ДНК-матрицы, имеющей последовательность-мишень протеломеразы, которая расщепляется и повторно соединяется протеломеразой ТеШ с последовательностью 8ЕЦ ГО N0: 15, в контакт с ДНК-полимеразой рЫ29 с последовательностью 8ЕЦ ГО N0: 2 или ее вариантом при температуре от примерно 25 до примерно 35°С в условиях, способствующих амплификации указанной матрицы с помощью указанной ДНК-полимеразы;и (b) приведение конкатемеров, полученных на стадии (а), в контакт с указанной протеломеразой ТеШ или ее вариантом при температуре от примерно 25 до примерно 35°С в условиях, способствующих активности указанной протеломеразы;необязательно, в котором указанная последовательность-мишень протеломеразы включает последовательность §ЕЦ ГО N0: 25 или ее вариант.
- 8Бесклеточный способ получения фармацевтической композиции, содержащей закрытую линейную молекулу ДНК, включающий:(a) приведение ДНК-матрицы, содержащей по меньшей мере одну протеломеразную последовательность-мишень, в контакт по меньшей мере с одной ДНК-полимеразой в присутствии одного или более праймеров в условиях, способствующих амплификации указанной матрицы;(b) приведение амплифицированной ДНК, полученной в (а), в контакт по меньшей мере с одной протеломеразой в условиях, способствующих получению закрытой линейной ДНК;и (c) введение полученной закрытой линейной ДНК в состав с фармацевтически приемлемым носителем или эксципиентом.
- 9Набор, содержащий по меньшей мере одну ДНК-полимеразу и по меньшей мере одну протеломе- 28 021069 разу и, необязательно, инструкции по применению в способе по любому одному из предшествующих пунктов.
- 10Способ индукции иммунного ответа против антигена в организме хозяина, причем способ включает осуществление способа по любому из пп.1-8 с использованием указанной ДНК-матрицы, кодирующей указанный антиген, и введение полученной закрытой линейной ДНК, кодирующей указанный антиген, указанному хозяину так, что указанный антиген экспрессируется в указанном хозяине и вызывает иммунный ответ против указанного антигена. - 29 021069 Фиг. 3 С 1 1 1 11 Ογ Ζ Β Фиг. 5 - 30 021069 А. В. 1 2345 67 89 Фиг. 6 - 31 021069 Список последовательностей тоиснионт сеыет1С5 ыштеб ПОЛУЧЕНИЕ ЗАКРЫТОЙ ЛИНЕЙНОЙ ДНК Ν106698Α РСТ/СВ10/000165 2010-02-01 СВ 0901593.4 2009-01-30 42 РабепЫп νβτβίοη 3.5 1 1728 ДНК Искусственная последовательность Нуклеотидная последовательность ДНК-полимеразы бактериофага рЫ29 из ВасШив 1 абдаадсаба бдссдадааа дасдсабадб бдбдасбббд адасаасбас бааадбддаа 60 дасбдбаддд бабдддсдба бддббабабд аабабадаад абсасадбда дбасааааба 12 0 ддбаабадсс бддабдадбб баСддсдбдд дбдбСдаадд басаадсбда СсбабаССбс 180 сабаассбса аабббдасдд адсббббабс аббаасбддб Ьддаасдбаа бддббббаад 240 бддбсддсбд асддаббдсс ааасасасаб аабасдабса Сассссдсас дддасаабдд 300 басабдаббд абабабдббб аддсбасааа дддааасдба адабасабас адбдабабаб 360 дасадсббаа адааасбасс дбСбссбдбб аадаадабад сбааадасбб Сааасбаасб 420 дббсббааад дбдабаббда ббассасааа дааадассад бсддсбабаа дабаасассс 480 даадаабасд ссбабаббаа ааасдабабб садаСбаСбд сддаасдбсб дббааббсад 54 0 бббаадсаад дбббадассд даСдасадса ддсадбдаса дбсбаааадд бббсааддаб 600 аббабаасса сбаадааабб саааааддбд СбСссбасаб сдадбсббдд асСсдабаад 660 даадбдадаб асдссбабад аддбддбббб асабддббаа абдабаддбб сааадааааа 720 дааабсддад ааддсасддс есссдабдбб аабадбсбаб ассссдсаса дабдбабадс 780 сдбсбссббс сабабддбда ассбабадба ббсдадддба аабасдбббд ддасдаадаб 840 басссассас асабасадса бабсадабдб дадббсдааб Сдааададдд сбабабассс 900 асбабасада бааааадаад баддббббаб аааддбаабд адбассбааа аадбадсддс 960 ддддадабад ссдаосбсбд дббдбсаааб дбадассбад ааббаабдаа адаасасбас 1020 дабббабаба асдббдааба бабсадсддс ббааааббба аадсаасбас аддбббдббб 1080 ааадабббба бадабааабд дасдбасабс аадасдасаб садааддадс дабсаадсаа 1140 сбадсаааас бдабдббааа садбсбабас ддбаааббсд сбадбаассс бдабдббаса 1200 дддааадбсс сббабббааа ададаабддд дсдсбаддбб бсадасббдд адаададдаа 1260 асаааадасс сбдбббабас ассбабдддс дббббсабса сбдсабдддс бадабасасд 1320 асааббасад сддсасаддс ббдббабдаб сддабаабаб асбдбдабас бдасадсаба 1380 сабббаасдд дбасададаб ассбдабдба абаааадаба бадббдассс баадаааббд 1440 ддабасбддд сасабдааад басаббсааа ададббаааб абсбдадаса даадассбаб 1500 абасаадаса бсбабабдаа адаадбадаб ддбаадббад бадааддбад бссадабдаб 1560 басасбдаба баааабббад бдббааабдб дсдддаабда сбдасаадаб баадааадад 1620 дббасдбббд адаабббсаа дбдссдддсд дддбддббсб адбсддаббс ддббдабдас адбсддаааа асаббсасаа бдаадссбаа бсааабаа дссбдбдсаа 1680 1728 2 575 БЕЛОК Искусственная последовательность Аминокислотная последовательность ДНК-полимеразы бактериофага рЫ29 из ВасШив 2 Меб Дуз Н1з Меб Рго Агд Дув Меб Туг Зег Сув Азр РЬе С1и ТЬг ТЬг 15 10 15 ТЬг Дуз Уа1 С1и Азр Суз Агд Уа1 Тгр А1а Туг С1у Туг Меб Азп Не 20 25 30 С1и Азр Шз Зег С1и Туг Дуз Не О1у Азп Зег Деи Азр С1и РЬе Меб 35 40 45 А1а Тгр Уа1 Деи Дуг Уа1 С1п А1а Азр Деи Туг РЬе Нхз Азп Деи Дуз 50 55 60 РЬе Авр С1у А1а РЬе Не Не Азп Тгр Деи С1и Агд Азп С1у РЬе Дуз 65 70 75 80 Тгр Зег А1а Азр С1у Деи Рго Азп ТЬг Туг Азп ТЬг Не Не Зег Агд 85 90 95 Меб С1у С1п Тгр Туг Меб Не Азр Не Сув Деи С1у Туг Дув С1у Дув 100 105 110 Агд Ьуз Не Ηίε ТЬг λ/а! 11е Туг Авр Зег Ьеи Ьуз Ьуз Ьеи Рго РЬе 115 120 125 Рго УаЬ Ьуз Ьуз 11е А1а Ьуз Азр РЬе Ьуз Ьеи ТЬг УаЬ Ьеи Ьуз СЬу 130 135 140 Азр Не Азр Туг Нхз Ьуз СЬи Агд Рго УаЬ СЬу Туг Ьуз 11е ТЬг Рго 145 150 155 160 <31и <31и Туг А1а Туг Не Ьуз Азп Азр 11е О1п Не Не А1а О1и Агд 165 170 175 Ьеи Ьеи Не С1п РЬе Ьуз С1п (Ну Ьеи Азр Агд МеС ТЬг А1а СЬу Зег 180 185 190 Азр Зег Ьеи Ьуз С1у РЬе Ьуз Азр 11е 11е ТЬг ТЬг Ьуз Ьуз РЬе Ьуз 195 200 205 Ьуз Уа1 РЬе Рго ТЬг Ьеи Зег Ьеи С1у Ьеи Азр Ьуз СЬи УаЬ Агд Туг 210 215 220 А1а Туг Агд СЬу С1у РЬе ТЬг Тгр Ьеи Азп Азр Агд РЬе Ьуз СЬи Ьуз 225 230 235 240 СЬи 11е СЬу СЬи СЬу МеС УаЬ РЬе Азр УаЬ Азп Зег Ьеи Туг Рго А1а 245 250 255 СЬп МеС Туг Зег Агд Ьеи Ьеи Рго Туг СЬу СЬи Рго Не УаЬ РЬе СЬи 260 265 270 СЬу Ьуз Туг УаЬ Тгр Азр СЬи Азр Туг Рго Ьеи НЬз Не СЬп Нхз Не 275 280 285 Агд Суз СЬи РЬе СЬи Ьеи Ьуз СЬи СЬу Туг 11е Рго ТЬг 11е СЬп 1Ье 290 295 300 Ьуз Агд Бег Агд РЬе Туг Ьуз С1у Азп СЬи Туг Ьеи Ьуз Зег Зег СЬу 305 310 315 320 СЬу СЬи 1Ье А1а Азр Ьеи Тгр ьеи Зег Азп УаЬ Азр Ьеи СЬи Ьеи МеС 325 330 335 Ьуз СЬи Н1з Туг Авр Ьеи Туг Азп УаЬ СЬи Туг Пе Зег СЬу Ьеи Ьуз 340 345 350 РЬе Ьуз АЬа ТЬг ТЬг СЬу Ьеи РЬе Ьуз Азр РЬе ХЬе Азр Ьуз Тгр ТЬг 355 360 365 Туг 1Ье Ьуз ТЬг ТЬг Зег СЬи СЬу АЬа 1Ье Ьуз СЬп Ьеи АЬа Ьуз Ьеи 370 375 380 МеС Ьеи Азп Зег Ьеи Туг СЬу Ьу8 РЬе АЬа Зег Азп Рго Азр УаЬ ТЬг 385 390 395 400 СЬу Ьуз УаЬ Рго Туг Ьеи Ьу8 СЬи Азп СЬу АЬа Ьеи СЬу РЬе Агд Ьеи 405 410 415 СЬу СЬи СЬи СЬи ТЬг Ьуз Азр Рго УаЬ Туг ТЬг Рго МеС СЬу УаЬ РЬе 420 425 430
- 1111е ТЬг АЬа Тгр АЬа Агд Туг ТЬг ТЬг 11е ТЬг А1а АЬа СЬп АЬа Суз 435 440 445 Туг Азр Агд Не Не Туг Суз Азр ТЬг Азр Зег Не НЬз Ьеи ТЬг СЬу 450 455 460 ТЬг СЬи Не Рго Азр УаЬ Не Ьуз Азр Не УаЬ Азр Рго Ьуз Ьуз Ьеи 465 470 475 480 СЬу Туг Тгр АЬа Ηίε СЬи Зег ТЬг РЬе Ьуз Агд УаЬ Ьуз Туг Ьеи Агд 485 490 495 СЬп Ьуз ТЬг Туг Не СЬп Азр Не Туг МеС Ьуз СЬи УаЬ Азр СЬу Ьуз 500 505 510 Ьеи УаЬ СЬи СЬу Зег Рго Азр Азр Туг ТЬг Азр Не Ьуз РЬе Зег УаЬ 515 520 525 Ьуз Суз АЬа С1у МеС ТЬг Азр Ьуз Не Ьуз Ьуз СЬи Уа1 ТЬг РЬе СЬи 530 535 540 Азп РЬе Ьуз УаЬ СЬу РЬе Зег Агд Ьуз МеС Ьуз Рго Ьуз Рго УаЬ СЬп 545 550 555 560 УаЬ Рго СЬу СЬу Уа! УаЬ Ьеи УаЬ Азр Азр ТЬг РЬе ТЬг Не Ьуз 565 570 575 :210> 3 775 БЕЛОК Искусственная последовательность Аминокислотная последовательность ДНК-полимеразы Ьеер УепС из Ругососсиз ер. 3 МеС Не Ьеи Азр А1а Азр Туг Не ТНг СЬи Азр СЬу Ьуз Рго Не Не 15 10 15 Агд Не РЬе Ьуз Ьуз СЬи Азп С1у СЬи РЬе Ьуз Уа1 <31и Туг Азр Агд 20 25 30 Азп РЬе Агд Рго Туг Не Туг А1а Ьеи Ьеи Ьуз Азр Азр Зег СЬп Не 35 40 45 Азр СЬи УаЬ Агд Ьуз Не ТЬг А1а <31и Агд Ηίβ С1у Ьуз Не УаЬ Агд 50 55 60 Не Не Азр А1а СЬи Ьуз УаЬ Агд Ьуз Ьуз РЬе Ьеи СЬу Агд Рго Не 65 70 75 80 СЬи УаЬ Тгр Агд Ьеи Туг РЬе СЬи НЬз Рго СЬп Азр УаЬ Рго А1а Не 85 90 95 Агд Азр Ьуз 11е Агд СЬи НЬз Зег А1а УаЬ Не Азр Не РЬе СЬи Туг 100 105 НО Азр Не Рго РЬе АЬа Ьуз Агд Туг Ьеи Не Азр Ьуз СЬу Ьеи Пе Рго 115 120 125 МеС С1и СЬу Азр СЬи СЬи Ьеи Ьуз Ьеи Ьеи АЬа РЬе Азр Пе СЬи ТЬг 130 135 140 Ьеи Туг НЬз СЬи СЬу СЬи СЬи РЬе АЬа Ьуз СЬу Рго Пе Пе МеС Пе 145 150 155 160 Зег Туг АЬа Азр СЬи СЬи СЬи АЬа Ьуз УаЬ Пе ТЬг Тгр Ьуз Ьуз Пе 165 170 175 Азр Ьеи Рго Туг УаЬ СЬи УаЬ УаЬ Зег Зег СЬи Агд СЬи Мес Пе Ьуз 180 185 190 Агд РЬе Ьеи Ьуз УаЬ Пе Агд СЬи Ьуз Азр Рго Азр УаЬ Пе Пе ТЬг 195 200 205 Туг Азп СЬу Азр Зег РЬе Азр Ьеи Рго Туг Ьеи УаЬ Ьуз Агд А1а СЬи 210 215 220 Ьуз Ьеи СЬу Пе Ьуз Ьеи Рго Ьеи СЬу Агд Азр СЬу Зег СЬи Рго Ьуз 225 230 235 240 МеС. СЬп Агд Ьеи СЬу Азр МеС ТЬг АЬа УаЬ СЬи 11е Ьуз СЬу Агд Пе 245 250 255 НЬз РЬе Азр Ьеи Туг Наз УаЬ 11е Агд Агд ТЬг Пе Азп Ьеи Рго ТНг 260 265 270 Гуг ТНг Ьеи СЬи АЬа УаЬ Туг СЬи АЬа Пе РЬе СЬу Ьуз Рго Ьуз СЬи 275 280 285 Ьуз УаЬ Туг АЬа Наз СЬи Не А1а СЬи АЬа Тгр СЬи ТЬг СЬу Ьуз СЬу 290 295 300 Ьеи СЬи Агд УаЬ АЬа Ьуз Туг Зег МеС СЬи Азр АЬа Ьуз УаЬ ТНг Туг 305 310 315 320 СЬи Ьеи СЬу Агд СЬи РЬе РЬе Рго Мес СЬи АЬа СЬп Ьеи Зег Агд Ьеи 325 330 335 УаЬ СЬу СЬп Рго Ьеи Тгр Азр УаЬ Зег Агд Зег Зег ТЬг СЬу Азп Ьеи 340 345 350 УаЬ СЬи Тгр Туг Ьеи ьеи Агд Ьуз АЬа Туг СЬи Агд Агп СЬи Ьеи АЬа 355 360 365 Рго А5П Ьуг Рго Авр СЬи Агд СЬи Туг СЬи Агд Агд Ьеи Агд СЬи Зег 370 375 380 Туг АЬа СЬу СЬу Туг УаЬ Ьуз СЬи Рго СЬи Ьуз СЬу Ьеи Тгр СЬи СЬу 385 390 395 400 Ьеи УаЬ Зег Ьеи Азр РНе Агд Зег Ьеи Туг Рго Зег Ые 1Ье Не ТНг 405 410 415 НЬз Азп УаЬ Зег Рго Азр ТНг Ьеи Азп Агд СЬи СЬу Суз Агд СЬи Туг 420 425 430 Азр УаЬ АЬа Рго СЬи УаЬ СЬу НЬз Ьуз РНе Суз Ьуз Азр РНе Рго СЬу 435 440 445 34 021069 11е Ьеи 480 С1у <31и О1и С1у Ьуз 560 Ьеи Ьуз С1у □1п А1а 640 11е Низ А1а А1а Агд С1у Уа1 Ьуз Уа1 Агд Рго <31у Мес Уа1 Не <31у Туг Не Уа1 690 695 700 Ьеи Агд С1у Азр <31у Рго Не Зег Ьуз Агд А1а 11е Ьеи А1а С1и 01и 705 710 715 720 РЬе Азр Ьеи Агд Ьуз Ηίβ Ьуз Туг Азр А1а С1и Туг Туг Не <31и Азп 725 730 735 <31п Уа1 Ьеи Рго А1а Уа1 Ьеи Агд 11е Ьеи <31и А1а РЬе С1у Туг Агд 740 745 750 Ьуз С1и Азр Ьеи Агд Тгр С1п Ьуз ТЬг Ьуз О1п ТЬг С1у Ьеи ТЬг А1а 755 760 765 Тгр Ьеи Азп Не Ьуз Ьуз Ьуз 770 775 4 2631 ДНК Искусственная последовательность Нуклеотидная последовательность ДНК-полимеразц I (ροϊΑ) из ВасШиз зЬеагоскегторйНиз 4 асдаадаада адсСадСасС аассдасддс аасадСдСдд саСассдсдс сССССССдсс ССдссасССС СдсаСаасда саааддсаСС сасасдаасд сддСЫзасдд дсССасдаСд асдС-Сдааса аааССССддс ддаадаасаа ссдасссаСС сасССдСадс дСССдасдсс ддааааасда сдссссддса сдааасдссс саададсаса ааддсддасд дсаасааасС сссссддаас СдСссдадса дсссссдссд сСдсдсдадс СаСсаааадс дСассдсаСС сссдсССаСд аасССдаСса ССасдаадсд дасдасасса Ссдддасдсс сдсСдсссдс дсСдадсаад аадддсссда адсдаааасс аСССссддсд ассдсдаССС аасссадсес дссссссдсс асдсдасддс сдаСаССасд ааааааддда ссассдасас сдадссдСас асдссадада ссдССсдсда аааасасддс сСдасСссдд адсаааСадс ддаСССаааа ддаССдаСдд дсдаСаааСс сдасаасасс ссдддсдсдс ссддсаСсдд ддаааааасд дсддСсаадс СдсСдаадса аСССддсасд дсддаааасд СдсСсдсаСс даССдаСдад дсдааадддд ааааасСдаа адаааасссд сдссаасасс дддаСССадс ссссссдадс 120 180 240 300 360 420 480 540 600 660 720 - 35 021069 ааасадседд Сасдааддас ССсССддааа СССдссаСсд даддсдасдд саСдддсдаС сссдссдасд аадсддааад сСсаасссдд дсддСдсддС садасдсссд сссаСддасд сСддсддсда даасадаедд ссадссддсс аадссдсадс сссдадаадс ддсааассдс ааадсдсаса ссдаассСдс дСсссдСсад дсссссдссс аССсасасаа асдсдссдсс ССддсдсааа дссадсСССс ддаСаСдСда сссаасдссс дсСдасаССа саддсссдсс аССдадсдас ссдсСдааад сдсссаСЕСд ссдсдасдсс ссддССдадс СдСсдССада СдасаССдСс аадассдсда аааадссаСс дсдссассса аадаасссдд дсссеадссд аааСддссдс дссддсадсс дааддддада аассдсссда ддадасддад ССдасдСсас Сассдаадад аСдсССдссд асааддсадс дссСдСсдСС аадаааасса ссасдаСдсс ссдаССдссд дааСсдсасс адсдаасдад СССССаСдсд сссддадасс дсдссддсСд асесдсааСС сссадсасдд ааасдаадаа ааааадсаСд СССдасдсса адсдддсадС сдССдссССа даассдадсс ссдсддсдсс дсссссдасс сассдсссдс сдессасссд сСсаадаСдс сддсдаСаСс дсСдсддСдд сдааааСдаа асааСаСдаа сддасдаадс ддссСаСддс аааддсдСса адсддСсдсс дссддасдаа сСдадсаСсС сдССсдсааа дсддсадсса СССдддсдсС Сдадсадссд аСССдсддаа саасдаасаа дассаассас СаасдаадсС Сдадсадссд ссссддссда аасддаассс ассддддсда асдСддаСас ааадсддсСС дсссддадсс сдссдаасаа ссдсдсдсса ссдадсадсд еасссасдад аададССсаа саССаассса ссаааасадс СсддадссаС СССаСССдаа СассддСдсС даадаадасд аааасаддсс асссдассСс ддсСдасдсд ССдсдссдса ссаСдаааСс дСсдааааса ССССдсасса ссдссадссс аассаасдса сассдаадда ссдссдааад ссдсдсдссс СдаСассддс сдасдсссаа ссаадсдссд асдсааассд ддсддсссад сСсддссдад аааасаССсс даССсддсСс даададдддс ддааааСссд ссаадсдССс адссддасСд дсссассссс дссдссдасс асссасааас сдаассдсдс асаСсдссда сдасдасааС сСааССдаад сдССссаасд сдаСССддаС ааасддсдас ддасассссс сасдсдадсд аададдаадс сасддссаас аддсаааддс сдсСаассСс ддсассдССС асддааССад сдассасдда асССдаасаС Сасдсдсааа даадссдссд аасссассда асдССасССс сдддсдСааа дсадСаСасд даааасаССд сдсаадаадс дааасадааа саасдссдСС дсаСсддсдс сдсСасссдс сСдасассас аадссдсааС дсадССССдс ададсддасд дссасдааса сдссаассса аддаадсдсс ссаааааадс даСдаССдас ссадсддсас ддесдааада ададсадсСС ССССдсСдса адсдсаСдас дадсСсаССС сддаадсдсс аааададдаа сасдсдадсс сдССссддаа дсдаСддадс аддссдссас дсСссдсдсд ссдассасса ссасддссса асасддсасд аСдссаааСа а 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2631 5 876 БЕЛОК Искусственная последовательность Аминокислотная последовательность ДНК-полимеразы I (ро!А) из ВасШив вСеагоСЪегторЬИиз 5 МеС Ьуз Ьуз Ьуз Ьеи Уа1 Ьеи Не Азр <31у Азп Зег Уа1 А1а Туг Агд 15 10 15 А1а РЬе РЬе А1а Ьеи Рго Ьеи Ьеи Ηίε Аеп Азр Ьуе С1у 11е Н1з ТЬг 20 25 30 Азп А1а Уа1 Туг <31у рЬе ТЬг МеС Мес Ьеи Азп Ьуз Не Ьеи А1а О1и 35 40 45 С1и αΐη Рго ТЬг Ηίδ Ьеи Ьеи Уа1 А1а РЬе Азр А1а <31у Ьуз ТЬг ТЬг 50 55 60 РЬе Агд Наз О1и ТЬг РЬе С31п С1и Туг Ьуз 61у О1у Агд С1п <31п ТЬг 65 70 75 80 Рго Рго С1и Ьеи Зег О1и С1п РЬе Рго Ьеи Ьеи Агд С1и Ьеи Ьеи Ьуз 85 90 95 А1а Туг Агд Не Рго А1а Туг <31и Ьеи Азр Ηίδ Туг О1и А1а Авр Азр ЮО 105 110 Не Не О1у ТЬг Ьеи А1а А1а Агд А1а <31и <31п С1и С1у РЬе <31и Уа1 115 120 125 Ьуз Не Не Зег С1у Азр Агд Азр Ьеи ТЬг С1п Ьеи А1а Зег Агд ΗΪ3 130 135 140 Уа1 ТЬг Уа1 Азр Не ТЬг Ьуз Ьуз О1у Не ТЬг Азр 11е <31и Рго Туг 145 150 155 160 ТЬг Рго О1и ТЬг Уа1 Агд С1и Ьуз Туг <31у Ьеи ТЬг Рго С1и <31п Не 165 170 175 - 36 021069 37 021069 Не С1и Ьеи Агд 6 6 0 Уа1 Ьеи А1а ΗΪ3 Пе А1а Азр Азр Азр Азп Ьеи 11е 665 670 С1и А1а РЬе С1п Агд Азр Ьеи Азр Пе Ηϊ3 ТЬг Ьуз ТЬг А1а Мес Азр 675 680 685 11е РЬе Н1з Уа1 Зег 31и С1и 31и Уа1 ТЬг А1а Азп Мес Агд Агд 31п 690 695 700 А1а Ьуз А1а Уа1 Азп РЬе С1у Пе Уа1 Туг С1у Пе Зег Аер Туг С1у 705 710 715 720 Ьеи А1а 31п Аап Ьеи Азп Не ТЬг Агд ЬуЗ С1и А1а А1а О1и РЬе Пе 725 730 735 О1и Агд туг РЬе А1а Зег РЬе Рго С1у Уа1 Ьуз σΐη Туг Мес С1и Азп 740 74 5 750 11е Уа1 С1п С1и А1а Ьуз О1п Ьуз С1у Туг Уа1 ТЬг ТЬг Ьеи Ьеи Ηϊβ 755 760 765 Агд Агд Агд Туг Ьеи Рго Азр Не ТЬг Зег Агд Азп РЬе Азп Уа1 Агд 770 775 780 Зег РЬе А1а С1и Агд ТЬг А1а Мес Азп ТЬг Рго Пе С1п С1у Зег А1а 785 790 795 800 А1а Азр Пе Не Ьуз Ьуз А1а Мес Пе Авр Ьеи А1а А1а Агд Ьеи Ьуз 805 810 815 31и О1и О1п Ьеи С1п А1а Агд Ьеи ьеи Ьеи С1п Уа1 ΗΪ3 Азр С1и Ьеи 820 825 830 11е Ьеи б1и А1а Рго Ьуе 31и 31и Пе 31и Агд Ьеи Суз 31и Ьеи Уа1 835 840 84 5 Рго С1и Уа1 МеС ЗХи 31п А1а Уа1 ТЬг Ьеи Агд Уа1 Рго Ьеи Ьуз Уа1 850 855 660 Аер Туг Ηΐ§ Туг 31у Рго ТЬг Тгр Туг Азр А1а Ьуз 865 870 875 21С ) ί 213 . : 1563 212 : ДНК 213 ι Искусственная последовательность Нуклеотидная последовательность протеломеразы фага ρ1ιίΗΑΡ-1 из На1отопа5 6 асдадсддсд адСсасдСад аааддСсдаС ссадсддааС ЪдаСададСд дссдсссадс 60 дадаСсааад адаСсдасдс сдасдасдад асдссасдСа аададаааас саадсдсасд 120 дсдсддсСдд сасдСадсСС саааасдсдс сСдсаСдаСд асаадсдссд сааддаССсс 180 дадсддаСсд сддссасдас сссссдссдс Сасасдасад аадсдсдсаа ддсддСдасС 240 дсдсадаасс ддсдссаСса садсССсдас садсадаСсд адсддсСддс садссдсСас 300 ссддсССасд ссадсаадсС ддаадсдсСс ддсаадсСда ссдаСаСсад сдссаССсдС 360 аСддсссасс дсдадсСдсС сдассадаСс сдсаасдаСд асдасдсССа СдаддасаСс 420 сдддсдасда адссддасса СдаааСсасд сдссассСда сдссдадсСс Сдсасадааа 480 адсасдссдд седаададдс садсдадасд седдаададс дедеддсдаа сасддссдад 540 аСсаасСасс ассддССдаС ддадасддСС сасдадсСдс Сдадсаассд ддададаасд 600 дСсдаСдддд адСаСсдсдд сСССССсадС сассСадсдс ССдддсСддс дсСддссасс 660 дддсдСсдсС сдаСсдаддС дсСдаадасс ддасддаСса сдааддСддд сдадСаСдад 720 ссддадссса дсддссаддс дааааадсдс ддсддсдСсд ассасадсда ддсссассас 780 асССаСассс сддсдааадс СдасссддСд аСсдаадсдС дддасдадсс ссдсссдссд 840 ссддаадсСд сСдадсСдса дддсаСддас аасадсдаСд Сдаассдссд сасддсдаад 900 асдсссааса сдсСсасСаа дсддаСсссе аасаасдасд адсдсдсссс сааддасадс 960 сдддсдассе дддсдсддсС ддСдСССдад есдсассссс сдсдсдасаа дсдссддаад 1020 ааадссассд аддасдСдСС сСддсдсдад асдссддддс асдаддасаС ддасасасад 1080 сдсадссасс дсдссСССаа аассдассас дасдадссдд аСсаадссда ссаддаадаС 1140 сасдаасасд ссадссдссс сдссдсдсСд саддсдсСдд асддссасда дсадсссдад 1200 адсадсдасд сссаддсдсд СдСдсасдсс СдддСдааад сдсадассда дсаддадссс 1260 дасдсдаааа ССасдсадСс СсСдаСсадс сдддадссдд дсдсссассд сссСдссаса 1320 ааадсдсасс СддадсСддс дсдададдсд сссдасдсдс сдаасдссда сссддасаад 1380 дСсдсддсдд садсдссдаа ддаадсадсс даддсдаадс сссддсСдаа сдсссассса 1440 сааддддаСд дсаддсдддс сддддсддсС СсааСсаасд дддСддаадС СдсасдддСд 1500 ддсаассадд саддссддаС сдаадсдасд ааадсддссс асааадсддс дддсдддсдс 1560 Сда 1563 7 520 БЕЛОК Искусственная последовательность Аминокислотная последовательность протеломеразы фага рЫНАР-1 из На1отопаз 7 Мес Зег С1у СЬи Зег Агд Агд Ьуз УаЬ Азр Ьеи АЬа С1и Ьеи Не СЬи Тгр Ьеи Ьеи Зег О1и 11е Ьуз СЬи 11е Азр А1а Азр Азр С1и МеС Рго Агд Ьуз О1и Ьуз ТНг Ьуз Агд МеС А1а Агд Ьеи АЬа Агд Зег РЬе Ьуз ТЬг Агд Ьеи Шз Азр Азр Ьуз Агд Агд Ьуз Азр Зег СЬи Агд 11е А1а УаЬ ТЬг ТЬг РЬе Агд Агд Туг МеС ТЬг С1и А1а Агд Ьуз А1а УаЬ ТЬг А1а С1п Азп Тгр Агд Ηί3 Ηί3 Зег РЬе Азр СЬп СЬп 11е СЬи Агд Ьеи Ьеи ТЬг Азр 11е Зег АЬа 11е Агд МеС АЬа Шз Агд СЬи Ьеи Ьеи Азр 115 120 125 СЬп 1Ье Агд Азп Азр Азр Азр АЬа Туг СЬи Азр 11е Агд АЬа Мес Ьуз 130 135 140 Ьеи Азр НЬз СЬи 1Ье Мес Агд НЬз Ьеи ТЬг Ьеи Зег Зег АЬа СЬп Ьуз 145 150 155 160 Зег ТЬг Ьеи АЬа СЬи СЬи АЬа Зег СЬи ТЬг Ьеи СЬи СЬи Агд АЬа УаЬ 165 170 175 Азп ТЬг УаЬ СЬи 11е Азп Туг Шз Тгр Ьеи МеС СЬи ТЬг УаЬ Туг СЬи 180 185 190 - 39 021069 Ьеи С1у Уа1 Туг Агд Рго А1а Не Ьуз А1а Туг Ьеи <31и Ьеи А1а Агд 435 440 445 С1и А1а Ьеи Азр А1а Рго Азп Уа1 Азр Ьеи Азр Ьуз Уа1 А1а А1а А1а 450 455 460 Уа1 Рго Ьу5 О1и Уа1 А1а О1и А1а Ьуз Рго Агд Ьеи Азп А1а ΗΪ3 Рго 465 470 475 480 С1п С1у Азр О1у Агд Тгр Уа1 01у Уа1 А1а Зег Не Азп С1у Уа1 О1и 485 490 495 8 1854 ДНК Искусственная последовательность Нуклеотидная последовательность протеломеразы фага ΡΥ54 из Уегз2П2а 8 аСдааааСсс аССССсдсда СССадССадС ддСССадСса аададаСсда сдаааСадаа 60 аааСсадасс дддсдсаддд СдасаааасС сддсдссасс адддсдсддс садааадссс 120 аааааСдссд СдСССаСдда Сааасддааа СаСсдсддСа асддСаСдаа даасадааса 180 ссдССаасаа саСССааСаа аСаСССаадС сдадсасдСС сссддСССда адаааддсСС 240 сассаСадсс ССссСсаасс сасадсаасс аСсссаааСа аасасссСдс аССсадсдаа 300 асаасаааад асссддасаа садасссдсС саСдаадССа дааСаааасс сааадаасса 360 асаасСсаСс ССдааСссдд СдССааЬССа ССадаааааа СаддСадсСС адддааааса 420 ааассассса садссааааа аасадссадс ссаааааааа сдСасссаСс асдддссаас 480 даСссадаСа сСССааССад СасСдаадаС дсСасадаас Сасаасаааа дССададсаа 540 дддассдасс СасССаасдс аССасаССсС сСаааадСаа ассаСдаадС сасдСасдса 600 ссаасдасдс адссССссда сададссдса ССаааадсСа ддсасдасдс сдсссССсас 660 сссааааадс дсаасассдс асесассдас СассссддсС асасдсаасд аасдасддас 720 асассасаСс ССссадаСаС адсССССдаа даССсдаСдд сассасССдс сссСССадса 780 сссдссссад садссдссад сддссдсада сааассдааа сассааССас сддсдадссс 840 дасдссаааа аСаааадсаС саССаааССС СсСддасаад саааааааад ааСддссдСС 900 ссаддсддас ассасдааас асасадссса ассдасссад адссасссас ссаасддсса 960 дадСССССас дсСсссаСад сссаасасСС сдассасааа аСССддаааС адсасаСдаС 1020 даасассдса ссдаасСаСс сдссассаас ддссссдсад ссаааесссс аааСдаСдса 1080 дсаааасадС ссСССдСсда сдасадаада дСаСССааад аСасссдСдс аасссасдсс 1140 сдсасадсас асдааааасд дсссадааса дасссссдсс дддсдаадсд сдасдаадас 1200 дСССссССсС сСдааССаСС аддссаСдас дасссадаСа сСсадсСддс аСаСааасаа 1260 ССсаадсСдд СаааСССсаа сссааааСдд асасссааСа СаСсадасда ааасссСсдд 1320 ссадссдсас сссаададсс сдасаасдас асдсссддсс садсасдсдд сдасдсддса 1380 дССсдсаСас асдадсдддс сааададсаа ссддсдсада ассссдсддс аааааСаасС 1440 дсаСассааа Ссаадааааа СССааасСдС сдаааСдасС Сддссадссд аСасаСддса 1500 СддСдСдсСд асдсдсСадд ддССдССаСС ддСдаСдаСд дасаддсаад дссадаадаа 1560 сСсссассаС сдсСсдСдсС СдаСаССаас дсСдаСдаса сСдасдсСда адаадасдаа 1620 асададдаад асСССассда сдаддаааса дасдасассд ааССсдасдС аСсадаСаас 1680 дссадСдаСд аадаСаадсс сдаадасааа ссссдсСССд садсассааС ссдсадаадс 1740 даддассссс ддссдассаа асссдаассс дсСддсаадс ааСаСадсСд ддадддСааС 1800 дссдааадСд ссассдасдс дасдааасаа дсасддасСд аааасасдда дсаа 1854 9 617 БЕЛОК Искусственная последовательность Аминокислотная последовательность протеломераз! Уегзгпла МеС Ьуз Не ΗΪ3 РЬе Агд Азр Ьеи Уа1 Зег <31у Ьеи Уа1 Ьуз С1и 11е Авр <31и Не О1и. ьув Зег Авр Агд А1а 01η. <31у Азр Ьуз ТЬг Агд Агд Туг О1п <31у А1а А1а Агд Ьуз РЬе Ьуз Азп А1а 17а1 РЬе МеЬ Азр Ьуз Агд Ьуз Туг Агд <31у Азп С1у МеС Ьуз Азп Агд Не Зег Ьеи ТЬг ТЬг - 41 021069 РЬе ТЬг Азр С1и СЬи 11е Азр Азр ТЬг СЬи РЬе Азр УаЬ Зег Азр Азп 545 550 555 560 Ьуз СЬп Туг Зег Тгр СЬи СЬу Азп А1а СЬи Зег УаЬ Не Азр А1а МеС 595 600 605 10 1923 ДНК Искусственная последовательность Нуклеотидная последовательность протеломераз, К1еЬв1е11а фага рЫК02 ! асдсдсаадд СдааааЪСдд СдадсСааСс ааССсдсССд СдадсдаддС сдаддсааСс даСдссссСд аСсдСссдса аддсдаСааа асдаадаааа ССааадссдс адсаССаааа еасаадаасд саССаСССаа сдасаааада аадссссдсд дсаааддссе адаааааада аСССсСдсса асасдССсаа сСсдСаСаСд адСсдддсаа ддаааадаСС сдаСдасада есдсассаса асСССдаааа даасдсаасс ааассассад ааааасассс сссасасадс даадаассас ссссдСддсс ссссасдссс дсддсаСсаа ссадасадса сасдссаада ссдсаадсса адсСаааада даСааСдсса ССддсадаад асССаСссаа СаСааадаСС ддСасааааа аСадсдаадС аааааСаааС ааасСсдсСа аСаааСаСсс СдааСддсаа ССсдсСаССа дсдасссааа садсдаадас СддааддаСа ааададаССа СсСССаСааа сСасСссаас ааддССсССс дсСсссддаа дасссдааса асссдааадс ааассасдад дсссСссасс аСсСдсадсС Садссссдсс дадсдаассс сСаСссадса дсдссдддсс аасдСссСса дсдадааааа дсдсаасдСС дссдСдассд асеаСссдсд сСаСаСдсад дссаСсСасд аСаСааСсаа саадссСаса дСССсдССсд аСССдасЬас СсдссдСддС асддссссдс СддсдССсдс ссССдссдсд сСаСссддСс дссдаасдаС СдаааСсасд сСссадддсд ааССССссдС сдсаддСааа саСасадСаа саССссСддд дсаадсСааа ааасдсСсдд аадасааадд СаСаСсаадд ааааСаСаСа ссССасдсда сдсСасСССа сссдссадсс СддСаааСда асССсдсСса Сдссссдссд ссдсддассс СдаСдаадСа асааааддас аСддсдаааа СдасасСсдс СсадааааСд ддсдсаССаа СдсааССсСс дсСасадссС ССааСссдСд ддСааааасС ССсССаддсд аСдассдссд сдсссасааа даСадссдсд ссасссасдс ссдсассдсс СасдаааСдС ссссссдсдС СдасссСсдд сддаадаасд ССдаСдадда сдСаССсССс аСддадаССс Ссддссасда сдаСдаааас асссаассдс асСаСаадса дСССаааССд дссаассссс ссадаасаСд дсдассааас дСсддсдадд адаасдсссд ссСадсддсд сСдсаааадс СддаСадсаС даСдссадаС СССдссаддд дсдасдссдд ддССсдСаСС саСдадассд СдаадсадсС ддсддадсад дасссассда еааааассас ааасадсасс ссдсдассдС ссаассссад сассаддссд аССссесдсС ассСддадСС Сдссдссдас дсассдддсс адССсдСсдд сдаааасддд саасддсаас СдааддаСда ддсдсссдса аСадСссСдс ССдаСдадда аасссссдад сссаСддасд асдСсдаСсС сдаСдасдаа аассасдасд асдааасдсс ддасдасдас дадаСсдаад Сддасдааад сдааддадад даасСддадд аадсдддсда сдссдаадад дссдаддсдд сСдаасадда ададаадсас ссСддсаадс сааасСССаа адсдссдадд дасаасддсд асддсассса саСддсддаа сссдаасссд дсддссдсса ссасдсссдд РссддСдссд ссддСааСсд ддсададдса асдсааСсСд ссСддадСдс ссассСсаад еда 11 640 БЕЛОК Искусственная последовательность Аминокислотная последовательность протеломеразы фага рМК02 К1еЬз1е11а 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1923 МеС Агд Ьуз Уа1 Ьуз Не СЬу СЬи Ьеи Не Азп Зег Ьеи УаЬ Зег СЬи 15 10 15 УаЬ СЬи АЬа Не Азр АЬа Зег Азр Агд Рго СЬп СЬу Азр Ьуз ТЬг Ьуз Ьуз Не Ьуз А1а АЬа АЬа Ьеи Ьуз Туг Ьуз Азп АЬа Ьеи РЬе Азп Азр 12 1617 ДНК Искусственная последовательность Нуклеотидная последовательность протеломеразы фага УР882 из даддСдаааа ссассдасда саасдаддсд ассасссддс сСдаааааас саадссдасс 120 ассадддсдд сдасСаааес саадассаад сСдсасдасд асаадсдссд дааддасдсд 180 ассадаассд сСсСдадсас ссассдсаад Сасасдасаа сддссадддс адсадссасс 240 дадсадаасс ддааасасса садссссдад садсадаСад адсддссддс сааааадсас 300 ссдсаасасд ссдадсадсс ддсддссасс ддддссаедд асаасассас сдадССдсдс 360 ссддсдсаСс дсдассСссС даададсаСс ааддасаасд аСдаадссСС сдаддасасс 420 сдсадсасда адссадасса сдаддСаасд сдссаСссда сдссасссад сдсдсаааад 480 дсдадасСдд сададдаадс сдссдаддсд ССдассдада адаааассдс сасддСсдас 540 аСсаасСаСс асдадссдас ддссддсдСд дСддадсСдС сдассаадаа дассаадасд 600 дСсддсадсд асадсассСа садсССсадс сддссддсдс ССддсаССдд сссддссасс 660 ддСсдссдСС сСаСсдадас асСдаадсад ддсдадссса ааааддСдда Сдадсадсдд 720 сСсдадССсс сСддссаадс дааааадсдс ддсддсдссд ассасссада дасссасасс 780 аСССасассс СддСсдасСс сдассСддСа ссдасддсдс СдаадаассС дсдададССд 840 ссадаадССс дсдсасСдда СдадСасдас саасСдддсд адассаадсд даасдасдсс 900 ассаасааас дссдсдсааа аасдсссаас сааассдсса адсадссссс Сддсадсдас 960 дадсдсдсдС ссааадасад ссдсдссасс СдддсдсдСс сддсссасда дссдсссссс 1020 саасдСдаСс сдсдссддаа ааадааадас даддасдссс сссддсадда дасдссдддс 1080 сасдаддаса ссдадасСса дааадссСаС аадсааССса аддСсдасСа садсдаассС 1140 дадсадссдд сдсасаадсс СддсаааССС аададсадад ссдаадсссс сдсддсдссс 1200 дасСсааасд аддасаССас сасссдссса СссаСддсса адасссасда ссдддсдааа 1260 дадсдсассд сддаадассс сдаддсдаас ассасасадс сасСсаСсас ссдддаасСд 1320 ддсссаддсс дсааддсдас сааддасСас сСсдасссдд ссдасдаСдс ссССдссдсд 1380 дсдаасассс ссдСсдасда сдсадссдсс даддссссад сСдаСдСдсс ддсадсадаа 1440 ааасадссда адааадсдса даадсссада сСсдСддссс ассаддссда Сдасдадсас 1500 сдддаадссс дддсдсСддС ддааддсдад даддсддсса дддСдаааас саадддсасс 1560 сдсдссдадд сааСдасадс сдсасдддад дссадссааа аддсасСсда сдасСаа 1617 13 538 БЕЛОК Искусственная последовательность Аминокислотная последовательность протеломеразы фага УР882 из УЬЬгЬо 13 МеС Зег <31у С1и Зег Агд СЬп Ьуз УаЬ Азп Ьеи СЬи СЬи Ьеи 11е Азп СЬи Ьеи УаЬ СЬи СЬи УаЬ Ьуз ТЬг Не Азр Азр Азп СЬи АЬа Не ТЬг Агд Зег СЬи Ьуз ТЬг Ьуз Ьеи Не ТЬг Агд АЬа АЬа ТЬг Ьуз РЬе Ьуз ТЬг Ьуз Ьеи НЬз Азр Азр Ьуз Агд Агд Ьуз Азр АЬа ТЬг Агд Не АЬа Ьеи Зег ТЬг Туг Агд Ьуз Туг Мес ТЬг МеС АЬа Агд АЬа АЬа УаЬ ТЬг - 44 021069 65 70 75 80 С1и С1п Азп Тгр Ьуз НЬз НЬз Зег Ьеи СЬи СЬп СЬп 11е СЬи Агд Ьеи 85 90 95 АЬа Ьуз Ьуз НЬз Рго СЬп Туг АЬа СЬи СЬп Ьеи УаЬ АЬа Ые СЬу АЬа 100 105 110 МеС Азр Азп 11е ТНг СЬи Ьеи Агд Ьеи АЬа НЬз Агд Азр Ьеи Ьеи Ьуз 115 120 125 Зег 11е Ьуз Азр Азп Азр С1и А1а РЬе СЬи Азр 11е Агд Зег МеС Ьуз 130 135 140 Ьеи Азр НЬз СЬи УаЬ МеС Агд НЬз Ьеи ТНг Ьеи Рго Зег АЬа СЬп Ьуз 145 150 155 160 АЬа Агд Ьеи АЬа СЬи СЬи АЬа АЬа СЬи АЬа Ьеи ТНг СЬи Ьуз Ьуз ТНг 165 170 175 АЬа ТНг УаЬ Азр 11е Азп Туг НЬз СЬи Ьеи МеС АЬа СЬу УаЬ УаЬ СЬи 180 185 190 Ьеи Ьеи ТНг Ьуз Ьуз ТНг Ьуз ТНг УаЬ СЬу Зег Азр Зег ТНг Туг Зег 195 200 205 РНе Зег Агд Ьеи А1а Ьеи С1у 11е С1у Ьеи АЬа ТНг СЬу Агд Агд Зег 210 215 220 11е СЬи 11е Ьеи Ьуз СЬп СЬу СЬи РНе Ьуз Ьуз УаЬ Азр СЬи СЬп Агд 225 230 235 240 Ьеи СЬи РНе Зег СЬу СЬп АЬа Ьуз Ьуз Агд СЬу СЬу АЬа Азр Туг Зег 245 250 255 СЬи ТНг Туг ТНг 11е Туг ТНг Ьеи УаЬ Азр Зег Азр Ьеи УаЬ Ьеи МеС 260 265 270 А1а Ьеи Ьуз Азп Ьеи Агд СЬи Ьеи Рго СЬи УаЬ Агд АЬа Ьеи Азр СЬи 275 280 285 Туг Азр СЬп Ьеи СЬу СЬи 11е Ьуз Агд Азп Азр АЬа 11е Азп Ьуз Агд 290 295 300 Суз АЬа Ьуз ТНг Ьеи Азп СЬп ТНг АЬа Ьуз СЬп РНе РЬе СЬу Зег Азр 305 310 315 320 СЬи Агд УаЬ РНе Ьуз Азр Зег Агд АЬа 11е Тгр АЬа Агд Ьеи АЬа Туг 325 330 335 СЬи Ьеи РНе РНе СЬп Агд Азр Рго Агд Тгр Ьуз Ьуз Ьуз Азр СЬи Азр 340 345 350 УаЬ РНе Тгр СЬп СЬи МеС Ьеи СЬу НЬз СЬи Азр 11е СЬи ТНг СЬп Ьуз 355 360 365 АЬа Туг Ьуз СЬп РНе Ьуз УаЬ Азр Туг Зег СЬи Рго СЬи СЬп Рго УаЬ 370 375 380 НЬз Ьуз Рго СЬу Ьуз РНе Ьуз Зег Агд АЬа СЬи АЬа Ьеи АЬа АЬа Ьеи 385 390 395 400 Азр Зег Азп СЬи Азр 11е ТНг ТНг Агд Зег Зег МеС АЬа Ьуз 11е НЬз 405 410 415 Азр Тгр УаЬ Ьуз СЬи Агд 11е АЬа СЬи Азр Рго СЬи АЬа Азп 11е ТНг 420 425 430 СЬп Зег Ьеи 11е ТНг Агд С1и Ьеи СЬу Зег СЬу Агд Ьуз УаЬ Ые Ьуз 435 440 445 Азр Туг Ьеи Азр Ьеи АЬа Азр Азр АЬа Ьеи АЬа УаЬ УаЬ Азп ТНг Рго 450 455 460 УаЬ Азр Азр АЬа УаЬ УаЬ СЬи УаЬ Рго А1а Азр УаЬ Рго АЬа АЬа СЬи 465 470 475 480 Ьуз СЬп Рго Ьуз Ьуз АЬа СЬп Ьуз Рго Агд Ьеи УаЬ АЬа Н1з СЬп УаЬ 485 490 495 Азр Азр СЬи Ηίε Тгр СЬи АЬа Тгр А1а Ьеи УаЬ СЬи СЬу СЬи СЬи УаЬ 500 505 510 АЬа Агд УаЬ Ьуз 11е Ьуз СЬу ТНг Агд УаЬ СЬи АЬа МеС ТНг АЬа АЬа 515 520 525 Тгр СЬи АЬа Зег СЬп Ьуз АЬа Ьеи Азр Азр 530 535 14 4055 ДНК Искусственная последовательность Нуклеотидная последовательность теломеразы (ΡβίΝ) и вторичного иммунного репрессора (сА) бактериофага N15 из ЕзсЛег/сЫа соН 14 сасасдсасс асассасасс ссаассасдд аасасассад сасасаассд сссассасас 60 дсдсдСаСаа СддасСаССд СдсдссдаСа аддадаасаС аадсдсадаа саасасдсас 120 сСаССссддС дссдсдсссс СССдССаССс СдсСаССаСд ССсСсССаСа дсдсдасдаа 180 адсадсасаа ссаассдсса ессдсссссс дассдсдсса сдаСаСссад адаессадаа 240 асдддддаас сдддасдадс ааддсааааа Ссддсдадсс даСсаасасд сссдсдаасд 300 аддсададдс аассдасдсс ссадассдсс сасааддсда саааасдаад адаассааад 360 ссдсадссдс асддСаСаад аасдсдссас ссааСдаСаа аадааадССс сдСдддааад 420 дассдсадаа аадаасаасс дсдаасассс ССаасдссса сасдадсадд дсаадааадс 480 ддсссдасда сааассасас саСадсСССд асааааасас сааСаааССа СсддаааадС 540 асссСессса садсдаадаа ссасссссас ддсСССсСаС дссСасддсС ааСаССсдсс 600 адсасаСдСс аСсдССасаа Сссааассда аадаааСааС дссдсссдсс даададссас 660 саааСдСаад ааСаддсСсС аааддсадСд аСдсаааааС адсаадасСа аСаааааааС 720 асссадассд дадССССдсС сССадСдаСС СааасадСда СдаССддаад дадсдссдСд 780 асСаСсСССа саадссассс саасааддсС сСдсдССдсс адаадаасса сассадссса 840 аддссаасса сдаддссссд сассассСдс адссаадссс сдсддадсдС асаСсСаСас 900 адсаасдасд ддссдасдсь ссдсдсдада адаадедсаа сдссдсддсс ассдасСасс 960 саасасасаС дсадСсСаСс СаСдаСаССС сдаасаассс сдсдассССа СССадСССаа 1020 асасСсдССс сддаасддса сссссддсес ссдссссддс сдсддсасса дддсдаадаа 1080 сдассдадас аасдссссад ддсдааСССд ссдСССсадд ааадСаСасд дссаассссс 1140 садддсаадс саааааасдс сссдаадаСа ааадсдсаас садаасдасс саСасСССаС 1200 дсдаадсааа ассасссдсс дааССассаа садаассдсд ССсССдсСсС дссдсасссд 1260 ассссдасда ддССдССааа ддаСаСддаа аддаСдаСас ааддСсСдад аасддсадда 1320 сааасдссас СССадсаааа дсасссаасс сССдддССаа асоасссссс ддсдасдасс 1380 дСсдсдСССа СааадаСадс сдсдсСассс асдсСсдсаС сдсССаСдад асдссссссс 1440 дсдСсдаСсс асддсддааа аасдСсдасд аддасдсдСС сССсасддад аССсссддас 1500 асдасдасда даасасссад сСдсассаСа адсадсссаа дссддссаас ССсСссадаа 1560 сссддсдасс сдаадссддд дасдааааса ссаддссддС ддссссдсад аааседдасд 1620 аСдаааСдсс аддссссдсс ададдсдасд сСддсдсссд ссСссасдаа ассдССаадс 1680 адседдедда дсаддассса ссадсааааа Саассаасад сасссессдд дсссССаааС 1740 ССадсссдас дасдассадс сддсасседд адсссдссдс сдасдсассд дддсадсссд 1800 ссддсдадаа сдддсадедд садседаада сададасасс сдсаассдсс ссдсссдасд 1860 аадаасссдС Сдадассаес дасдаассдд аСдаСдадСс ссаадасдас дадсСддаСд 1920 аадасдааас сдадсссдас дадддсддсд дсдасдаасс аассдаадад даадддссад 1980 аадаасаСса дссааседсе сбаааасссд СсССсаадсс сдсааааааС аасддддасд 2040 даасдсасаа дасададссс дааеасдасд дааадсасса сдсссддСсс ддссссдссд 2100 аСадсссеаС ддссдсааед сдасссдсае дддааасдСа ссасадсСаа аадаааадсс 2160 ассддедсса ассддсддсе сссссассда ддсседессс сасссасссс седсааддда 2220 сддааддаСС аддсддааас сдсадседса асСасддаса СсдссдСссс дасСдсаддд 2280 асссссссдс дсааадсддд дсссааассс дддседдсса ассссасссе СсСдсаассд 2340 ссддсдаСдс СадСССсдСд даСадсдССС ссадсССССс аасддссадс СсааааСдСд 2400 сСддсадсас сССссссаде сссдеассаа сассддсдас сддсадсссе ссасаадаса 2460 сассссддсд ассдссасда ассасассдс дсадсадсес ссдсесдеад асасдсаСдС 2520 Сдсссададс сдСССсСдса дссдССааСа Сссддсдсас дСсддсдаед ассдссддда 2580 даСсасссас ддссассддд сссддсдасд ддсссссдса ддсдсддсдд ададссаесс 2640 адасдссдсс аасссаСдсд ссасддсаес даааасСССд СдсСаСдСсд СССаСсаддс 2700 ссдаадССсС есСССсСдсс дссадсссад СддССсассд дсдССсССад дсСсаддсСс 2760 дасаааадса СасСсдссдс ССССссддаС адсСддсада ассСсдССсд СсасссасСС 2820 дсддаассдс саддсСдСсд ссссссдссс сассдсдСсд сддсадсдда ддаССаСддС 2880 дСададасса даеессдаСа ссасаСССас сссссСддсс аСссдаСсаа дСССССдСдс 2940 сссддссааа ссдадддсса ассссссасс аСдаСссадс ССасдсааСд саСсадаадд 3000 дседдсСаСа сссааСдсад сасадасасс садсдссаса аассасдддс сассассдас 3060 аадаассасс сдсасадддс ддссссссед ааасдаааад асддададад ссССсаССдс 3120 дсссссссдд ассссадсСд сСсадааадд дасадддадс адссдсдадс ССсссдсдсд 3180 адсссдсдсд сдассСдсад аадССссдса дсССсссдса ааСасадсдС ддссСсаСаа 3240 сСддадаСад СдсддСдадс ададсссаса адсдссссаа ссСдсадсад дсдССссСса 3300 ассдссссса дсаддссссд ддсдсссаас сдаасссддс есасдсдасс ассссдсСда 3360 - 46 021069 ссдддаСасд ддссдасада асдаддасаа аасддссддс даассддсда сдадсССссс дсссддасда сдсаасддсд даааддсддс ддаСаСддда ссссссдссс дсдсддасда садссдсааа СССдааСССд аасасддсас дсассссСаС сссдсасадд дСдсСассас сададССдад ааСсСсСаСа ддддсддсад сссадасадд дССсСсааса ссддСасаад аадааассдд сссаассдаа дССддсссса ссСдадссас саСаасссад дСаСдсдсад асссаасаса саааааааса сдсСддсдсд СдССдСдсдс ССсССдСсаС Ссддддссда даддсссддс СдсадаСССС дссдсадсдд ддсаасссса ссдссааадс адаасдсасд СсааСааССС аддСддаСаС СССассссдС дассадСсас дсдсасаддс дсссссаСад сссдссссас Сдассдасса даасссдасс адссассдда дСссддсааС сССаССдасд ассдсадсса ссссадасдс сдссСсааас сссасасддс сасдаасдад ссасСддаас ддаасадсса дсаддСасад сддаасдаас сасааасддС Ссадасдссд ссадаасдСс дсассасдас дССссаСсса ССсддсаССд ссдас 15 631 БЕЛОК Искусственная последовательность Аминокислотная последовательность теломеразы бактериофага N15 Е5сЬег1сЫа соН 15 Мес Зег Ьуз Уа1 Ьуз 11е С1у СЬи Ьеи 11е Азп ТЪг Ьеи Уа1 Азп С1и 15 10 15 УаЬ СЬи АЬа 11е Азр А1а Зег Азр Агд Рго <31п СЬу Азр Ьуз ТНг Ьуз 20 25 30 Агд 11е Ьуз А1а А1а АХа А1а Агд Туг Ьуз Авп А1а Ьеи РЬе Азп Азр 35 40 45 Ьуз Агд Ьуз РЬе Агд СЬу Ьуз СЬу Ьеи СЬп Ьуз Агд 1Ье ТЬг АЬа Азп 50 55 60 ТЬг РЬе Авп АЬа Туг МеС Зег Агд АЬа Агд Ьуз Агд РЬе Азр Азр Ьуз 65 70 75 80 Ьеи ΗΪ3 НЬз Зег РЬе Азр Ьуз Азп 1Ье Азп Ьуз Ьеи Зег СЬи Ьуз Туг 85 90 95 Рго ьеи Туг зег С1и С1и ьеи зег Зег тгр ьеи зег мес Рго тьг ЬОО 105 110 Азп 11е Агд <31п Нхз МеС Зег Зег Ьеи ОЬп Зег Ьуз Ьеи Ьуз СЬи 115 120 125 МеС Рго Ьеи А1а С1и С1и Ьеи Зег Азп УаЬ Агд 11е СЬу Зег Ьуз 130 135 140 Зег Адр А1а Ьуз 11е А1а Агд Ьеи 11е Ьуз Ьуз Туг Рго Авр Тгр 145 150 155 РЬе АЬа Ьеи Зег Азр Ьеи Азп Зег Азр Азр Тгр Ьуз СЬи Агд Агд 165 170 175 Туг Ьеи Туг Ьуз Ьеи РЬе СЬп С1п СЬу Зег АЬа Ьеи Ьеи СЬи СЬи 180 185 190 Нхз С1п Ьеи Ьуз Уа1 Азп Нхз СЬи Уа1 Ьеи Туг Нхз Ьеи СЬп Ьеи 195 200 205 Рго А1а СЬи Агд ТЪг Зег Не С1п <31п Агд Тгр А1а Азр УаЬ Ьеи 210 215 220 <31и Ьуз Ьуз Агд Азп Уа1 УаЬ Уа1 11е Азр Туг Рго ТЪг Туг Мес 225 230 235 Зег 11е Туг Азр Г1е Ьеи Азп Азп Рго А1а ТЪг Ьеи РЬе Зег Ьеи 245 250 255 ТЬг Агд Зег СЬу МеС АЬа Рго Ьеи А1а РЬе АЬа Ьеи АЬа АЬа Уа1 260 265 270 СЬу Агд Агд Мес Не СЬи Не Мес РЬе СЬп СЬу С1и РЬе АЬа УаЬ 275 280 285 СЬу Ьуз Туг ТЬг УаЬ Азп РЬе Зег СЬу С1п А1а Ьуз Ьуз Агд Зег 290 295 300 Азр Ьуз Зег Уа1 ТЬг Агд ТЬг Не Туг тЬг Ьеи Суз СЬи А1а Ьуз 305 310 315 РЬе ν«ι1 <31и Ьеи Ьеи ТЬг СЬи ьеи Агд Зег Суз Зег АЬа АЬа Зег 325 330 335 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4055 АЬа 11е СЬу Зег 160 Азр Ьеи Зег Агд ОЬп 240 Азп Зег зег СЬи Ьеи 320 Азр - 47 021069 РЬе Азр Ии Уа1 Уа1 Ьуз С1у Туг <31у Ьуз Азр Азр ТЬг Агд Зег О1и 340 345 350 Азп Иу Агд Не Азп А1а Не Ьеи А1а ьуз АЛа РЬе Азп Рго Тгр Уа1 355 360 365 Ьуз Зег РЬе РЬе С1у Азр Азр Агд Агд Уа1 Туг Ьуз Азр Зег Агд А1а 370 375 360 11е Туг А1а Агд Не А1а Туг Пи МеС РЬе РЬе Агд Уа1 Азр Рго Агд 385 390 395 400 Тгр Ьуз Аеп Уа1 Азр СЬи Азр Уа1 РЬе РЬе МеС 01и Не Ьеи О1у Кхэ 405 410 415 Аер Азр (31и Азп ТЬг Ип Ьеи ΗΪ3 Туг Ьуз С1п РЬе Ьуз Ьеи А1а Азп 420 425 430 РЬе Зег Агд ТЬг Тгр Агд Рго СЯи Уа1 О1у Азр С1и Азп ТЬг Агд Ьеи 435 440 445 Уа1 А1а Ьеи Нп Ьуз Ьеи Азр Азр С1и Мес Рго С1у РЬе А1а Агд С1у 450 455 460 Авр А1а Ну Уа1 Агд Ьеи Нхз С1и ТЬг Уа1 Ьуз Ип Ьеи Уа1 С1и С1п 465 470 475 480 Азр Рго Зег А1а Ьуз Не ТЬг Азп Зег ТЬг Ьеи Агд А1а РЬе Ьув РЬе 485 490 495 Зег Рго ТЬг МеС Не Зег Агд Туг ьеи С1и РЬе А1а А1а Азр А1а Ьеи 500 505 510 СЯу Пп РЬе Уа1 СЯу Ии Азп О1у С1п Тгр С1п Ьеи Ьуз Не С1и ТЬг 515 520 525 Рго А1а Пе Уа1 Ьеи Рго Азр Ни С1и Зег Уа! С1и ТЬг 11е Азр Ни 530 535 540 Рго Азр Азр Ии Зег Ип Азр Азр Ии Ьеи Азр Пи Азр Ии Пе Ии 545 550 555 560 Ьеи Азр Ии Иу Иу Иу Азр Ии Рго ТЬг Ни Ии Ии Иу Рго Ии 565 570 575 С1и Нхв С1п Рго ТЬг А1а Ьеи Ьуз Рго Уа1 РЬе Ьуз Рго А1а Ьуз Азп 580 585 590 Азп Иу Азр С1у ТЬг Туг Ьуз 11е С1и РЬе 01и Туг Азр СЯу Ьуз Ηίδ 595 600 605 Туг А1а Тгр Зег Иу Рго А1а Азр Зег Рго МеС А1а А1а МеС Агд Зег 610 615 620 А1а Тгр С1и ТЬг Туг Туг Зег 16 22 ДНК Искусственная последовательность 22-нуклеотидная консенсусная последовательность совершенного инвертированного повтора из мезофильного бактериофага прочие признаки (1)..(1) η представляет собой а, с, д или С прочие признаки (5)..(6) η представляет собой а, с, д или с прочие признаки (9)..(10) η представляет собой а, с, д или С прочие признаки (13)..(14) п представляет собой а, с, д или С прочие признаки (17)..(18) п представляет собой а, с, д или с прочие признаки (22) . . (22) η представляет собой а, с, д или С 16 псаСппсапп сдппСаппаС дп 17 22 ДНК Искусе 'венная последовательн Особенно предпочтительная последовательность совершенного инвертированного повтора для использования с протеломеразами фага N15 из ЕзсЪеггсЫа соН и фага РЫ КО2 из К1еЪз1е11а Особенно предпочтительная последовательность совершенного инвертированного повтора для использования с протеломеразой фага ΡΥ54 из Уег52П2а 18 дсабасбасд сдсдбадбаб дс 19 22 ДНК Искусственная последовательность Особенно предпочтительная последовательность совершенного инвертированного повтора для использования с протеломеразой фага ρΚίΗΑΡ-1 из На1отопаз 19 ссабасбаба едбабадбаб дд 20 22 ДНК Искусственная последовательност Особенно предпочтительная последовательность совершенного инвертированного повтора для использования с протеломеразо: фага УР882 из νίύΓίο 20 дсабасбаба едбабадбаб дс Особенно предпочтительная последовательность совершенного инвертированного повтора для использования с протеломеразой из ВоггеНа Ьигд0ог£ег1 22 24 ДНК Искусственная последовательность Особенно предпочтительная последовательность совершенного инвертированного повтора для использования с протеломераз' фага νρθ82 из УхЬгго 23 42 ДНК Искусственная последовательность Особенно предпочтительная последовательность совершенного инвертированного повтора для использования с протеломеразой фага ΡΥ54 из УегзтЫа 23 ассбабсбса дсабасбасд сдсдбадбаб дсбдааабад дб 24 90 ДНК Искусственная последовательность Особенно предпочтительная последовательность совершенного инвертированного повтора для использования с протеломеразой фага рЫНАР-1 из На1отопав 24 ссбабаббдд дссассбабд бабдсасадб бсдсссабас бабаедбаба дбабдддсда асбдбдсаба сабаддбддс ссаабабадд - 49 021069 25 56 ДНК Искусственная последовательность Особенно предпочтительная последовательность-мишень протеломеразы 25 бабсадсаса сааббдссса ббабасдсдс дбабаабдда сбаббдбдбд сбдаба 5ι 26 42 ДНК Искусственная последовательность Особенно предпочтите. последовательность-мишень протеломеразы абдсдсдсаб ссаббабасд сдсдбабааб ддсдабааба са 4: 27 52 ДНК Искусственная последовательность Особенно предпочтительная последовательность-мишень протеломеразы 27 бадбсассба бббсадсаба сбасдсдсдб адбабдсбда аабаддббас Од 5;ДНК Искусственная Особенно прелпочтитель последовательность : последовательность-мишень протеломераз! дддабсссдб сссабасаба сабдбабсса бдбддсабас бабасдбаба дбабдссдаб дббасабабд дбабсаббсд ддабсссдбб 29 38 ДНК Искусственная последовательность Особенно предпочтительная последовательность-мишень протеломеразы 29 басбааабаа абаббабаба бабааббббб баббадба - 50 021069 Зас рСЬ 34 дСдсаадСдс аддСдссада ас 22 35 27 ДНК Искусственная последовательность Ваш рОЬ 35 даСааадаад асадСсаСаа дСдсддс 27 36 56 ДНК Искусственная последовательность Последовательность, комплементарная ЗЕф 10 N0: 25 36 СаСсадсаса сааСадСсса ССасасдсдс дсасаасддд саассдсдсд сСдаСа 56 37 56 ДНК Искусственная последовательность Се1К 37 СаСсадсаса сааССдссса ССасасдсдс дсасаасддд саассдсдсд сСдаСа 56 38 56 ДНК Искусственная последовательность Се1Ь 38 саСсадсаса сааСадСсса ССасасдсдс дсасаасдда есассдсдсд седаСа 56 39 42 ДНК Искусственная последовательность Последовательность, комплементарная ЗЕО Ю N0: 26 39 СдСаЬСаСсд ссаССаСасд сдсдСаСааС ддаСдсдсдс аС 42 40 52 ДНК Искусственная последовательность Последовательность, комплементарная ЗЕ(2 Ю N0: 27 40 садСаассса ссссадсаса сСасдсдсдС адсасдссда аатаддСдас Са 52 41 90 ДНК Искусственная последовательность Последовательность, комплементарная 5Е0 Ю N0: 28 41 аасдддассс сдааСдаСас сасасдсаас аСсддсаСас сасасдсаса дСаСдссаса 60 СддасасаСд СаСдСаСдда асдддасссс эо 42 за ДНК Искусственная последовательность Последовательность, комплементарная ЗЕф ю N0: 29 42 сассаасааа ааассасаса сасаасассс асссадса 38
Independent claims11
413 paragraphs in 13 sections, as filed
The present invention relates to a ίη νίίτο cell-free method for producing closed linear deoxyribonucleic acid (DNA).
BACKGROUND OF THE INVENTION
Traditional methods for large-scale amplification of DNA using cells are costly. For example, the use of bacteria requires their growth in large volumes in expensive fermenters, which must be kept under sterile conditions to prevent contamination of the culture. Bacteria also need to be lysed to release amplified DNA, and the DNA needs to be cleaned of other bacterial components. In particular, the production of DNA vaccines or other therapeutic DNA agents requires a high degree of purity to remove endotoxins that are toxic to mammals.
In addition to the cost of the process, the use of bacteria in many cases can hamper the accuracy of amplification. In the complex biochemical environment of a bacterial cell, it is difficult to control the quality and yield of the desired DNA product. A bacterium can accidentally alter a gene of interest cloned in amplified DNA and render it useless for a given purpose. Recombination can also cause problems in the accuracy of the products of DNA of interest. Cell-free enzymatic DNA amplification methods avoid the need for host cells and are therefore preferred.
For example, the production of DNA cassettes for medical use, almost without exception, is based on their incorporation into bacterial plasmids and their amplification in bacterial fermentation processes.
The current state of DNA production methods in this area in various ways limits the possibilities for improving the production of such DNA-based therapeutic agents. In addition, the plasmid product is essentially a crude DNA molecule, in the sense that it contains nucleotide sequences that are not required for the therapeutic function of the DNA molecule. Accordingly, in the field of manufacturing DNA products, such as DNA-based therapeutic agents, there is a need for improved methods for amplifying DNA in large quantities. In particular, there is a need for improved methods for amplification of specific forms of DNA, such as closed linear DNA. Closed linear DNA molecules are particularly suitable for use in therapy due to their higher stability and better safety relative to other forms of DNA.
Summary of the invention
The present invention relates to a method of ίη νίίτο cell-free linear covalently closed DNA (closed linear DNA). This method allows to improve the production of linear covalently closed DNA in comparison with current methods, including cellular methods and amplification in plasmids. This method significantly increases the yield of the product, while reducing the cost of cleaning it.
According to the present invention, a linear covalently closed DNA based on a DNA template is obtained enzymatically in the absence of host cells. The DNA template contains at least one protelomerase target sequence. Matrix DNA is contacted with at least one DNA polymerase in the presence of one or more primers under conditions conducive to amplification of the template. DNA amplified from the matrix is contacted with at least one protelomerase under conditions conducive to the production of closed linear DNA.
Accordingly, the present invention relates to a ίη νίίτο cell-free method for producing a closed linear deoxyribonucleic acid (DNA), including:
(a) contacting a DNA matrix containing at least one protelomerase target with at least one DNA polymerase in the presence of one or more primers under conditions conducive to amplification of said matrix; and (b) contacting the amplified DNA obtained in (a) with at least one protelomerase under conditions conducive to the production of closed linear DNA.
The invention further relates to a kit providing the components necessary in the method of the invention. Thus, the invention relates to a kit containing at least one DNA polymerase and at least one protelomerase and instructions for use in the method of the invention.
Brief Description of the Drawings
FIG. 1 - replication of linear covalently closed DNA in bacteriophages and the role of protelomerase.
A. Image of the extrachromosomal linear covalently closed bacteriophage DNA; * - the center of the palindromic sequence of telomeres; The K sequence is an inverted palindromic repeat of the L sequence.
B. Replication of bacteriophage DNA in the host: the oval indicates DNA chain replication; synthesis of a chain complementary to K and b leads to identical double-stranded KB sequences.
C. Products resulting from the action of protelomerase. Protelomerase binds to
- 1 021069
The K-sequence both cuts and ligates opposite chains at the central point of the palindromic sequence, re-forming telomeres and completing the replication of the original linear covalently closed DNA.
FIG. 2 - effect of phage N15 protelomerase from ExceisPase (Teh) on a circular double-stranded DNA containing its target site 1e1Kb. Te1Kb is an inverted palindrome with 28 nucleotide right (1e1K) and left (1e1b) arms indicated by arrows. The underlined sequences indicate imperfection in the palindrome 1e1Kb. The central 22-nucleotide perfect inverted Te1O palindrome is necessary for binding of the TeSh enzyme. TeS cleaves this 22nucleotide sequence in the center and connects the ends of complementary chains, forming covalently closed ends.
FIG. 3 is a comparison of protelomerase target sequences found in various organisms. Boxed sequences indicate the length of a perfect or imperfect palindromic sequence. Imperfections in the palindrome are highlighted. Highlighted base pair sequences are common to all protelomerase target sequences, which indicates their importance for the binding and action of protelomerase. A. Phage N15 from Exiley Pais. B. Phage RP K02 from K1eB81e11a. FROM. Phage Ru54 from VegaPa. Ό. Phage RP NAR from Na1otoia8. E. Phage UR882 from UPpo. R. Plasmid 1pB31.16 from Votgeya LigdbogGsp. Boxed sequences show the length of the perfect or imperfect palindromic sequence for each bacteriophage. C. A consensus inverted palindromic sequence is shown for the binding and action of bacteriophage protelomerase. It is a sequence of a 22-nucleotide perfect inverted repeat (11 base pairs to either side of the cleavage site). The consensus sequence is deduced from the conserved residues shown in color shown on AE. Conservative base pairs and their positions in the palindrome are indicated. Dashes indicate the flexibility of the composition of the sequence, that is, when the bases can be any nucleotide N (A, T, C or C).
FIG. 4 is a specific method for йη narrowly amplifying a linear double-stranded covalently closed DNA using a DNA polymerase with a chain displacement activity of the rolling ring type (KSA) in combination with TeSh protelomerase. A. Closed linear DNA template. K and b are DNA sequences of the right or left shoulder of the TeH protelomerase binding sequence. B. Denaturation of the starting matrix to form circular single stranded DNA. C. Primer binding. Ό-E. Amplification by the type of a rolling ring on a single-stranded DNA matrix using DNA polymerase with KSA-like displacement activity. P. The formation of long concatemer double-stranded DNA containing single elements of the amplified matrix, separated by protelomerase (KE) binding sequences. C. Contact with TeS protelomerase specific for the Kb sequence. Protelomerase cleaves concatemeric DNA according to the Ksite site and ligates to complementary strands, giving amplified copies of the original linear covalently closed DNA template.
FIG. 5 shows the cutting of a DNA cassette expressing a gene of interest from a long double-stranded DNA molecule to create a closed linear DNA cassette. A. A linear double-stranded DNA molecule containing a DNA cassette comprising a gene of interest flanked by protelomerase target sequences. B. Cutting the DNA cassette as a linear covalently closed DNA molecule.
FIG. 6 shows amplification of closed linear DNA and expression of a reporter gene using a dog bone expression cassette.
A. Confirmation by agarose gel electrophoresis cleavage using TES KSA amplified concatemers with the formation of closed linear DNA. Lanes 1-3 show the KSA amplified plasmid pIS18. Lane 1: 3 μl KSA-amplified undigested RIS18. Lane 2: 2 μl of KSA-amplified plasmid pIS18 digested with RuiT Lane 3: 2 μl of KSA-amplified plasmid pIS18 treated with TeS (negative control). Lanes 4-6 show the KSA-amplified plasmid pC18-1e1Kb. Lane 4: 3 μl of KSA amplified non-cleaved rIS18-1e1KE Lane 5: 1 μl KSA-amplified plasmid riC18-1e1KE cleaved with RuT Lane 6: 4 μl KSA-amplified plasmid pIS181e1KE treated with TeSh. Indicated linear linear DNA of 2.7 kbp resulting from TeS processing is indicated. Flanking tracks are markers of DNA size.
B. LOC analysis (LAL-Op-A-CHP, microarray analysis) showing the resistance of closed linear DNA to temperature denaturation. Lane 1: DNA size marker. Lanes 2 and 3: 100 ng of RSK OOS product. Lanes 4 and 5: 100 ng of the denatured RSK OOS product. Lanes 6 and 7: DNA in the form of a dog bone - 100 ng of pCOS OOS treated with TeS. Lanes 6 and 7: DNA in the form of a dog bone - 100 ng of pCOS OOS, treated with TeS and denatured.
C. Confirmation of expression of closed linear DNA in cells by transfection. Axis y: average ratio of luminescence intensity of firefly luciferase (Ryeuu) and coral polyp luciferase (Kesh11a); x axis: linear DNA constructs used in transfection. RSK pCb: open linear PCR fragment from the plasmid pCb4.13, capturing the gene 1IS (luciferase).
- 2 021069
CSC ΌΘΟ: an open linear PCR fragment amplified on a pCb ΌΘΟ template using primers flanking the 1e1Kb sites. Dog bone, mini prep: closed linear DNA from the plasmid pCb ΌΘΟ isolated using a mini DNA extraction kit (mini prep), digested Rui1 (to remove contaminating vector DNA) and split TeS. Dog bone, KSA: closed linear DNA from plasmid pCOS EOS. amplified by KSA, split Rui1 and split Te.
Description of sequences
8ES GO N0: 1 represents the nucleotide sequence of the DNA polymerase of the bacteriophage pSh29 from VasShik.
8ES GO N0: 2 is the amino acid sequence of the bacteriophage rSh29 DNA polymerase from VasShik encoded by 8ES GO N0: 1.
8ES GO N0: 3 is the amino acid sequence of the DNA polymerase Eeeer Uep1 from Rugosossik cr.
8ES GO N0: 4 represents the nucleotide sequence of DNA polymerase I from Bashik k1eatoShegtorSh1ik.
8EC GO N0: 5 is the amino acid sequence of DNA polymerase I of Bacillus k1eatoShegtoriyik encoded by 8ES GO N0: 4.
8ES GO N0: 6 represents the nucleotide sequence of the protelomerase of phage pSHAP1 from Na1topack.
8EC GO N0: 7 is the amino acid sequence of prothelomerase of phage rSHNAP-1 from Na1topack encoded by 8ES GO N0: 6.
8ES GO N0: 8 represents the nucleotide sequence of the protelomerase of phage RU54 from Vegkia.
8ES GO N0: 9 is the amino acid sequence of the protelomerase of phage RU54 from Vegkia, encoded by 8ES GO N0: 8.
8ES GO N0: 10 represents the nucleotide sequence of the protelomerase of phage pKK02 from K1eKk1e11a.
8EC GO N0: 11 is the amino acid sequence of phage rShK02 protelomerase from K1eBk1e11a encoded by 8ES GO N0: 10.
8ES GO N0: 12 represents the nucleotide sequence of the protelomerase of phage UR882 from UST.
8ES GO N0: 13 is the amino acid sequence of protelomerase of phage UR882 from USU, encoded by 8ES GO N0: 12.
8ES GO N0: 14 is the nucleotide sequence of the protelomerase of bacteriophage N15 from ExNepsecois (1eS) and the nucleotide sequence of a secondary immune repressor (CA).
8ES GO N0: 15 is the amino acid sequence of the protelomerase of bacteriophage N15 from Excepsois soya (1eS), encoded by 8ES GO N0: 14.
8ES GO N0: 16 is the consensus nucleotide sequence of a perfect inverted repeat present in the target sequence of the bacteriophage protelomerase.
8ES GO N0: 17 is the 22-nucleotide sequence of a perfect inverted repeat from phage N15 from E. coli and phA pKK02 from Kliebie11a.
8ES GO N0: 18 represents the 22-nucleotide sequence of a perfect inverted repeat from phage RU54 from Vegkia.
8ES GO N0: 19 represents the 22-nucleotide sequence of a perfect inverted repeat from the phage rShNAP-1 from Na1otopak.
8ES GO N0: 20 represents the 22-nucleotide sequence of a perfect inverted repeat from phage UR882 from UShtyu.
8ES GO N0: 21 represents the 14-nucleotide sequence of a perfect inverted repeat from plasmid 1pB31.16 from Wotteya DogDogGegE
8ES GO N0: 22 represents the 24-nucleotide sequence of a perfect inverted repeat from phage UR882 from UShtyu.
8ES GO N0: 23 represents the 42-nucleotide sequence of a perfect inverted repeat from phage RU54 from Vegkia.
8EC ΕΌ N0: 24 represents the 90-nucleotide sequence of a perfect inverted repeat from the phage rShNAP-1 from Na1otopak.
8ES GO N0: 25 represents the nucleotide sequence of phage N15 from E. soybean containing the protelomerase sequence of the target.
8ES GO N0: 26 is the nucleotide sequence from phA pKK02 from K1eKk1e11a containing the protelomerase target sequence.
8ES GO N0: 27 is the nucleotide sequence from phage RU54 from Vegkia containing the protelomerase target sequence.
8EC GO N0: 28 represents the nucleotide sequence from phage UR882 from UVpo. containing protelomerase target sequence.
8EC GO N0: 29 is the nucleotide sequence from the plasmid 1pB31.16 from VotneHybogdogGez containing the protelomerase target sequence.
8EC GO 0: 30 is a modified oligonucleotide primer used in TeS amplification.
8EC GO N0: 31 is a modified oligonucleotide primer used in TeS amplification.
8EC EC GO N0: 32 is a synthetic oligonucleotide containing a recognition site Teh, 1e1Kb.
8EC EC GO N0: 33 is a synthetic oligonucleotide containing the recognition site TeS, 1e1Kb.
8EC EC GO N0: 34 is a sequence of a primer used in amplification of a fragment of RAC Ό00.
8EC GO N0: 35 represents the sequence of the primer used in the amplification of the fragment RAC Ό00.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods for producing linear double-stranded covalently closed DNA, that is, closed linear DNA molecules. Closed linear DNA molecules typically contain covalently linked ends, also described as hairpin loops in which there is no pairing between the bases of the complementary DNA chains. Hairpin loops connect the ends of complementary DNA chains. Structures of this type are usually formed at the telomeric ends of chromosomes to protect against loss or damage of chromosomal DNA by connecting terminal nucleotides into a closed structure. In the examples of closed linear DNA molecules described herein, hairpin loops flank complementary paired DNA strands to form a dog bone structure (shown in FIG. 1).
The methods of the present invention relate to the high-throughput production of closed linear DNA molecules by incorporating a separate processing step that converts the amplified DNA into closed linear DNA. In addition, the methods of the present invention carry out ίη νίίτο in a cell-free medium, and as such they are not limited to the use of DNA matrices with additional sequences necessary for the propagation of bacteria. Therefore, as indicated below, the method according to the invention can be used to obtain closed linear DNA molecules that do not have vector sequences that cause problems, and are particularly suitable for therapeutic use.
Closed DNA molecules are particularly useful as therapeutic agents, that is, DNA-based drugs that can be used to экспрессη νίνο express a gene product. This is due to the fact that their covalently closed structure prevents the attack by enzymes such as exonuclease, leading to increased stability and duration of gene expression compared to open DNA molecules with exposed DNA ends. It has been demonstrated that linear double-stranded open-ended cassettes are not effective for gene expression when these cassettes are introduced into host tissues. It is believed that the cause is cartridge instability due to the action of exonucleases in the extracellular space.
Isolation of DNA ends in covalently closed structures also has other advantages. The integration of DNA ends into genomic DNA is prevented, so closed linear DNA molecules are more secure. Also, the closed linear structure prevents the concatemerization of DNA molecules in host cells, and therefore the expression level of the gene product can be controlled more precisely. The present invention relates to a ίη νίίτο cell-free method for producing closed linear DNA molecules, which includes DNA amplification on a matrix and specific treatment of amplified DNA with protelomerase.
Typically, the method of the invention can be used to produce DNA for expression of ίη νίίτο in a host cell, in particular in DNA vaccines. DNA vaccines usually encode a modified DNA form of an infectious organism. DNA vaccines are administered to a subject in which they then express a specific protein of an infectious organism, eliciting an immune response against a protein that is usually protective. DNA vaccines can also encode a tumor antigen in anti-cancer immunotherapy.
A DNA vaccine may include a nucleotide sequence encoding an antigen for treating or preventing a number of pathological conditions, including, but not limited to, cancer, allergic reactions, toxic effects and infection of pathogens, such as, but not limited to, fungi viruses, including human papillomavirus (NRU), H1U, H8U2 / H8U1, influenza virus (types A, B and C), poliovirus, respiratory syncytial virus, rhinoviruses, rotaviruses, hepatitis A virus, Norvolk virus group, enteroviruses, astroviruses, measles virus, parainfluenza virus, virus
- 4 021069 mumps, chickenpox virus, cytomegalovirus, Epstein-Barr virus, adenoviruses, rubella virus, type 1 T-cell lymphoma virus (NTLU-Ι), hepatitis B virus (IVU), hepatitis C virus (NSU), hepatitis Ό virus, poxvirus, Marburg and Ebola viruses, bacteria, including
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Rzeyosopav, Vog4e (E11a regGizzy, Briselia, Rangenza I1 and Argus lucidae, Liveris erythroderisopensis, Undisensis
Toxorrhiza op (W, Satulobacillas 515, Mogacheis (bacilli, bacteria that cause donovanosis (Kliebacidae dapiota Ibiv) and actinomycetes; fungal pathogens, including those that cause candidiasis and aspergillosis; parasitic pathogens, including parasitic pathogens, pathogens, , SgurJuvropysht. ZsvyuVuvota, Ppeitus sapia, causative agent of trichomoniasis and trichinosis.
DNA vaccines can contain nucleotide sequences encoding a virus antigen from the adenovirus family (including, for example, human adenovirus), herpes viruses (including, for example, H8U-1, H8U-2, EVU, SMU and U2U), papovaviruses (including, for example, NRU), poxviruses (including, for example, smallpox virus and vaccine virus), parvoviruses (including, for example, parvovirus B19), reoviruses (including, for example, rotavirus), coronaviruses (including, for example, 8AR8 virus), flaviviruses (including, for example , yellow fever virus, West Nile virus, dengue virus, hepatitis C virus and tick-borne encephalitis virus), picornaviruses (including poliovirus, rhinovirus and hepatitis A virus), togaviruses (including, for example, rubella virus), filoviruses (including, for example, virus Marburg and Ebola), paramyxoviruses (including, for example, parainfluenza virus, respiratory syncytial virus, mumps and measles viruses), rhabdoviruses (including, for example, rabies virus), bunyaviruses (including, for example, Gantt virus), orthomyxoviruses (including, for example, influenza A, B, and C viruses), retroviruses (including, for example, H1U and NTLU), and hepatadaviruses (including, for example, hepatitis B virus).
An antigen may refer to a pathogen responsible for a veterinary disease, and in particular relates to a viral pathogen, including, for example, reovirus (such as African horse disease virus or sheep infectious catarrh fever virus) and herpes viruses (including horse herpes virus). The antigen can be the antigen of the virus of the disease of the legs and mouth, the tick virus borne encephalitis virus, dengue virus, 8AK virus 8, West Nile fever virus and Huntaan virus. The antigen may refer to an immunodeficiency virus and may, for example, be from 81U or a feline immunodeficiency virus.
DNA vaccines obtained by the method of the invention may also contain a nucleotide sequence encoding tumor antigens. Examples of antigens associated with tumors include, but are not limited to, testicular cancer antigens, such as members of the MACE family (MACE 1, 2, 3, etc.), ΝΥ-Ε8Θ-1 and 88X-2, differentiation antigens, such as tyrosinase, dr100, P8A, Neg-2 and CEA, mutated intrinsic antigens and viral tumor antigens, such as E6 and / or E7 from oncogenic types of NRU. Additional examples of specific tumor antigens include ΜΆΡΤΗ Me1ap-A, p97, beta-HCC, SAShas, MACE-1, MACE-2, MACE-4, MACE-12, MIS1, MIS2, MIS3, MiS4, MIS18, CEA, NES, P1A , EpCat, dr75 melanoma antigen, Nkeg 8, high molecular weight melanoma antigen, K19, Ty1, Ty2, members of the pMe1 17 gene family, c-Me1, P8M (prostate mucin antigen), P8MA (prostate-specific membrane antigen), prostate secretory protein, alpha-fetoprotein, CA125, CA19.9, TAC-72, antigens VRSA-1 and VRSA-2.
Also, using the method of the invention, other types of therapeutic DNA molecules can be obtained, for example, those used in gene therapy. For example, such DNA molecules can be used to express a functional gene when the subject has a genetic disorder caused by a non-functional version of this gene. Examples of such diseases include Duchenne muscular dystrophy, cystic fibrosis, Gaucher disease, and adenosine deaminase deficiency (AEA). Other diseases where gene therapy can be used include inflammatory diseases, autoimmune, chronic and infectious diseases, including diseases such as AIDS, cancer, neurological diseases, cardiovascular diseases, hypercholesterolemia, various blood diseases, including various anemia, thalassemia and hemophilia, and emphysema. For the treatment of solid tumors, genes encoding toxic peptides (e.g. chemotherapeutic agents such as ricin, diphtheria toxin and cobra venom factor), tumor suppressor genes such as p53, genes encoding mRNA sequences that are antisense to transforming oncogenes can be expressed, antineoplastic peptides, such as tumor necrosis factor (ΤNΡ) and other cytokines, or transdominantly negative mutants of transforming oncogenes.
Also provided is the preparation of other types of therapeutic molecules by the method of the invention.
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DNA For example, using the method of the invention, it is possible to obtain DNA molecules that are transcribed into active forms of RNA, for example, small interfering RNA (siRNA).
In embodiments of the invention directed to the production of DNA molecules having therapeutic uses, the DNA matrix will typically include an expression cassette containing one or more promoter or enhancer elements and a gene or other coding sequence that encodes an RNA or protein of interest. In specific embodiments of the invention aimed at creating DNA vaccine molecules or DNA molecules for gene therapy, the DNA matrix includes an expression cassette consisting of a eukaryotic promoter operably linked to a sequence encoding a protein of interest and, optionally, an enhancer sequence and / or eukaryotic transcription termination sequence. Typically, the DNA template may be in the form of a vector, typically used to contain a gene, for example, an extrachromosomal genetic element, such as a plasmid.
A promoter is a nucleotide sequence that initiates and regulates the transcription of a polynucleotide. Promoters may include inducible promoters (when expression of a polynucleotide sequence operably linked to a promoter is induced by a substance determined by analysis, a cofactor, regulatory protein, etc.), repressed promoters (when expression of a polynucleotide sequence operably linked to a promoter is suppressed (repressed) by a chemical compound, cofactor, regulatory protein, etc.) and constitutive promoters. The term promoter or control element is intended to include the full-sized promoter regions and functional segments of these regions (for example, which control transcription or translation).
Functionally connected refers to the arrangement of elements in which the described components are arranged to perform their usual functions. Thus, this promoter, operably linked to a nucleotide sequence, is capable of expressing this sequence in the presence of appropriate enzymes. The promoter does not have to be connected directly to the sequence, provided that it expresses it. Therefore, for example, the presence of intermediate untranslated but transcribed sequences between the promoter sequence and the nucleotide sequence is possible, and the promoter sequence can still be considered functionally linked to the coding sequence. Therefore, the term functionally connected is intended to encompass any spatial arrangement or orientation of the promoter element and the DNA sequence of interest, which allows the transcription of the DNA sequence of interest to be initiated after the promoter element is recognized by the transcription complex.
According to the present invention, closed linear DNA molecules are obtained by the action of protelomerase on DNA amplified on a DNA matrix containing at least one protelomerase target sequence.
The protelomerase target sequence is a DNA sequence, the presence of which in the DNA matrix allows its transformation into closed linear DNA as a result of the enzymatic activity of protelomerase. In other words, the protelomerase target sequence is necessary for the cleavage and subsequent ligation of double-stranded DNA with protelomerase to form covalently closed linear DNA.
Typically, the protelomerase target sequence contains any perfect palindromic sequences, that is, any double-stranded DNA sequence having axial symmetry of second order rotation, also described herein as a perfect inverted repeat. As shown in FIG. 3, all protelomerase target sequences from various mesophilic bacteriophages and bacterial plasmids have a common feature - the presence of a perfect inverted repeat. The length of a perfect inverted repeat varies depending on the particular organism. In Votgeya BigbogGSP, a perfect inverted repeat has a length of 14 base pairs. In various mesophilic bacteriophages, a perfect inverted repeat has a length of 22 base pairs or more. Also in some cases, for example in the case of bacteriophage N15 from E. Thus, the central perfect inverted palindrome is flanked by sequences of inverted repeats, that is, it is the central part of the larger imperfect inverted palindrome (see Figs. 2 and 3; underlined bases indicate places in which the symmetry of inverted repeats is broken).
The protelomerase target sequence used in the invention preferably comprises a double-stranded palindromic sequence (perfect inverted repeat) of at least 14 base pairs. Preferred sequences of perfect inverted repeats include sequences 8E0 GO N0: 16-21 and their variants. 8E0 GO N0: 16 (ΝΟΑΤΝΝΤΑΝΝΟΟΝΝΤΑΝΝΑΤΟΝ) represents the 22-nucleotide consensus sequence of a perfect inverted repeat from a mesophilic bacteriophage. As shown in FIG.
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3, certain nucleotide positions of a perfect inverted repeat are conserved between different bacteriophages, while in other positions the sequence may differ. Thus, §Eph GO N0: 16 represents the minimum consensus sequence of a perfect inverted repeat for use with bacteriophage protelomerase in the method of the present invention.
For the consensus sequence defined by §Eph of GO N0: 16, §Eph of GO N0: 17 (SSATTATASOSOTSOTAATOO) is a particularly preferred perfect inverted repeat sequence for use with protomeraserase phage N15 from E. soi (§Eph GO N0: 15) and phage RyK02 from 1 <1Ebie11a (§Eph GO N0: 11). Also for the consensus defined by §Ef GO N0: 16, the sequence of §Ef GO N0: 18-20
ZEP GO N0: 18 (OSATASTASASOSOSSTAOOTATOS),
ZEF GO ΝΟ: 19 (SSATASTATASSTATAOATATSO),
Zef GO ΝΟ: 20 (OSATASTATASOTATATOATATOS), are particularly preferred sequences of perfect inverted repeats for use, respectively, with protelomerase phage ΡΥ54 from Usgkia (§Eph GO N0: 9), phage pYNAR-1 from Na1otopak (8Ef GO N0: 7) and phage UR882 from ULpo (8Eph ΙΌ N0: 13). 8Eph ΙΌ N0: 21 (ATTATATATATAAAT) is a particularly preferred perfect inverted repeat sequence for use with the proteomerase from Votgeia ligdyogsp. This sequence of perfect inverted repeat is located in the linear covalently closed plasmid 1BB31.16 contained in Votgeya LigyogGep. This 14-nucleotide sequence is shorter than the 22nucleotide consensus perfect inverted repeat for bacteriophages (8Eph GO N0: 16), which indicates that bacterial protelomerases may differ in specific target sequences from bacterial protelomerases. However, all protelomerase target sequences carry a common structural motif for a perfect inverted repeat.
A perfect inverted repeat sequence may be longer than 22 nucleotides in length, depending on the requirements of the particular protelomerase used in the method of the invention. Therefore, in some embodiments, the length of the perfect inverted repeat can be at least 30, at least 40, at least 60, at least 80, or at least 100 base pairs. Examples of such perfect inverted repeat sequences include 8Eph GO N0: 22-24 and variants thereof.
ZEF GO N0: 22 (OSSATASTATASOTATAOATATSSS)
ZEFGOYO: 23 (ASSTATTTSAOOSATASTASASOSOTAOTATOSTOSTOAAAOOOOT)
ZEF GO ΝΟ: 24 (STATATTOOOOSSASSTATOTATOSASAOOTTSOSSSSATASTATASOT
ATAOOTATOOOOOAAASTOTOSATASATAOOOOTSSSSAATATASO)
8Eph GO N0: 22-24 and their variants are especially preferred for use, respectively, with protelomerases of phage UR882 from U1bpo (8Eph GO no: 13), phage ΡΥ54 from Uegysh (8Eph GO no0: 9) and phage pYNAR-1 from Na1otopak ( 8Eph GO N0: 7).
A perfect inverted repeat can be flanked by additional sequences of inverted repeats. Flanking inverted repeats can be perfect or imperfect repeats, that is, they can be completely symmetric or partially symmetrical.
Flanking inverted repeats can adjoin directly to the central palindrome or can be separated from it. The protelomerase target sequence may include an imperfect inverted repeat sequence that contains a perfect inverted repeat sequence of at least 14 base pairs in length. An example is 8Eph GO N0: 29. The sequence of an imperfect inverted repeat may comprise a sequence of a perfect inverted repeat of at least 22 base pairs in length. An example is 8Eph GO N0: 25.
Particularly preferred protelomerase target sequences include 8Eph GO sequences N0: 25-29 or variants thereof.
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5E <? Yu N0: 25:
(TATSAOOSASAAATTOSSSSATTATASSSSSSSTATAATOSASTATTO
TOTOSTATATA)
5E <3 GO ΝΟ: 26 (ATSSOSSSSATSSATTATASOSOSSTATAATSSSSATAAATASA)
5Е0 GO ΝΟ: 27 (TAOOTSASSTATTTSAOOSATASTASOSOSSTAOOTATSSTOAAAAOO
TTASTS)
5Ε (} ΙΟΝΟ: 28:
(OSOATSSSOTTSSATASATASATOTATSSATSTSSSSATASTATASO
TATAOTATSSSSATOTTASATATOOOTATSATTSSSSATSSSOTT)
8Εζ> GO ΝΟ: 29 (TASTAAAAAATATTATATATATAATTTTTTATTASTA)
The sequences δΕΟ GO N0: 25-29 contain sequences of perfect inverted repeats described above, and additionally contain flanking sequences from the corresponding organisms.
The protelomerase target sequence containing the sequence δΕΟ GO N0: 25 or a variant thereof is preferred for use in conjunction with the TeШ protelomerase (phage N15 from E. soi) with the sequence δΕΟ GO N0: 15 or its variants. The protelomerase target sequence containing the sequence δΕΟ GO N0: 26 or a variant thereof is preferred for use in conjunction with the protelomerase of the phage РЫК02 from К1еЬ51е11а with the sequence δΕΟ Н0: 11 or its variants. The protelomerase target sequence containing the δΕΟ GO sequence N0: 27 or a variant thereof is preferable for use with the phage из54 protelomerase from Υе ^ 5 ^ η ^ а and the δΕΟ GO sequence N0: 9 or its variants. The protelomerase target sequence containing the sequence δΕΟ GO N0: 28 or a variant thereof is preferable for use with the protelomerase phage UR882 from U1bpo with the sequence δΕΟ GO # 13 or its variants. The protelomerase target sequence containing the sequence δΕΟ GO N0: 29 or a variant thereof is preferable for use in conjunction with the Votte nigella nigella protelomerase.
Variants of any palindromic or protelomerase target sequences described above include their homologs and mutants. Mutants include truncated sequences, substitutions or deletions in the sequence relative to the native sequence. A sequence variant is any sequence whose presence in the DNA matrix allows its conversion into closed linear DNA as a result of the enzymatic activity of protelomerase. This can easily be determined using the appropriate closed linear DNA formation assay. You can use any suitable analysis method described in this area. An example of a suitable analysis method is described in Epek e1 a1., ΡNΑδ (2000) 97, 7721-7726. Preferably, this option provides for the binding and activity of protelomerase, which are comparable with the properties and activity observed in the case of the native sequence. Examples of preferred variants of the palindromic sequences described herein include shortened palindromic sequences that retain the perfect repeat structure and which retain the ability to produce closed linear DNA.
However, variants of protelomerase target sequences can be modified so that they do not preserve the structure of a perfect palindrome, provided that they are able to act as substrates for the action of protelomerase.
It should be understood that an experienced person will easily determine the appropriate protelomerase targets for use in the invention based on the structural principles indicated above. Potential protelomerase target sequences can be screened for their ability to generate closed linear DNA using the assay methods described above.
The DNA template may contain more than one protelomerase target sequence, for example, two, three, four, five, ten or more target protelomerase sequences. The use of multiple protelomerase target sequences can allow short, closed, linear DNAs containing sequences of interest to be cut from a larger DNA molecule. In particular, one or more of the sequences of interest in the DNA matrix can be flanked on both sides (i.e., 5 ′ and 3 ′) by the protelomerase target sequence. The two flanking protelomerase target sequences can then mediate the excision of each short sequence of interest from amplified DNA as closed linear DNA, the object of action of protelomerase (as shown in Fig. 5). The DNA matrix may contain one or more sequences of interest (preferably expression cassettes) flanked on both sides by protelomerase target sequences. The DNA template may include two, three, four, five or more sequences of interest flanked by the protelomerase target sequences described above.
In a preferred embodiment of the invention, the method of the invention uses a DNA template containing an expression cassette flanked on both sides by a protelomerase target sequence. Preferably, the expression cassette contains a eukaryotic promoter operably linked to a coding sequence of interest and, optionally, a eukaryotic transcription termination sequence. In this embodiment, after amplification of the DNA template and contacting with the protelomerase of the invention, the expression cassette is released from the amplified matrix as closed linear DNA. As a result, excess sequences in the DNA matrix are simultaneously removed from the product.
Such redundant or additional sequences (also described as bacterial or vector sequences) may include a bacterial replication origin, bacterial selective markers (e.g., antibiotic resistance genes), and unmethylated CPO dinucleotides. Removal of such sequences creates a minimal expression cassette that does not contain additional genetic material. Also, the bacterial sequences described above may be problematic for some therapeutic approaches. For example, in mammalian cells, bacterial / plasmid DNA can cause the expression of the cloned gene to be turned off, making it impossible to obtain continuous expression of the protein of interest. Antibiotic resistance genes also used in bacterial growth can be a risk factor for human health. In addition, bacterial plasmid / vector DNA can trigger an unwanted non-specific immune response. Certain characteristics of bacterial DNA sequences, the presence of unmethylated cytosine-guanine dinucleotides, commonly known as CPO motifs, can also lead to undesirable immune responses.
In some embodiments, especially if the product — closed linear DNA — is a DNA vaccine, CPO motifs can be stored in the product sequence. This is because they provide a beneficial adjuvant effect on the immune response to the encoded protein.
Therefore, the invention relates to a ίη νίίτο method for producing a closed linear expression DNA cassette. This method includes: a) contacting a DNA matrix containing at least one expression cassette flanked on both sides by a protelomerase target sequence with at least one DNA polymerase in the presence of one or more primers under conditions conducive to amplification of the specified matrix ; and b) contacting the amplified DNA obtained in a) with at least one protelomerase under conditions conducive to the formation of a closed linear expression DNA cassette. The product — a closed linear expression DNA cassette — may comprise, consist of, or consist essentially of a eukaryotic promoter operably linked to a coding sequence of interest and, optionally, a transcription termination eukaryotic sequence. In the product - a closed linear expression DNA cassette - one or more bacterial or vector sequences, usually selected from the group consisting of:
(ί) the bacterial point of initiation of replication; (ίί) bacterial selective markers (usually antibiotic resistance genes); and (ίίί) unmethylated CPO motifs.
As indicated above, any DNA matrix containing at least one protelomerase target sequence can be amplified by the method of the invention. Therefore, although the production of DNA vaccines and other therapeutic DNA molecules is preferred, the method of the invention can be used to produce any type of closed linear DNA. The DNA matrix may be double-stranded (ds) or single-stranded (sc) DNA. The double-stranded DNA matrix may be an open circular double-stranded DNA, a closed circular double-stranded DNA, an open linear double-stranded DNA, or a closed linear double-stranded DNA. Preferably, the matrix is a closed circular double-stranded DNA. Closed ring dsDNA templates are particularly preferred for use with KSL DNA polymerases. The ring dsDNA template can be a plasmid or other vector commonly used to contain the gene when bacteria multiply. Therefore, the method of the invention can be used to amplify any commercially available plasmid or other vector, such as a commercially available DNA-based drug, and then to convert the amplified vector DNA into closed linear DNA.
Open ring dsDNA can be used as a template when the DNA polymerase is a polymerase with a chain displacement activity that can initiate amplification from a DNA strand having a break. In this embodiment, the template can be preincubated with one or more enzymes that introduce single stranded breaks in the DNA strand in the template in one or more places. Closed linear dsDNA can also be used as a template. The closed linear dsDNA template (starting material) may be identical to the product of the closed linear DNA. When used as a template, closed linear DNA can be incubated under denaturing conditions to form single-stranded circular DNA before being introduced into conditions or under conditions conducive to amplification of template DNA.
As indicated above, the DNA template typically includes the expression cassette described above, that is, contains, consists of, or essentially consists of a eukaryotic promoter, operably linked to a sequence encoding a protein of interest, and, optionally, a eukaryotic sequence transcription termination. Optionally, the expression cassette may be a minimal expression cassette as defined above, that is, one or more bacterial or vector sequences typically selected from the group consisting of: (ί) a bacterial replication initiation point are missing; (ίί) bacterial selective markers (usually antibiotic resistance genes); and (ίίί) unmethylated CpSmotives.
The DNA template may be provided in an amount sufficient for use in the process by any method known in the art. For example, a DNA template can be obtained using polymerase chain reaction (PCR). If the DNA matrix is dsDNA, then it is used in amplification in the form of denatured single-stranded DNA obtained by preliminary incubation at a temperature of at least 94 ° C. Therefore, the method of the invention preferably includes the step of denaturing the dsDNA template to produce single stranded DNA. Alternatively, dsDNA may be provided in double-stranded form. In the reaction, the entire DNA template or its selected region can be amplified.
Contact the DNA matrix with at least one DNA polymerase under conditions conducive to amplification of the specified matrix. Any DNA polymerase may be used. Any commercially available DNA polymerase is suitable for use in the method of the invention. You can use two, three, four, five or more different DNA polymerases, for example one that provides a corrective function, and one or more others that do not have a corrective function. DNA polymerases with different mechanisms of action can be used, for example polymerases with chain displacement activity, and DNA polymerases that replicate DNA in other ways. A suitable example of a DNA polymerase that does not have chain displacement activity is T4 phage DNA polymerase.
High stability of the DNA polymerase is preferable so that its activity is not significantly reduced as a result of prolonged incubation under the conditions of the method. Therefore, it is preferable that the enzyme has a long half-life in the range of process conditions, including, but not limited to, temperature and pH. It is also preferred that the DNA polymerase has one or more characteristics suitable for the manufacturing method. Preferably, the DNA polymerase has high accuracy, for example, due to corrective activity. In addition, high processability of DNA polymerase, high chain displacement activity and low Kt values for 6ΝΤΡ and ΌΝΆ are preferred. DNA polymerase is capable of using circular and / or linear DNA as a template. DNA polymerase is able to use dsDNA or ssDNA as a template. Preferably, the DNA polymerase does not exhibit non-specific exonuclease activity.
An experienced professional can determine whether a given DNA polymerase has the characteristics described above by comparing it with the properties exhibited by commercially available DNA polymerases, for example, pY29, Eser Ushch® and DNA polymerase I from Vasilik ChsagoShsgtorNPik (ΒδΙ). the sequences of which are given in §EC EC GO N0: 2, 3 and 5, respectively. HF DNA polymerase I is commercially available from the company №θ№ Ep§1apb Vy1a8, 1ps. High processivity usually refers to the average number of nucleotides added by the DNA polymerase enzyme per association / dissociation with the matrix, i.e. the length of the fragment synthesized from the primer obtained in one association event.
Polymerases with chain displacement activity are preferred. Preferred polymerases with chain displacement activity are Ρ1ιί29 (8EC ΙΌ N0: 2), Eser Usp1 (8EC ГО GO N0: 3) and Hf DNA polymerase I (8EC ΙΌ N0: 5) or any of them. Sequence Variants §EC EC GO N0: 2, 3, and 5 may be variants that are described below for protelomerase enzymes. The term chain displacement is used herein to describe the ability of a DNA polymerase to replace complementary chains when it encounters a double-stranded DNA region during DNA synthesis. It should be understood that amplification methods with chain displacement differ from PCR based methods in that denaturation cycles are not required for efficient DNA amplification, since double-stranded DNA is not an obstacle to the continuous synthesis of new DNA chains. In contrast, PCR requires denaturation cycles during the amplification process (that is, raising the temperature to 94 ° C or higher) to melt double-stranded DNA and produce new single-stranded matrices.
Preferably, the DNA polymerase with chain displacement activity used in the method of the invention has a processivity (length of the fragment synthesized from the primer) of at least 20 kb, more preferably at least 30 kb at least 50 kb or at least 70 kb or more. In particularly preferred embodiments of the invention, a DNA polymerase with a chain displacement activity has a processivity comparable to that of a DNA polymerase pH29 or a higher processivity.
Preferred chain extrusion replication is rolling ring amplification (KSA). The term KSA describes the ability of KSA-type DNA polymerases (also referred to as KSA polymerases in the present description) to continuously move along the ring chain of a DNA matrix, while extending the hybridized primer. This leads to the formation of linear single-stranded products with multiple repeats of amplified DNA. These linear single-stranded products serve as the basis for multiple events of hybridization, primer extension and chain displacement, resulting in the formation of concatemeric double-stranded DNA products also containing multiple repeats of amplified DNA. Thus, multiple copies of each amplified single DNA element are present in the products of concatemer double-stranded DNA.
KSA polymerases are particularly preferred for use in the method of the present invention. KSL type chain replication replication products typically require complex processing to release single DNA elements. The present invention is advantageous in that the use of the catalytic functions of protelomerase allows this treatment to be carried out in a single step. The use of protelomerase also directly generates the desired closed DNA structure without the need for additional processing steps to form molecules having this structure.
For amplification according to the invention, it is preferable that the DNA template is also in contact with one or more primers. Primers may be non-specific (e.g., with a random sequence) or may be specific to one or more sequences contained in the DNA template. Preferably, the primers have a random sequence for the possibility of non-specific initiation in any part of the DNA template. This gives a high amplification efficiency as a result of multiple initiation reactions with each matrix chain. Examples of random sequence primers are hexamers, heptamers, octamers, nonamers, decamers, or longer sequences, for example 12, 15, 18, 20, or 30 nucleotides in length. A primer with a random sequence can be from 6 to 30, from 8 to 30, or from 12 to 30 nucleotides in length. Random sequence primers are typically supplied as a mixture of oligonucleotides, which are all possible combinations, for example, hexamers, heptamers, octamers, or nonamers in a DNA matrix.
In other embodiments, the primers are specific. This means that they have a sequence complementary to the sequence of the DNA template with which it is desirable to initiate amplification. In this embodiment, a pair of primers can be used to specifically amplify a portion of a DNA template that is internal to the binding sites of the two primers. Primers may be unlabeled or may include one or more labels, for example, radionuclides or fluorescent dyes. Primers may also contain chemically modified nucleotides. The length and sequence of primers can usually be selected based on the annealing temperature, that is, the ability to bind the matrix at the temperature used in the amplification step.
Contacting the DNA template with DNA polymerase and one or more primers occurs under conditions conducive to annealing of the primers on the DNA matrix. Conditions include the presence of single-stranded DNA, allowing hybridization of the primers. Conditions also include temperature and a buffer allowing annealing of the primer on the matrix. Appropriate annealing / hybridization conditions can be selected depending on the nature of the primer. An example of preferred annealing conditions used in the present invention includes a buffer: 30 mM Tg5-HC1. pH 7.5, 20 mM KCl, 8 mM MgCl<sub>2</sub>. Annealing can be carried out after denaturation by gradual cooling to the desired reaction temperature.
After contact of the DNA template with the DNA polymerase and one or more primers, an incubation step follows under conditions conducive to amplification of the template. Preferably, the conditions contribute to the amplification of the matrix by displacing the replicated chains by replicating another chain according to the type of chain displacement. Conditions include the use of any temperature that allows amplification of DNA, usually in the range from 20 to 90 ° C. A preferred temperature range may be from about 20 to about 40, or from about 25 to about 35 ° C.
Typically, the appropriate temperature is selected based on the temperature at which a particular DNA polymerase has optimal activity. This information is usually known and is included in the general knowledge of an experienced professional. For example, when using p! 29 DNA polymerase, a suitable temperature range will be from about 25 to about 35 ° C, preferably about 30 ° C. An experienced specialist will simply determine the appropriate temperature for effective amplification according to the method according to the invention. For example, the method can be carried out in a temperature range and control the output of amplified DNA to identify the optimal temperature range for a given DNA polymerase.
Other conditions conducive to amplification of the DNA template include the presence of DNA polymerase and one or more primers. Conditions also include the presence of all four deoxyribonucleotides (άΝΤΡ), ATP, TTP, CTP and OTP, the corresponding buffering agents / pH, and other factors that are required for enzyme function or stability. Suitable conditions include any conditions that ensure the activity of DNA polymerases known in this field.
For example, the pH may range from 3 to 10, preferably from 5 to 8, or be about 7, for example about 7.5. The pH can be maintained in this range using one or more buffering agents. Such buffers include, but are not limited to, ME8, B18-TG18, AEA, ACE8, P1PE8, MOB8, MOR8, MOR8O, B18-Tg18 Rgorape, BE8, TE8, HEPE8, ΌΙΡ8Ο, TAR8O, Tp / ta, HEPPO, POP8O, TEM, EPP8, Tpsche, O1u-O1u, Vyusha, NERV8, TAP8, AMPO, TAV8, AMP8O, CHE8, САР8О, АМР, САР8, САВ8, phosphate buffer, citric acid-sodium hydrogen phosphate buffer, sodium acetate acetic acid buffer, sodium acetate buffer -acetic acid, imidazole buffer and sodium carbonate sodium bicarbonate buffer. The reaction may also contain divalent metal salts, such as, but not limited to, magnesium salts (MD<sup>2</sup>') and manganese (MP<sup>2</sup>'), including chlorides, acetates and sulfates. Monovalent metal salts such as sodium salts and potassium salts, for example potassium chloride, may also be included. Other salts that may be included are ammonium salts, in particular ammonium sulfate.
Detergents may also be included. Examples of suitable detergents include Thyop X100, T \\ ehp 20 and derivatives of any of them. Stabilizing agents may also be included in the reaction. You can use any stabilizing agent, in particular bovine serum albumin (BSA), and other stabilizing proteins. Reaction conditions can also be improved by the addition of agents that unwind the DNA helix and make matrix denaturation easier. Such agents include, for example, dimethyl sulfoxide (DMSO), formamide, glycerin and betaine.
It should be understood that an experienced specialist is able to modify and optimize the amplification and incubation conditions for the method according to the invention, based on their general knowledge. Similarly, specific concentrations of specific agents can be selected based on previous examples in the art and further optimized based on general information. For example, a suitable reaction buffer in KSA-based methods in this area is a buffer: 50 mM TY8-HC1, pH 7.5, 10 mM MdC1<sub>2</sub>, 20 mM (NH4)<sub>2</sub>8Ο4, 5% glycerol, 0.2 mM BSA, 1 mM άΝΊΚ The preferred reaction buffer used in the KSA amplification according to the invention is a buffer: 35 mM TG18-HC1, 50 mM KS1, 14 mM MDS1<sub>2</sub>, 10 mm ^<sub>4</sub>)<sub>2</sub>8O<sub>4</sub>, 4 mM OTT, 1 mM YETR. This buffer is particularly suitable for use with the KSA polymerase pY29.
Reaction conditions may also include the use of one or more additional proteins. The DNA template can be amplified in the presence of at least one pyrophosphatase, such as inorganic yeast pyrophosphatase. Two, three, four, five or more different pyrophosphatases may be used. These enzymes are capable of destroying the pyrophosphate produced by DNA polymerase from DKTR during chain replication. The accumulation of pyrophosphate in the reaction can inhibit DNA polymerase and reduce the speed and effectiveness of DNA amplification. Pyrophosphatases can break down pyrophosphate to non-inhibitory phosphate. An example of a suitable pyrophosphatase for use in the method of the present invention is pyrophosphatase from 8assyagotus 8 sesuv ^ 8 ^ ae, commercially available from the company No. ν д д 1 1 Д В ю,,,,, 1 ps.
In the method of the invention, any single-stranded DNA binding protein (88BP) can be used to stabilize single-stranded DNA. 88BP proteins are essential components of living cells and are involved in all processes including ssDNA, such as DNA replication, repair, and recombination. In these processes, 88BP binds the temporarily formed ssDNA and can help stabilize the structure of ssDNA. An example of a suitable 88BP for use in the process of the present invention is the T4 phage 32 gene protein, commercially available from # v EpnapAnBy1a8, 1ps.
In addition to the amplification step, the method of the invention also includes the step of producing closed linear DNA. Amplified DNA contacts at least one protelomerase under conditions conducive to the production of closed linear DNA. This simple processing step, based on the properties of protelomerase, gives an advantage over other methods used to produce closed linear DNA molecules. The amplification and processing steps can be carried out simultaneously or sequentially. However, it is preferable that the amplification and processing steps are carried out sequentially, the processing step being carried out after the amplification step (i.e., on amplified DNA).
The protelomerase used in the invention is any polypeptide capable of cleaving
- 12 021069 and reconnect the matrix containing the target site of the protelomerase to obtain a molecule of covalently closed linear DNA. Thus, protelomerase has the functions of cleavage and ligation of DNA. Enzymes with protelomerase activity have also been described as telomereresolvases (for example, in Votgea ligidogep). A typical protelomerase substrate is circular double-stranded DNA. If such DNA contains a protelomerase target site, the enzyme can cut DNA at this site and ligate the ends, creating a linear double-stranded covalently closed DNA molecule. Requirements for target sites of protelomerase are described above. Also, as indicated above, the ability of a given polypeptide to catalyze the production of closed linear DNA from a matrix containing a protelomerase target site can be determined using any appropriate assay method described in this field.
Protelomerases are also described in bacteriophages. In some lysogenic bacteria, bacteriophages exist as extrachromosomal DNA, which is a linear double-stranded molecule with covalently closed ends. The replication of this DNA and the preservation of covalently closed ends (or telomeric ends) depend on the activity of the protelomerase enzyme. The role of protelomerase in viral DNA replication is illustrated in FIG. one. An example of this catalytic activity is the activity of the Te1Y enzyme from bacteriophage N15, which infects Exxepsia co11. TES recognizes a specific nucleotide sequence in circular double-stranded DNA. This sequence is a slightly imperfect inverted palindromic structure called 1e1Pb containing two halves, 1e1P and 1e1b, flanking a 22-nucleotide inverted perfect repeat (1e1O) (see Fig. 2). Two 1e1Pb sites are formed in circular double-stranded DNA as a result of the initial activity of a specific DNA polymerase acting on linear prophage DNA. TeS turns this circular DNA into two identical linear prophage DNA molecules, completing the replication cycle. 1e1P and 1e1b contain the closed ends of the linear prophage DNA, allowing further DNA replication in a similar way.
The method according to the invention requires the use of at least one protelomerase. The method according to the invention may include the use of more than one protelomerase, for example two, three, four, five or more different protelomerases. Examples of suitable protelomerases include protelomerases from bacteriophages, such as RNLR-1 from Na1 topotactic escatappa (SEC GO No. 0: 7), ΡΥ54 from 51σ51t ep1go1u11sa (SEC Go GO0: 9), pYK02 from K1ebk1e11a oxyuza (§11) and UR882 from UVpo cr. (§ETs GO N0: 13) and N15 from Excepsoi Soi (§ETS GO N0: 15) or variants of any of them. Especially preferred is the use of bacteriophage N15 protelomerase (§EC GO 0: 15) or a variant thereof. Options 8E0 GO N0: 7, 9, 11, 13 and 15 include their homologues and mutants. Mutants include truncated sequences, substitutions or deletions in the sequence relative to the native sequence. The variant should produce a closed linear DNA from a matrix containing the site of the protelomerase target described above.
Any homologues referred to herein are typically functional homologs and typically have a homology of at least 40% with the corresponding region of the native protein. Homology can be determined using known methods. For example, in the IAOSO Raskade software package there is the BE8TP1T program, which can be used to calculate homology (for example, using its default settings) (Eeeeeeee! A1. (1984) No. s1eyu Asyyk Rekeagsy 12, 387395). The algorithms RGOEIR and BABT can be used to calculate homology or sequence alignment (usually with their default settings), for example, as described in Aiksi1 8.P. (1993) 1. Mo1. Euo1. 36: 290-300; Aiksi1, 8.R. e! a1. (1990) 1. Mo1. Vu1. 215: 403-10. Sequence analysis software using the BABA algorithm is generally available at the National Center for Biotechnological Information (S1p: // \ y \ y \ UL1sY.n1tlpido \ 7).
Algorithm BAGT performs a statistical analysis of the similarity between two sequences; see, for example, Katyi apy Aiksiyi1 (1993) Propos. No. And. Asai. §СЕ И8А 90: 5873-5787. One of the similarity parameters provided by the BABA algorithm is the least probability of the sum (P (C)), which indicates the probability with which a coincidence between two nucleotide or amino acid sequences will occur by chance. For example, a sequence is considered similar to another sequence if the least probability of the sum in comparing the first sequence with the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A polypeptide variant contains a sequence (or consists of a sequence) that has at least 40% identity with the native protein. In preferred embodiments of the invention, the variant sequence may have at least 55, 65, 70, 75, 80, 85, 90%, and more preferably at least 95, 97 or 99% homology with a particular region of the native protein for at least 20 preferably at least 30, for example at least 40, 60, 100, 200, 300, 400 or more continuous amino acids, or even throughout the sequence of the variant. Alternatively, the sequence variant may have at least 55, 65, 70, 75, 80, 85, 90%, and more preferably at least 95, 97 or 99%
- 13 021069 homology with a full-sized native protein. Typically, a sequence variant differs from a significant region of a native protein by at least or not more than 2, 5, 10, 20, 40, 50, or 60 mutations (each of which may be a substitution, insertion, or deletion). A variant of the sequence of the invention may have a percentage of identity with a particular region of a full-sized native protein that matches any of a certain percentage of homology (i.e., it may have at least 40, 55, 80 or 90%, and more preferably at least 95, 97 or 99% identity) over any length of the sequence described above.
Options for native protein also include shortening the sequence. Any shortening of the sequence may be used provided that the variant is still capable of producing the closed linear DNA described above. The shortening of the sequence is usually carried out to remove sequences that are not necessary for catalytic activity and / or do not affect the conformation of the folded protein, in particular the folding of the active site. Shortening the sequence is also chosen to improve the solubility of the protelomerase polypeptide. Suitable truncated sequences can be easily identified by the systematic removal of sequences of different lengths from the Ν- or C-terminus.
Variants of the native protein further include mutants that have one or more, for example 2, 3, 4, 5-10, 10-20, 20-40 or more amino acid insertions, substitutions, or deletions relative to a particular region of the native protein. Deletions and insertions are preferably carried out outside the catalytic domain. The inserts are usually carried out at the Ν- or C-ends of the sequence obtained from the native protein, for example, for recombinant expression. Substitutions are also usually carried out in areas that are not necessary for catalytic activity and / or do not affect the conformation of the folded protein. Such substitutions can be made to improve the solubility or other characteristics of the enzyme. Although this is usually not preferred, substitutions can also be made in the active site or in the second sphere, that is, substitutions can be made for residues that affect or are in contact with the position or orientation of one or more amino acids in the active site. These replacements may be made to improve catalytic properties.
Substitutions are preferably introduced into one or more conservative changes in which the amino acids are replaced by other amino acids with a similar chemical structure, similar chemical properties or similar side chains. The introduced amino acids can have polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge, similar to the amino acids that they replace. Alternatively, a conservative substitution may introduce another amino acid that is aromatic or aliphatic, instead of the aromatic or aliphatic amino acid previously present. Conservative amino acid substitutions are well known in the art and can be selected in accordance with the properties of the 20 essential amino acids described in Table. BUT.
Table a
Chemical properties of amino acids
<td>A1a</td><td>Aliphatic, hydrophobic, neutral</td><td>Honey</td><td>Hydrophobic, neutral</td>
<td>Suz</td><td>Polar, hydrophobic, neutral</td><td>Azp</td><td>Polar, hydrophilic, neutral</td>
<td>Agr</td><td>Polar, hydrophilic, charged {-)</td><td>Rgo</td><td>Hydrophobic, neutral</td>
<td>31and</td><td>Polar, hydrophilic, charged (-)</td><td>C1p</td><td>Polar, hydrophilic, neutral</td>
<td>RNe</td><td>Aromatic hydrophobic, neutral</td><td>Agd</td><td>Polar, hydrophilic, charged (+)</td>
<td>61y</td><td>Aliphatic, neutral</td><td>Zeg</td><td>Polar, hydrophilic, neutral</td>
<td>ΗΪ3</td><td>Aromatic, polar, hydrophilic, charged (+)</td><td>Thr</td><td>Polar, hydrophilic, neutral</td>
- 14 021069
<td>Not</td><td>Aliphatic, hydrophobic, neutral</td><td>Ya1</td><td>Aliphatic, hydrophobic, neutral</td>
<td>Bz</td><td>Polar, hydrophilic, charged {+)</td><td>Tgr</td><td>Aromatic hydrophobic, neutral</td>
<td>Bie</td><td>Aliphatic, hydrophobic, neutral</td><td>Tug</td><td>Aromatic, polar, hydrophobic</td>
It is particularly preferred that the variant is capable of producing the closed linear DNA described above with efficacy comparable to or identical to the native protein.
As indicated above, it is preferable that the amplification of the DNA according to the method according to the invention was carried out by DNA polymerase with chain displacement activity, more preferably with K. CL-DNA polymerase. The combination of KSL-DNA polymerase and protelomerase in the ίη νίΐτο cell-free method provides amazing efficiency and ease of obtaining closed linear DNA.
As noted above, long linear single-stranded DNA molecules are initially formed in chain displacement reactions, which then serve as new matrices, resulting in the formation of double-stranded molecules (Fig. 4). Double-stranded molecules contain a continuous series of tandem units of amplified DNA formed as a result of the processive action of polymerases with chain displacement activity (concatemer). These concatemer DNA products contain numerous repeats of amplified template DNA. The concatemer generated in the method of the invention therefore contains many units of a sequence amplified from a DNA template. The concatemer may contain 10, 20, 50, 100, 200, 500, 1000 or more units of the amplified sequence, depending on the length of one amplified unit. The size of the concatemer may be at least 5 kbp, at least 10 kbp, at least 20 kbp, more preferably at least 30 kbp, at least 50 kbp; at least 70 kbp or more.
In many embodiments, for example, in the manufacture of DNA-based drugs, the use of single elements of amplified DNA is required. Therefore, for the release of single elements of amplified DNA, the processing of such concatemers is necessary. To convert this concatemer DNA into single elements of amplified DNA, it must be cut in a certain position, and the ends of paired chains must be re-ligated. This can usually be done by including restriction endonuclease sites in the DNA template. Thus, for the cleavage of restriction endonuclease recognition sites and the release of single elements, restriction endonucleases with a concatemer can be incubated. Open linear double-stranded DNA resulting from restriction endonucleases can then be incubated with the DNA ligase enzyme in order to covalently connect (close) single DNA elements.
In accordance with the present invention, the conversion of concatemeric DNA into closed linear single DNA elements is achieved using only one protelomerase enzyme. This provides the preferred simplicity and economy of the method for producing closed linear DNA molecules. First, the cleavage and subsequent ligation of single elements is achieved by incubation with a single enzyme. Secondly, single elements are also released having the desired closed linear structure, and thus, no additional processing steps are required to obtain this structure (i.e., from a covalently closed ring single DNA element).
DNA amplified on a DNA template is incubated with at least one protelomerase under conditions conducive to the production of closed linear DNA. In other words, the conditions facilitate the cleavage and subsequent ligation of double-stranded DNA containing the protelomerase target sequence, with the formation of a covalently closed linear DNA with hairpin-shaped ends. Conditions conducive to the production of closed linear DNA include the use of any temperature that allows the production of closed linear DNA, usually in the range from 20 to 90 ° C. Preferably, the temperature may be in the range from 25 to 40 ° C, for example from about 25 to about 35 ° C, or to be about 30 ° C. The appropriate temperature for a particular protelomerase can be selected based on the above principles regarding the temperature conditions for DNA polymerases. A suitable temperature for using protelomerase ΤΟΝ from the bacteriophage E. soy (sequence 8EO GO ΝΟ: 15) is from about 25 to about 35 ° C, for example about 30 ° C.
Conditions conducive to the production of closed linear DNA also include the presence of protelomerase and the corresponding buffering agents / pH and other factors that are necessary for the enzyme to function and be stable. Suitable conditions include any conditions used to ensure the activity of protelomerase enzymes known in the art. For example, when using protelomerase ΤΟΝ of bacteriophage N15 from E. soy, a suitable buffer may be: 20
- 15 021069 mM TP8-HC1, pH 7.6; 5 mm CaCl<sub>2</sub>; 50 mM potassium glutamate; 0.1 mM EETA; 1 mM dithiothreitol (ETT). Agents and conditions for maintaining optimal activity and stability can also be selected from the conditions given for DNA polymerases.
In some embodiments of the invention, it is possible to use conditions that provide protelomerase activity that are the same as those used for DNA amplification. In particular, the use of the same conditions is described for the case where DNA amplification and protelomerase treatment are carried out simultaneously or sequentially. In other embodiments of the invention, it may be necessary to change the reaction conditions if the conditions used to ensure optimal DNA polymerase activity result in suboptimal protelomerase activity. Removing specific agents and changing reaction conditions can be accomplished by filtration, dialysis, and other methods known in the art. It will be easy for an experienced person to determine conditions giving optimal DNA polymerase activity and / or protelomerase activity.
In a particularly preferred embodiment, when using KSA DNA polymerase, preferably pY29, in DNA amplification, the DNA amplification is carried out in a buffer essentially identical or essentially consisting of 35 mM TY8-HCl, 50 mM KCl, 14 mM M § C1<sub>2</sub>10 mM (NN<sub>4</sub>)<sub>2</sub>§0<sub>4</sub>, 4 mM OTT, 1 mM 6TR; at a temperature of 25 to 35 ° C, for example about 30 ° C. The protelomerase treatment step can then be carried out, preferably using TeS, and / or preferably under buffer conditions substantially identical or essentially consisting of 20 mM TgE-HC'k pH 7.6; 5 mm CaCl<sub>2</sub>; 50 mM potassium glutamate; 0.1 mM EETA; 1 mM dithiothreitol (OTT); at a temperature of 25 to 35 ° C, for example about 30 ° C.
All enzymes and proteins for use in the method according to the invention can be obtained recombinantly, for example, in bacteria. All recombinant expression methods known to the skilled person can be used. A plasmid or other form of recombinant vector containing a nucleotide sequence encoding a protein of interest can be introduced into bacteria to express the encoded protein. For example, for the expression of sequences of §ETs GO N0: 2, 5, 7, 9, 11, 13 or 15, the vector, respectively, may contain sequences of §ETs GO N0: 1, 4, 6, 8, 10, 12 or 14. Then the expressed protein is usually purified, for example, using an affinity tag, in a sufficient amount and transferred to a form suitable for use in the method of the invention. Methods for producing recombinant proteins are included in the total amount of knowledge of an experienced specialist. The foregoing applies to any protein described herein.
Amplified DNA obtained by contacting the DNA template with DNA polymerase can be purified before contact with protelomerase. Thus, the method of the invention may further include the step of purifying the DNA amplified on the DNA template. However, in a preferred embodiment of the invention, the method is carried out without purification of the amplified DNA before contacting with protelomerase. This means that the amplification and processing steps can be carried out sequentially, usually in a single container or solution. In some such embodiments of the invention, the method includes adding a buffer that provides protelomerase activity, that is, provides conditions conducive to the formation of closed linear DNA.
After obtaining closed linear DNA as a result of the action of protelomerase, the method of the invention may further include the step of purifying the linear covalently closed DNA product. The above purification is usually carried out to remove unwanted products. Cleaning can be carried out by any means known in the art. For example, processing amplified DNA or linear covalently closed DNA may include phenol / chloroform purification of nucleic acids or the use of selectively nucleic acid binding columns, such as those available from Οίη ^ η. An experienced person can easily determine the appropriate purification techniques for use in the isolation of amplified DNA.
After obtaining and purifying the linear covalently closed DNA in a sufficient amount, the method may further include introducing it into the DNA composition (a composition containing DNA), for example, into a therapeutic DNA composition.
The therapeutic DNA composition will be the type of therapeutic DNA molecule mentioned above. Such a composition will contain a therapeutically effective amount of DNA in a form suitable for administration by the desired route, for example, as an aerosol, as an injection composition or a composition suitable for oral administration, mucosal or topical administration.
The introduction of DNA into a standard pharmaceutical preparation can be carried out using standard chemical methods and techniques for the manufacture of pharmaceutical compositions that are known to an experienced specialist in this field. Any pharmaceutically acceptable carrier or excipient may be used.
Auxiliary materials, such as wetting or emulsifying agents, pH buffering agents and the like, may be present in the excipient or carrier. These excipients, carriers and auxiliary materials are generally pharmaceutical agents that can be administered without nonspecific toxicity and which, in the case of vaccine compositions, will not elicit an immune response in the individual receiving the composition. A suitable carrier may be a liposome.
Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Also pharmaceutically acceptable salts, for example, salts of inorganic acids, such as hydrochlorides, hydrobromides, phosphates, sulfates and the like, and salts of organic acids, such as acetates, propionates, malonates, benzoates and the like, can be included in the present composition. It is also preferred, although not necessary, that the preparation contains a pharmaceutically acceptable excipient that serves as a stabilizer, in particular for a peptide, protein or other similar molecules, if they are to be included in the composition. Examples of suitable carriers that also act as stabilizers for peptides include, without limitation, pharmaceutical grade dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran and the like. Other suitable carriers include, but are not limited to, starch, cellulose, sodium or calcium phosphates, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEGs), and combinations thereof. A comprehensive description of pharmaceutically acceptable excipients, carriers, and excipients is found in the publication НΛ RNLNMLSETE 1LE 8C1EXX1L (Mask Rieb. Co., Co., 1991), which is incorporated herein by reference.
The method according to the invention is carried out ίη νίίΓο in a cell-free environment. Therefore, the method is carried out in the absence of host cells, and it usually involves the use of purified enzymatic components. Accordingly, amplification of template DNA and treatment with protelomerase are usually carried out by contacting the reaction components in solution in a suitable container. Optionally, the individual components may be in immobilized form, for example, may be attached to a solid support.
It should be understood that the method according to the invention can be carried out at any scale. However, it is preferable to carry out the method of DNA amplification on a commercial or industrial scale, that is, to obtain amplified DNA in milligrams or in larger quantities. Preferably, the method can produce at least 1 mg, at least 10 mg, at least 20 mg, at least 50 mg, or at least 100 mg of amplified DNA. It is also preferable to obtain the final product, closed linear DNA, from amplified DNA in milligram or large quantities. Preferably, using the method it is possible to obtain at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, or at least 100 mg closed linear DNA.
The invention further relates to a kit containing the components required for carrying out the method according to the invention. This kit includes at least one DNA polymerase, at least one protelomerase and, optionally, instructions for using them in the method described herein. A kit may contain two, three, four, five or more different DNA polymerases.
Preferably, the kit contains at least one DNA polymerase with chain displacement activity, even more preferably ΚCΑ DNA polymerase. It is particularly preferred that the kit contains p1y29 DNA polymerase (LES ΙΌ ΝΟ: 2), Veer VeS® DNA polymerase (LES GO ΝΟ: 3) or Ββί 1 DNA polymerase (LES GO 5: 5), or a variant of any of them. In some embodiments, DNA polymerases that replicate DNA by other methods may also be included. The kit includes at least one protelomerase. A kit may include two, three, four or more different protelomerases. Protelomerase can be chosen from any of 8EC GO ΝΟ: 5, 7, 9, 11, 13 or 15, or their variants. It is particularly preferred that the kit includes включе1N bacteriophage Ν15 protelomerase from E. soy (LES GO ΝΟ: 15) or a variant thereof.
The kit may also include at least one single-stranded DNA binding protein (88BP). Preferred 88BP is the protein of gene 32 of the T4 phage, commercially available from No. \ ν Epn1apy Vy1a5, 1ps. Two, three, four or more different 88BPs may be included in the kit. The kit may further include pyrophosphatase. A preferred pyrophosphatase is pyrophosphatase of 8. ce1 ^ 151ae, commercially available from No. v Epnapia Vy1ab, 1ps. Two, three, four, five or more different pyrophosphatases may be included in the kit. The kit may include any DNA polymerase, protelomerase, 88BP or pyrophosphatase described herein. The kit may also include ## GR, appropriate buffers, and other factors that are necessary for the activity or stability of the DNA polymerase and / or protelomerase described above.
Examples
Example 1. The expression of Τе1N and the creation of vector constructs containing sequences of protelomerase targets.
Τе1N was amplified from a commercially available cloning vector, ρίΑΖΖ (bis1dep), using modified oligonucleotide primers
- 17 021069
RT1R 5 'ATOASSAAOOTAAAAATSOOTO 3' (Zef GO ΝΟ: 30)
RT1K 5 'TTAOOSTOTAOOTASSTTTSSSSAT 3' (ZE <5 GO ΝΟ: 31) for directional cloning while maintaining the reading frame into a commercially available vector, pOE30 (01adep). This system allows the induced expression of proteins with 6 Ηίδ residues at the Ν end under the control of the 1ac promoter, while simultaneously providing strong trans-repression from the plasmid pKEP4 expressing the 1ac1 gene. A number of possible recombinant clones have been identified in E. coli strain M15. and by sequencing, the insertion of ΤβΙΝ was confirmed while maintaining the reading frame. Six clones were further characterized in small-scale expression induction experiments. All clones expressed a protein with a weight of 74.5 kDa, which corresponded to the molecular weight of the recombinant protelomerase Τβ1Ν.
Τβ1Ν was expressed in strain M15 E. coP / pEPEP4, inducing protein expression from plasmid pOE30 with ΙΡΤΟ; and the cells after induction were treated with ultrasound (6 pulses of 30 s at 100% power) and centrifuged (30 min at 25000 d), which gave an insoluble and soluble fraction of the cell lysate. Gel electrophoresis analysis revealed the presence of Το1Ν in the soluble fraction. Purification of O2O2 was carried out on a Shk ^ a column using the Ak1a Prnne chromatography system (OE NeBaSate) with elution in a 0-100% (0.5 M) imidazole gradient. Purified Το1Ν was dialyzed to remove imidazole and stored in buffer: 10 mM ίτίδ-ΗΟ, pH 7.4, 75 mM IaCl, 1 mM 0,1, 0.1 mM ΕΌΤΑ and 50% glycerol.
Vector constructs to confirm the activity of Νο1 созданы were created by directed cloning of synthetic oligonucleotides containing the recognition site Το1Ν, 1с1РБ
KY
Z'ASSTTTATSAOOSASASAATGSSSSATTATASOSOSOTATAAOOOOSTATT OSTSTSTOATAO 3 '(ERL) GO ΝΟ: 32)
CT2
5'OATSSTATSAOOSASASAATAOOTSSATTATASSSSOTOTAATSSOSAATT OTOTSSTOATAA 3 '(5Е <} ГО ΝΟ: 33) at the sites of WatH1 and ΗίηάΙΙΙ of plasmids pIS18 and pBK329. RIS18 has an access number in the database ОепЬапк б09136, and it can be purchased at RegteShak, catalog number 8 №0051; pVK329 has an access number in the Opepap database - 101753, and it can be purchased from the company Ό8ΜΖ, catalog number 5590.
Additionally, for transfection studies, two copies of recognition sites (1e1RB) were cloned into a vector for luciferase expression, plasmid pOB4.13 (Rgoteda) into unique restriction sites 8Ai and BATH1 flanking the expression cassette for the firefly luciferase gene. The first 1e1BB site was cloned into a unique 8ac1 site above the 8Y40 promoter after annealing the synthetic 1e1BB oligonucleotides with overlapping ends for 8ac1. The second 1e1RB site was cloned into a unique WatH1 site below the 8Y40 polyadenylation signal using synthetic 1e1RB oligonucleotides with overlapping ends for WatH1. The resulting construct was called pOb ΌΘΟ because it allows the formation of a covalently closed linear (in the form of a sugar dog bone) DNA encoding luciferase for expression in mammalian cells.
Example 2. Confirmation of the ability of 1е1N to cleave DNA.
The splitting of the supercoiled annular vector structures pIS18 1e1PB and pOb ΌΘΟ with Τе1N was confirmed. 100 ng of each substrate was incubated with 4.5 pmol Τе1N for 1 h 40 min at 30 ° С. The reaction was carried out in a buffer for Τе1N [10 mM Epc-NS! pH 7.6, 5 mM CaCl<sub>2</sub>, 50 mM potassium glutamate, 0.1 mM ΕΌΤΑ, 1 mM ΌΤΤ].
The cleavage products were visualized by agarose gel electrophoresis under native conditions. Incubation of the supercoiled annular pIS18 1e1RB with Τe1N released a linear fragment of 2.7 kb in size, indicating a cleavage. Incubation of the supercoiled ring pOBO LLC with He1N released two 2.4 kbp fragments, indicating cleavage at two sites
1е1КЬ.
Additionally, pIS18 1e1PB and pOBI LLC were linearized by restriction enzyme digestion, and then they were incubated with Τе1N to additionally confirm specific cleavage at the 1е1PE 100 ng site of pIS18 1e1РБ linearized with Xtp1 and then incubated with Τе1N. This reaction released the expected 1.9 kb fragments. and 0.8 kb 100 ng of pbO LLC was linearized with ΡνιιΙ and then incubated with 1е1N. This released the expected fragments of 2.4 kbp, 1.6 kbp. and 0.7 kb Likewise, linearization of pObOo with сκίΙ and subsequent incubation with Τе1N released 2.4 kbp fragments, 1.1 kbp, and another 1.1 kbp fragment. These experiments demonstrate the endonuclease activity of Τе1N on circular or linear DNA substrates containing the protelomerase target sequence.
In a preliminary assessment of the activity of DNA cleavage, it was found that an excess of Τе1N up to 3.4 pmol cuts at least 200 ng of pIS18 1e1RB for 1 h. In experiments to determine
- 18 021069 the dependence of the cleavage on time the same amount of DNA was cleaved for about 10 minutes
Example 3. Confirmation of the ligation activity of TeS and the formation of closed linear DNA.
Confirmation of the closed linear DNA structure of the cleavage products using TeS was performed using gel electrophoresis under denaturing conditions. pCOS OOS were incubated with TeS as in Example 3. A synthetic PCR product (RSK EOS) corresponding to the region contained in the dog bone sequence, but with open DNA ends, was used as a control. A linear fragment of RAC OOS was amplified using primers flanking sites 1e1Kb
ZAROZH 5 'OTOSAASTOSAOOSTOSSASAAS 3' (5EC GO ΝΟ: 34);
Your ROLE 5 'OATAAAOAOAOAOAOTSATAAAOOTSSOOS 3' (3Εζ> GO ΝΟ: 35).
On an agarose gel under native conditions [0.8% agarose in TAE buffer (40 mM TLK acetate, 1 mM EETA)], 2.4 kbp product obtained by incubating 100 ng pCOS OOS with Teh , migrated at the same distance as the RSK OOS (2.7 kb), since both products remained double-stranded.
However, when separated in a denaturing agarose gel [separation in 1% agarose in H<sub>2</sub>O in the presence of 50 mM NO, 0.1 mM EETA and neutralization after separation in 1 M TgF-HC'T pH 7.6, 1.5 M No C1], allowing denaturation and separation of double-stranded DNA into single-stranded DNA, a canine-shaped fragment the bones obtained by processing Teh migrated with a higher molecular weight [approximately 5 kbp] than open-ended PCR control or pIS18 1e1Kb linearized with Xtp1 (both fragments with a size of 2.7, etc. about.).
This difference in migration indicated the formation of a closed linear structure in the form of a dog bone as a result of TeS. Denaturing a dog-bone structure will produce single-stranded open rings that migrate much more slowly through the gel compared to linear single-stranded molecules released by denaturing a linear open-ended PCR product.
Confirmation of the formation of a closed linear structure of the products as a result of the action of Teh was also shown by analysis of temperature denaturation using capillary electrophoresis in a microchip (LOS electrophoresis). LOS analysis is a platform for capillary electrophoresis for the rapid separation of biological molecules. AdbeSh Wuapa1u / er with ^ NA 7500 chips (AdLep !, IR) can be used to separate and approximately determine the size of a DNA fragment up to 7000 bp long.
This system using a microchip does not detect single stranded DNA. Thermal denaturation (95 ° C for 5 min) and rapid (<1 ° C / s) cooling at a rate of 1 ° C / s of standard double-stranded DNA in low salt, for example, in N<sub>2</sub>Oh, results in single-stranded DNA, which cannot be visualized in the LOS system. However, DNA ends that are covalently linked into dog bone DNA (obtained by cleavage of Teh) cannot separate after denaturation and therefore are repeatedly annealed to form double-stranded DNA, which remains visible. Comparison of temperature-denatured and rapidly chilled DNA therefore makes it possible to distinguish between covalently closed dog bone DNA (cc1) and ordinary open linear (o1) double-stranded DNA.
DNA samples (100 ng) in N<sub>2</sub>О was denatured (95 ° С for 5 min), rapidly cooled (<1 ° С / s) to 4 ° С in thin-walled PCR tubes in a thermal cycler (Vyugab 1-ss1eg, Vyugab, IR). For comparison with TeS digestion, the samples were first incubated in IX TeS buffer with 1 μl of purified protelomerase at 30 ° С for 10 min. Control samples were also treated, but without enzyme. Samples (1 μl) were analyzed using AdLep! Vuapa1u8eg and chips ^ NA 7500, following the manufacturer's instructions.
The results are shown in FIG. 6B. They show that closed, dog-bone-shaped linear DNA obtained by incubating pCOS EOS with TeS is resistant to temperature denaturation compared to equivalent standard open linear DNA (CSC EOS). Equal resistance to thermal denaturation was also obtained using KSA amplified dog bone DNA obtained by KSA amplification and TeS digestion.
In other experiments, TeS cleavage was performed on an open-ended RSK EOS fragment. This led to the formation of a thermostable cleavage product, dog bone DNA with a size of 2.8 kb, and thermostable dog bone ends with sizes of 0.09 and 0.14 kb.
The sizes of the dog bone fragment and ESC EOS established in the LOC analysis were in the range from 2.8 to 3.0 kb. and 3.1-3.5 kbp respectively, compared with data based on the nucleotide sequence suggesting approximate sizes of 2.4 and 2.7 kb This reflects the size difference caused by conformational differences in migration that occur in
- 19 021069 non-denaturing L0C analysis.
Example 4. The formation of closed linear DNA from concatemeric DNA obtained using KSA (amplification by the type of a rolling ring).
A очныйη νίίτο cell-free method for amplifying a DNA matrix and converting amplified DNA into closed linear dog bone DNA molecules was performed. For amplification of covalently closed plasmid matrices in which the 1c1Pb site was present or absent, the KSL method was used using the p129 enzyme of the pK29 phage from Vasichik kishkk and random hexamers as primers under different reaction conditions. This resulted in amplification of concatemer DNA as a result of the processive activity of displacement of the polymerase chain pY29. The first experiments were carried out using the TrstryRA kit (OE HsaIsags) according to the manufacturer's instructions. However, this kit was subsequently replaced by a method developed by the authors (using polymerase p1y29 from the company NEB), which gave a higher yield of product with higher purity.
Denaturation of 40 pg - 200 ng of the closed ring matrix and annealing of the primers was carried out in 10 μl of annealing / denaturation buffer: 30 mM Tg18-HC1, pH 7.5, 20 mM KCl, 8 mM MdCl<sub>2</sub>, 20 μM hexamers with a random sequence. Denaturation and annealing were carried out by heating at 95 ° C for 1 min, followed by cooling to room temperature for 30 min.
Then, to 10 ml of the reaction mixture containing annealed DNA / primer, 10 μl of reaction buffer [35 mM Tpc-HCl, 50 mM KCl, 14 mM MdCl 1 was added<sub>2</sub>10 mM (1MN<sub>4</sub>)<sub>2</sub>80<sub>4</sub>, 4 mm ETT, 10 units. pY29, 0.002 units. ΡΡί (inorganic pyrophosphatase from yeast), 1 mM BETR].
The reaction mixture with a volume of 20 μl was incubated at 30 ° C for 18 hours. The sample was separated by gel electrophoresis to verify the formation of concatemers, and then the reaction mixture was digested with restriction enzyme or Tsc to test products.
Concatemer DNA amplified by KSA was then incubated with TcSh. Typically, the KSA-amplified DNA substrate was diluted in water and 10x TCB buffer to a final volume of 20 μl. The results for pIS18 1c1Kb are shown in FIG. 6A.
As can be seen from lane 1 of the gel, the undigested concatemer amplified DNA forms a cellular structure that is not included in the gel. However, TcSH was able to cleave the KSA material, resulting in the release of a 2.7 kbp fragment. in the shape of a dog bone (lane 6). Confirmation that DNA amplified by KSA was the original template used in the reaction was obtained by restriction with ΡνιιΙ (lanes 2 and 5). pIS18 (without 1c1Kb) served as a negative control for TCg activity (lane 3).
Similarly, in other experiments, the concatemers pOb Ь 0Ο obtained using KSA also split TcS. Accordingly, it was shown that the method according to the invention is effective for the amplification of closed linear DNA on the original matrix. In addition, it is possible to amplify closed linear DNA in a simple manner using sequential steps involving KSA polymerase and protelomerase without the need for intermediate purification of the amplified DNA.
Example 5. Expression of amplified closed linear DNA.
In order to study the expression of the luciferase reporter gene from closed linear dog bone bone obtained according to the invention, transfection experiments were carried out using Hcba cells. As a control, covalently closed circular DNA and a linear control fragment of CSC Ό0Ο were used.
Transfection was carried out at a confluence of 60% cells in wells with a diameter of 20 mm in KPM1 medium using TgapkGssyt® reagent (Rgottsda) in accordance with the manufacturer's instructions. 400 ng DNA constructs were used for each transfection. Transfection efficiency was normalized within one experiment and between experiments, including an internal control in each transfection, 40 ng of the plasmid pOb4.73 expressing CSPhA luciferase (containing the 1K1is gene from CSPhA cfGfGpeak). Firefly luciferase activity (an enzyme responsible for the luminescence of insects Ryoypik rugaNK) and KspSha luciferase were measured sequentially using the Accu8S1st (Rgottsda) kit Accu8Sgax® Xroisg (ECK ™). Relative luminescence units were measured using an O1oMax luminometer Lippipotrol (Rotsda) luminometer, and the results were expressed as the ratio of the luminescence intensity of firefly luciferase to KspSa luciferase. All experiments were performed in triplicate.
The following constructs were tested in transfection experiments:
Control DNA pOb4.13 1is pOb4.73 HH1is RSK Ό0Ο
PCR control (fragment from pOb4.13, overlapping gene 1c) pObO0 (pOb4.13 containing 2 sites 1c1Kb)
Dog bone mini prep (pOb-E0O isolated using the mini-selection kit
- 20 021069
DNA (mini prep) digested with Rui1 (to remove contaminating vector DNA) followed by TeU cleavage).
Dog bone, KSA (pOb-ΌΘΟ amplified by KSA, split Rui1 and split TeS).
KSA pOb ΌΘΟ (concatemer DNA obtained as a result of the initial KSA amplification of the plasmid pOb ΌΘΟ).
The results are shown in FIG. 6C. It was shown that closed linear DNA, including that which was amplified by KSA, expresses luciferase at a higher level relative to open linear PCR constructs. This demonstrates that the closed linear DNA obtained according to the invention can be used to successfully express luciferase when introduced into mammalian cells.
Sequences of the Invention
Table a
The nucleotide sequence of the DNA polymerase from bacteriophage rY29 VasNk1 £ (ZEO Yu N0: 1) aEdaadsaEa Edssdadaaa daEdEaSadE EdEdasEEEd adasaasEas EaaadEddaa 60 dasEdEaddd EaEdddsdEa EddEEaEaEd aaEaEadaad aEsasadEda dEasaaaaEa 120 ddEaaEadss EddaEdadEE EaEddsdEdd dEdEEdaadd EasaadsEda EsEaEaEEEs 180 saEaassEsa aaEEEdasdd adsEEEEaEs aEEaasEddE EddaasdEaa EddEEEEaad 240
Edddsd eddd edd aaasasaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa!
EasaEdaEEd aEaEaEdEEE addsEasaaa dddaaasdEa adaEasaEas adEdaEaEaE 360 dasadsEEaa adaaasEass dEEEssEdES aadaadaEad sEaaadasEE EaaasEaasE 420 dEEsEEaaad dEdaEaEEda EEassasaaa daaadassad SsddsEaEaa daEaasasss 480 daadaaEasd ssEaEaEEaa aaasdaEaEE sadaEEaEEd sddaasdEsE dEEaaEEsad 540
EEEaadsaad dEEEadassd daEdasadsa ddsadEdasa dEsEaaaadd EEEsaaddaE 600 aEEaEaassa sEaadaaaEE saaaaaddEd EEEssEasaE EdadEsEEdd asEsdaEaad 660 daadEdadaE asdssEaEad addEddEEEE asaEddEEaa aEdaEaddEE saaadaaaaa 720 daaaEsddad aaddsaEddE sEEsdaEdEE aaEadEsEaE aEssEdsasa daEdEaEads 780 sdEsEssEEs saEaEddEda assEaEadEa EEsdadddEa aaEasdEEEd ddasdaadaE 840
EasssasEas asaEasadsa EaEsadaEdE dadEEsdaaE Edaaadaddd sEaEaEasss 900 asEaEasada Eaaaaadaad EaddEEEEaE aaaddEaaEd adEassEaaa aadEadsdds 960 ddddadaEad ssdassEsEd dEEdEsaaaE dEadassEad aaEEaaEdaa adaasasEas 1020 daEEEaEaEa asdEEdaaEa EaEsadsdds EEaaaaEEEa aadsaasEas addEEEdEEE 1080 aaadaEEEEa EadaEaaaEd dasdEasaEs aadasdasaE sadaaddads daSsaadsaa 1140 sEadsaaaas EdaEdEEaaa sadEsEaEas ddEaaaEEsd stadEaasss EdaEdEEasa 1200 dddaaadEss sEEaEEEaaa adadaaEddd dsdsEaddEE EsadasEEdd adaadaddaa 1260 asaaaadass sEdEEEaEas assEaEddds dEEEEsaEsa sEdsaEddds EadaEasasd 1320 asaaEEasad sddsasadds EEdEEaEdaE sddaEaaEaE asEdEdaEas EdasadsaEa 1380 saEEEaasdd dEasadadaE assEdaEdEa aEaaaadaEa EadEEdasss EaadaaaEEd 1440 ddaEasEddd sasaEdaaad EasaEEsaaa adadEEaaaE aEsEdadasa daadassEaE 1500 aEasaadasa EsEaEaEdaa adaadEadaE ddEaadEEad EadaaddEad EssadaEdaE 1560
EasasEdaEa EaaaaEEEad EdEEaaaEdE dsdddaaEda sEdasaadaE Eaadaaadad 1620 dEEaedEEEd adaaEEEsaa adEsddaEEs adEsddaaaa EdeadssEaa dssEdEdsaa 1680 dEdssdddsd dddEddEEsE ddEEdaEdas asaEEsasaa EsaaaEaa 1728
Amino acid sequence of the DNA polymerase of bacteriophage pY29 from Vaschiz (ZEO Yu N0: 2)
ΜΚΗΜΡΚΚΜΥ3 SOGETTTKUE OSKUNAΥSUM ΝΙΕ0Η3ΕΥΚΙ SIZZOEGMAI UKUOAOUG 60
ΗΝΙΚΕϋΕΑΕΙ 1NIEEKISGK IZAOSRNTU ITIZKMSSOI UMYUYUSUK ΟΚΚΚΙΗΤνίΥ 120
OZKKRRRU KKTAKOGKT UTskEOYUUNK ΕΕΡνΰΥΚΙΤΡ ΕΕΥΑΥΙΚΝϋΙ ONEEEYO 180
RKOSYZHMTA sozoeksgko ΙΪΤΤΚΚΡΚΚν GRTESZOK EUROUAUKSSR ΤΗΕΝϋΡΕΚΕΚ 240
Е1СЕСМУГЭУ Ν51ΥΡΑ2ΜΥ5 ΕΙΧΡΥβΕΡίν GESKUUNOEO URNYUES YEREKESIR 300
ΤΙ0ΙΚΚ5ΚΕΥ KSMEUKZZS CE1АОНЬ5 and УЬЬЬМЬКЕН 0ЬУУНУЕУ13С ККЕКАТТСЬ 360
ΚΟΕΙΟΚΗΤΥΙ ΚΤΤ3Ε6ΑΙΚ2 1 AKKMNOU OKGAZNROUT eKURUKE NO ALGKГSEE 420
TKORUUTRMS UP TAMAR.ΥΤ T1TAA2ASU0 K11US0T081 NTSTE1RSU ΙΚΟίνΟΡΚΚΕ 480
SUIANESTGK KUKU.OKTU KDOUMKEUO SKEUES5ROO ΥΤΟΙΚΓενκε ASMTEKHKKE 540
UTGENGKUS ZKKMKRKRUO URSSUUURR ΤΪΤΙΚ 575
- 21 021069 _T Table B
Amino acid sequence of DNA polymerase Oeer Uep! from Rugosossis sp. (8E0 U N0: 3)
MIOAUHTE ϋΕΚΡΙΙΚΙΓΚ ΚΕΝ6ΕΓΚνΕΥ ΟΚΝΓΚΡΥΙΥΑ КО COSMOUS νκκν 60
ΚίνκίΙϋΑΕΚ νΚΚΚΓΙ, ΟΚΡΙ EUQUENGER βΟνΡΑΙΗΟΚΙ ΑΕΗ3ΑνΐΟΙΓ ΕΥΟΙΡΓΑΚΚΥ 120
YOKSYRME BOYEKY SHETUNESE EGKSR11M1 ΕΥΑΟΕΕΕΑΚν ΙΤΜΚΚΙΟΕΡΥ 180 νεννΒΒΕΕΕΜ ΙΚΕΪΈΚνίΚΕ ΚΟΡΡνίΙΤΥΝ βΟΒΓϋΕΡΥΕν TINKING CUTTER 240
MEKSEMTAU E1KSK1NROE ΥΗνίΚΚΤΙΝΕ ΡΤΥΤΙ, ΕΑνΥΕ ΑΙΕΌΚΡΚΕΚν ΥΑΗΕΙΑΕΑΗΕ 300
TSKVIEKUAK ΥΕΜΕϋΑΚνΤΥ ENERGERM OZMLbor ΕΜ0ν3Ρ.3ΞΤΰ ΝΙΛ'ΕΜΪΙΛΡ, Κ 360
ΑΥΕΚΝΕΙΑΡΝ ΚΡΟΕΚΕΥΕΚΕ RESEUASSUU KEREXNBS UZIORESU RZSTNUUZ 4 20
ROTIER.ESSE ΕΎβνΑΡΕνΟΗ KRSKOGRSE1 RZKEYE ΚβΕΙΚΚΚΜΚΑ ZORKHEKKM 480
ΟΥΚβΚΑΙΚΙΙ, ΜΙΕΥΥΕΥΥΰΥ AKAKIUSKES AEZUTANSKE ΥΙΕΓνΚΚΕΙ, Ε ECRSU / LU1 540
OTOSUAT1R SAKREE1KKK ΑΧ, ΕΕΫΟΥΙΝΑ KIRSJEU ESRUUKSRRU TKKKUAYO 600
ESKITKSIE 1UKKONSE1A KETOAKUYEA ΙΣΚΗΕΝνΕΕΑ νΚίνΚΕνΤΕΚ LZKUE1RREK 660
Ι, νΐΥΕβΙΤΚΡ N U U U U A A U 720 720
Table c
The nucleotide sequence of the DNA polymerase I (ροΙΑ) from Bacillus s (eago№eplorY1iz (ZEO Yu N0: 4) abdaadaada adsadbasb aaSRdabdds aasadbdbdd sabassdsds sSbbSEbdss bbdssasbSE EdsaEaasda saaaddsaUb sabasdaaTd sddYbasdd dbSEasdaEd abdEEdaasa aaabShdds ddaadaasaa ssdasssaM askEdTads dYbdasdss ddaaaaasda sdbbssddsa bdaaasdTTS saadadEaTa aaddsddasd dsaasaaas sssssddaas Sdbssdadsa? DYssssdsbd ssdsdsdads oatSaaaads dbassdsass sssdsbba'd aasbdabsa Yasdaadsd dasdaaa Esdddasds sdsdssds dsddsaad aaddd + SSda adEdaaaaTs aTTssddsd assdsdaEEE aasssadsrs dssbsssdTs abdbdasddE sdabaSSasd aaaaaaddda EEassdasaS SdadssdTaS asdssadada ssdbbsdsda aaaabasdds sbdasbssdd adsaaabad ddabYaaaa ddabbdabdd dsdaEaaaEs sdasaasabs ssdddsdTds ssddsabsdd ddaaaaaasd dsddSsaads Eds1daadsa abESddSasd dbddaaaabd SdsbsdsaTs dabdaTdad dSdaaadddd aaaaasbdaa adaaaasYd sdssaasass dddabbSads sSsSdads aaasadsbdd sdbssaTbbd ssdsdasdss esddbbdeds bdbsdTEada TdasabbdSs basdaaddas aadassdsda aeaadbsabs dsdbbabbba aadaasbsdd dssssadbsd BBC ^ bddaaa aaaddssds dssddsadss daaddddada aassdsbbda ddadaEddad "Sdssa ^ sd bbdasdSsaT sassdaadad aSdsSSdssd asaaddsads dsSSdSsdSS daddSdaSdd aadaaaasSa ssasdaSdss ssdaSSdSsd daaSsdsasS adSdaasdad saSdddsdaS SSSSSaSdsd sssddadass dsdsSddsSd aSSsdsaaSS SSSadsaSdd sSSdssdaSd aaasdaadaa aaaaadsaSd SSSdasdssa adsdddsadS sdSSdssSSa aadSddaaad daaSSdadsS SsdsddsdSs dssSSSdaSS SaSSdsSsds SdssSaSSSd sSsaaSssdd sSsaadaSds sddsdaSaSs dsSdsddSdd sdaaaaSdaa asaaSaSdaa dsddSdsddS sddaSdaads ddSsSaSdds aaaddsdSsa adsddSsdsS dssddasdaa sadasdsSSd sSdadsaSsS sdSSsdsaaa dsddsadssa SSSdddsdsS Sdadsadssd SSSaSddasd aSSSdsddaa saasdaasaa daSsaaSSaS saasdaadsS Sdadsadssd sSddsddsda SSSSddsSda aaSddaaSSs asSddddSda asdSddaas aaadsddsSS daasadaSdd dSSsddadsS sdssdaasaa sSdsdSdssa Ssdadsadsd saSSSasdad sSadssddss aadadSSsaa saSSaasSsa ssaaaasads SsddadSsaS SSSaSSSdaa aadsSdsads SassddSdsS daadaadasd aaaasaddsS aSSsdasSSs ddsSdaSdSd sSSdadaads SSdsdssdsa SsaSdaaaSs dSsdaaaasa SSSSdsaSSa ssdssadsSS ddsaaasSds aaSsaasdSa SaSSdaadda SSdSSdaaad SSdSdsdsss SdaSassdds aaadSdsaSa sdaSdSSsaa ssaadsdsSd asdsaaasSd ddsddsSsad sSsddssdad ssdaasSSds aaaasaSSss daSSsddsSs daadadddds ddaaaaSssd ssaadsdSSs dSsssdSsad adssddasSd dsSsaSSSSs dssdssdaSS asSsasaaaS SdaaSSdsds
120
180
240
300
360
420
480
540
600
660
720
780
840
900
960
1020
1080
1140
1200
1260
1320
1380
1440
1500
1560
1620
1680
1740
1800
1860
1920
1980
- 22 021069 dssEsdsss aaSsdssda dasdasaa s6aa16daad sdTSsseasd sdaSS ^ ddaT 2040 asasasaa aaasddsdab ddasaSTTSs saSdSdadsd aadaddaad sasddssaas 2100 aedsdssdss addsaaadds sdsaasSbs dd £ a1sdSTS asddaaYad sdatsasdda 2150 Iddsdsaaa asdaasa Sasdsdsaaa daadsdssd aa Easda asdTTasTEs 2220 dssadsTTTs sdddsdkaaa dsadSaSad daaaasad bdsaadaads daaasadaaa 2280 ddaTasdSda saasdsTgdY;! ACD * sddsds sdsSaY SDS s1daaT (: al aadssdsaa 2340 Msaasd1ss dsadEYLds adadsddasd dssaSdaasa sdssaaEsa addaadsdss 2400 dsdasaTa Yaaaaaads daSdaSTdaE EEadsddsas ddsEdaaada adadsadsEE 2460 saddsEsdEs EEEEdsEdsa adEdsaEdas dadsEsaEEE Eddaadsdss aaaadaddaa 2520 aEEdadsdaE EaEdEdadsE EdEEssddaa dEdaEddads addssdEEas dsEssdsdEd 2580 ssdsEdaaad EsdasEassa EEasddsssa asaEddEaEd aEdssaaaEa and 2531
Amino Acid Sequence of DNA Polymerase ρ (ρο Вас) from Vasekw $ 1ea # tegtorY1i8 (5E0 Yu N0: 5)
MKKKYUYOS NZUAUKAEEA RYNN0KS1 NTIAUUYRTM ΜΕΝΚΙΙΑΕΕ0 RTNYL'LGOA 60 YKTTENNETG СС KSS GROWTH RREZEOGRE RNEKOUNT RAUEONUEA ϋΟΤΙβΤΙΛΑΚ 120 ΑΕΟΕΕΓ! A5KNUTU01T KKS1TOGERU TRETUVEKUS TREOTUOK 180 SMSOKZON1 RSURS1SEKT AUKKSGST UEIUAZYUE UKSEKKEN KSNROAZ 240 K (ZA51SKOA RUEE8SH01U UE (ZDZHEKU1 AGKEbGOB GEKMAARAA ESBKREEME 300 ΓΑϊνονίΤΕΕ MAOKAAUU EUMEEIUNEA R1US1AUNE NSKGGMVRET AAOEOGAN 360 AOETKKKZM GOAKVAUUA KIKS1EVZU AGOAAU ΕΝΡΑΟΟΑΟΟΙ AAUAKZHOUE 420 AUVZOEAUUS KSUKKZROE OTAENUKK AAA1NAE (ER GMOONNNEO OOTKEOR 480 ΙΑΑΙ1ΑΕΜΕΓ TSUNUOTKK EOMYZEAES Ι , ΡΑΙΕΟΡΪΥΕ 1 ASOECOGY1VZ RCSUITB 540 KJOURUKKT KTSUZTZAYU LJKJARNET UECHNUVS SKOZTUTES KUUVROTS 600 KUNTMGNOA TOTSKZZAE RV0Y1R1K EESEKHKSAG URZEROMYG ΛΛΏΥ5Ι2ΙΕΙ.Κ 660 νίΑΚΙΑΟϋΟΝ YEAGOVOO ΙΗΤΚΤΑΜϋΙΕ NUZEEEUTAY MRESCHAKAUMG Y1UUS130UY 220 ASNSHTYAK ΕΑΑΕΕΙΕΚΥΓ AZERSUKOUM ΕΝίνβΕΑΚΟΚ SUUTTNKK KUR01TZKN 780 ELUKZEAEKT AMYTR10S5A ΑΟΙΙΚΚΑΜΙΟ AAEKEEO OAKOUNO EYEARKEE 840 TEVSEURE UMErAUTVU RKURUNUbR TIURAK 876
Table Ό
The nucleotide sequence of the protelomerase of the phage pYNAP- '! of Nakttopae (δΕΟ Yu N0: 6) aSdadsddd adksasdTad aaaddsda Nadsddaa daadadd ddssads 60 dadabsaaad hell + sdasds sdadabdad adssasdSa aadadaaaas saadsdsad 120 dsdsddstdd sasdTadsSS saaaasdsds with £ dsaSdad asaadsdssd saaddasS 180 dadsddabsd FBC-Ssasdas sSsdssds SasaSdasad aadsdsdsaa ddsdddaeS 240 dsdsadaasS ddsdssa ca sadssdas sadsadaSsd adsddsSdds! sadssdsdas 300 ssdsds1a6d ssadsaedss ddaadsdsbs ddsaadsSda ssda ^ aSsad ssdbcd! 360 aTddsssass dsdadsbds sdassadas sdsaasdad asdaedsa Tdaddasabs 420 sdddsdaSda adsddassa sdaaaSsad sdssassda sddadsSs Sdsasadaaa 480 adsasdsTdd sdaadadds sadsdadasd sddaadads dsdsdddaa sasddsdad 540 aEsaasTass aoSddTTdaS ddadasddTS asdadsEdo dadaassd ddadadaad 500 dSsda1dddd adTaTsdsdd s6TS1SsadT TassTadsds SSdddsSdds dsbddssass 660 dddsd1sds6 sdasdadd dsTdaadass ddasddaTsa sdaaddddd odadSadad 720 sTddadSKHsa dsddssadds daaaaadsds ddsddsdsd asSaSadoda ddsSSassas 180 aNaTasss ddTdaaads dassddTd aTsdaadsd dddabdadsT: bsdsTsdsd 840 ssddaadsTd sSdadsSdsa dddsaSddas aasadsdaSd Tdaassdssd sasddsdaad 900 asdsbsaasa sdsTsastaa dsddaTsSTT aasaasdad adsdsdSSS saaddasads 960 sdddsdas dddsdsdds ddTdTTTdad sTdsasS £ with £ sdsdsdasaa dsdsTddaad 1020 aaadTsassd addasdTdSS sTddsddad aSdsTdddds adaddasaT ddabasasad 1080 sdsadsass dsdssS ^ aa aaSsdasSas dasdadssdd asaadssda ssaddaada 1140
Tasdeasasd CT: adssdssS sdesdedsTd saddsdsTdd asddssada dsadsSdad 1200 adsadsdasd sssaddsdsd 1ddsadss SdddTdaaad sdsadaTsda dsaddadss 1260 dasdsdaaaa SSasdsadSs Tsdabsads sdddadsdd dsdSasd sssdssaa 1320 aaadsdass bddadsdds dsdadaddsd ssdasdsds sdaasdbsda tseddasaad 1380 dsdsddsdd garden dssdaa ddaadadss daddsdaads sssddsdaa sdsssasssa_1440 saaddddaTd dsaddSdddT sdddd £ SDS ± 1saabsaasd ddd1ddaad £ dsasddd'Sd! 1500 ddsaassadd saddssddda T sdaadsda ^ d aaadsdddb aaaaadsdds ddddddddsd 1560
Tda 1563
Amino acid sequence of protelomerase of phage RYNAR-1 from Nacttopases (5EO 10 N0: 7)
MZSEZKRKUO RAEEIEZZ ΕΤΚΕΐΌΛΟϋΕ MRVKEKTKKM AVAKAZGKTK Ι.ΗΏΟΚΚΒΚΟ3 60
ЕВ1АУТТЕКК УМТЕАККАУТ Α (3Ν «ΕΗΗΞΕ0« CHEEZVAU RAUAZKIEE 6KTO15A1K 120
ΜΑΗΚΕΕΕΟΟΙ ΒΒ000ΑΥΕ0Ι RAKK10NE1M EXTRACT 5ΤΙΑΕΕΑ3ΕΤ ΕΤENAUNAUNTUE 130
ΙΝΥΗΜΕΜΕΤν EZZKM UEYEYEYEYEYEYEYEYEYEYVVVVVVVV 240 240 240 240 240 240
LIEZSOAKKV 60νϋΥ5ΕΑΥΗ TUTUUKAOHU HEMAEMVZA RZAAYEOSMO VEUUVKKTLK 300
WRITING ΝΝΟΕΡνΕΚΟΚ RA1NEWING SOFTWARE KUTOUEEIKE MUSEPMOTO 360
Κ3ΥΒΑΓΚΙ0Υ ΟΕΡΠΟΑϋΟΕΟ UENAJEVAAJ OASIANEOE 5 5 OAHAKU TO IUKA01E0ER 420
0AK1T05Y5 ΚΕΪίδνΥΒΡΑΙ KAUYEAKEA BOARNOUYZH WAAAURKEUA EAKRVNANR 480
OSRSKIUSUA 31M5UEUWAU SI0ASK1EAM KAAUKAASSV_520
- 23 021069
Table e
The nucleotide sequence of protelomerazy phage ΡΥ54 Uegagna (ZEO Yu N0: b) abdaaaabss aEHEsdsda byyadyyad ddyadya aadadasda daaaadaa 60 aaasadass dddsdsaddd Sdasaaaas sdadsa ddadadadadadadada
Tsdbbaasaa sabbyaaTaa aTaTTaady sdadsassT sSsddySTda adaaaddsSG 240 sassatadet yyssbsaass tatadsaast atsTseazTa aaaysstds aSSsadsdaa 300 aSaaSaaaad aSsSddaSaa SadasssdsS saSdaadSSa daaSaaaasS SaaadaaSSa 360 aSaasSsaSs SSdaaSssdd SdSSaaSSSa SSadaaaaaa SaddSadsSS adddaaaaga 420 aaassaSsSa sadsSaaaaa aaSadSSads ssaaaaaaaa SdSasssaSs aSdddsSaaS 480 daSsSadaSa sSSSaaSSad SasSdaadaS dsSasadaaS Sasaasaaaa dSSadadsaa 540 dddassdass SasSSaasds aSSasaSSsS sSaaaadSaa assaSdaadS SaSdSaSdsa 600
SSaasdaSds ssssssssSda SadadsSdsa SSaaaadsSa ddsSdasds SdsssSSSSas 660
SSSaaaaads dSaasaSsdS assSaSdSaSaSsssddsS asaSaSdsaasd aaSdasddas 720aSasSasasaSssssasaSaS adsSSSSSSdaa daSSsdaSds sassasSSdss ssssssssa 780
SSSdsSsssSad GardensSdsSdsGarden sdsSdsdsada SaaaSSdaaa SesSaaSSas SdddSdadSSSS 840 dasdssaaaa SaaaaadsaS saSSaaaSSS Ssddasaa saaaaaaaaaaSddssdsss 900
SsaddSddas aSSaSdaaaS aSasadSsSa aSSdasSsad adsSaSSsaS SsaasddSSa 960 dadsssssas dsSsSsaSad sSsaaSasSS sdaSSasaaa asssddaaas adsasaSdaS 1020 daasaSsdSa sSdaasSaSs SdSSaSSaas ddSSSSdSad ssaaassSSS aaaSdaSdsa 1080 dsaaaasadS SsSSSdSsda Sdasadaada dSaSSSaaad aSasssdSds aaSSSasdsS 1140 sdsaSadsaS aSdaaaaaSd dSSSadaasa daSssSsdsS dddsdaadSd sdasdaadaS 1200 dSSSSsSSsS sSdaaSSaSS addssaSdas daessadaSa sSsadsSdds aEaEaaasaa 1260 sSsaadsSdd SaaaSSSsaa SssaaaaSdd asasseaaSa SaesadaSda aaasssSsdd 1320
SssadSdsas SssaadadssS FoodsaedaSedasdssaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ...
SddSdsdsSd asdsdsSadd ddSSdSSaSS ddEdaEdaEd dasaddsaad dssadaadaa 1560 sSsssassaS sdsSsdSdsS EdaEaEEaas dsSdaSdasa sEdasdsEda adaadaEdaa 1620 aEadaddaad asSSSasSda EdaddaaaEa dasdasassd aaSSsdasdS aSsadaSaas 1680 dssadEdaEd aadaSaadss sdaadaEaaa ssSsdsSSSd sadsassaaS EsdEadaadE 1740 daddasEsEE ddsEdaEEaa aSSSdaaSSS dsEddsaads aaEaEadsEd ddadddEaaE 1800 dssdaaadSd SSaSsdaSds daSdaaasaa dsaEddasEd aaaaEaEdda dEaa 1854
Amino acid sequence of phage el54 protelomerase from Uetz1P1a (ZEO Yu N0: 9)
MKNEGKOHL / W SUKEHSETE KZOKAOSyKT R.KUOSAAKKG ΚΝΑνΓΜΕΚΚΚ ΥΚ6Ν3ΜΚΝΚΙ 60 ZTTGMKUZ KANZKREEK ΗΗΞΚΡ03ΙΑΤ Ι8ΝΚΥΡΑΡ3Ε IKOOYKRA NEUIKKE 120 HTNEEZSTY EKHS5SK1 KRZTAKK1U5 KKMURZIA "SHLTYZTEO ATBYZOKEO 180 eTONANZ ΕΚνΝΗΞνΗΪΑ TMORZOKAA KAVNOAAN ΕΚΚΗΝΙΥΡΙΟ ΥΡΟΥΜΟΕΗΤϋ 240 1NROHAGE OZMAZARA GAAAAZSKK 01E1YTSEG 0ΑΚΝΚ3ΙΙΚΡ ZSOAKKVMAU 300 36 £ ΗΥΕΙΥΞΕ YUZERYUK EGKZNZZH KSNE1ANO ENKTEZUHY PO / AKRYOA 360 ΑΚΟΓΕνϋΟΕΚ νΡΚΟΤΡΑΙΥΑ ΒΙΑΥΕΚΜΓΚΤ ORVIAXOE URZEZNO ΟΡϋΤΟΧΛΥΚΟ 420 ΓΚΧ, νΝΓΝΡΚΜ ΤΡΝΙ50ΕΝΡΚ AALSEOM MRSЬKSKSKSA UNCHXXXXXXΝΡΑΑΚΙΤΝΡΑΑΚΙΤ 80ΛβΝΡΑΑΚΙΤ 4 80 AUSLKKMMS ΚΝΟ1Λ5ΕΥΜΑ ISAOAS-U'L SOSZOAVREE RZЬЬΑΟΟΤϋΑΕΕϋΕ ΑΟΟΤϋΑΕΕϋΕ 540 ΙΕΕΏΡΤΟΕΕΙ 00ΤΕΓ003030
- 24 021069
Table Ρ
The nucleotide sequence of the phage RyKO2 protelomerase from MeBeAn (ZeO 10 N0: 10)
Taaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
THESE ASEEEEDAaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
SSdsaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
SEsdsSaTTa dSdaSTSaaa SadsdaadaS sddaaddaSa aaadadasSa SsSSSaSaaa 540 sSaSSssaas aaddSSsSSs dsSssSddaa dasSSdaaSa assSdaaads aaassaSdad 600 dsssSsSaSs aSsSdsadsS SadSSsSdss dadsdaassS sSaSssadsa dsdsSdddss 660 aasdSssSsa dsdadaaaaa dsdsaasdSS dSsdSdaSSd asSaSssdsd sSaSaTdsad 720 dssaEsSasd aSaSaaSsaa saadssSaSa dESSsdSSsd aSESdasSas SsdSsdSddS 780 aSddssssds SddsdSSsds ssSSdssdsd sSaSsSddSs dssdaaEdaS SdaaaSsaSd 840 sSssadddTd aaSSTSssdS sdsaddSaaa SaSasadSaa saSSssSddd dsaadsSaaa 900 aaasdssSsdd aadaSaaadd SaSaSsaadd aaaaSaSaSa sssssaSdsda sdsSasessa 960
SSSdSSadSS SddSaaaSda asSSsdsSsa SdssssdsSd sdsddaSSS SdaSdaadSa 1020 aSaaaaddaS aSddsdaaaa SdasasSsds SsadaaaaSd ddsdSaSSaa SdsaaSSsSs 1080 dsEasadsSS EaaSssdSd ddSaaaaasS SSsSSaddsd aSdassdssd sdSSSaSaaa 1140 daSadssdsd saSSSasds ssdSaSSdss SaEdaaaEdS sSSssdsdS SdasssSsdd 1200
СддадааСд СссаСддда СдСаССсССССс аДддаССс СсддссаСда сдаСдааааа 1260 сссааасдс асеаСаасда ДСССаааССд Дсаасесс сдадасдддддддсдасаааС 1320 ДсдддддсадаааАС 1320
SESdssaddd dsdasdssdd ddSSsdSaS saSdadassd SdaadsadsS ddSddadsad 1440 dasssaSsda SaaaaaSsas aaasadsass sSdsdassdS SSaasSSsad SassaddsSd 1500 aSSssSsdsS assddadS SdssdssdaS dsaSSdddss adESsdSsdd SdaaaaSddd 1560 saaSddsaas SdaaddaSda ddsdssSdsa aSadSssSds SSdaSdadda aaSSsSSdad 1620 ssSaEddasd asdSsdaSsS sdaSdasdaa aassasdasd aSdaaasdsE ddaSdesdaS 1680 dadaEsdaad Sddasdaaad sdaaddadad daasEddadd aadsdddsda sdsSdaadad 1740 dssdaddSdd sSdaasadda adadaadsas ssSddsaads saaasSSSaa adsdssdadd 1800 yeah
Sssddsdssd ssddeaaesd ddsadadds aEdsaassdss ssdddadssds sSasSSsaad 1920
Food 1923
Amino acid sequence of the protelomerase of the phage pKK02 from K1eSb1e11a (ZEO 10: 11)
MKKUK1SEY MZUZUEUEA! OAZORROSOE TKKHKAAAK UKNARYOKK KRKSKSEKK 60 Ι5ΑΝΤΡΝΞΥΜ ZKAKKKGSOK Ι, ΗΗΝΕΈΚΜνί KZEKURUZ EEZZNZMR ΑΑ5ΙΕ0ΚΜ3Β 120 OAKKEHMR ΕΑΕϋΕ5ΝΙΚΙ STKVZEAKHY ΚΙΑΝΚΥΡΕΒΟ GAHZOVZEO ΜΚΟΚΚϋΥΙ.ΥΚ 180 ROOSZZE OKYKUYNE UUNOZZA EVT5100KIA VUZEKKRLU ννίΟΥΡΒΥΜΟ 240 ΑΙΥϋΙΧΒΚΡΙ UZGOTTVVS MARAGAAA 5SYAKM1E1M OEERZUASK UTUTRSOAK 300 KKZELKS13K KTUTSSAT EUZUNEIZ ΟΡΑΑΑβϊΌΕν ΙΚΕΥΕΕΝϋΤΚ 3ΕΝ2ΒΧΝΑΙΙ, 360 ATARVRNUKT ΡΣΕΟΟΚΚνΥΚ Ο3ΒΑΙΥΑΒΙΑ UEMRRKUORK NKNUOEOUEG ΜΕΙΙΌΗϋϋΕΝ 420 TONUKORK ΑΝΓ5ΚΤΗΚΡΝ USEENAVAA OKRZMMRO RAKSOASUK1 NETUVDUEO 4 80 0Ρ3ΙΚΙΤΝ3Τ VRRIRZTV ΙΡΚΥΕΕΓΑΑ0 ASORUEEYS ONOKOEARA 1UROEE1E 540 RMOOUOSOE NNOOETOOO E1EUOEZESE EEEASOAEE AEUAEOBEKN RSKRNRKARK 600 OMSOSTUMUE REGSSKNUAI ZSAASNKUEA Μ03ΑΗ3ΑΥΡΚ 640
- 25 021069
Table C
The nucleotide sequence protelomerazy phage νΡ882 of \ Dpo (8EO Yu N0: 12) aSdadsddsd daddSdaaaa assadddsdd assadaaSsd dadsadaasS ssdsaaasd sSddsdsaSs sdsadsaSda dsdadasSdd aSsaasSaSs d1sddsadsd ddSsdSsdSS sSsdadSSsS aSSaeasss ssadaadbss aSsaaSaaas dadsdsdSdS saasdSdaSs sasdaddasa dadsadssdd dasssaaaSd dadsdSaSSd ddsssaddss dSdaatasSs aaasadssda SdddaadssS sdsdSSdadd aaadSadasa ssaSsdaSda sdasSaaaSS sSsSdadsas ddaaasassa sSdadsadsS dsdassSssS adSSadassa Sadaddaads AssadSdaS AssassassSa sSaSsdadaS sSdddssaads sddsdassss dsdsasSdda dsSdSdsaaa
SsaaadaGarden sdsdsSdadaa
SsdadasSsa
Sdssasaadss addasaSas sddaadassa dSaaddSdaS sdcSddaSda adaaadsdsa dddsdsSddsSaaSdasads aaaddsaaas saaSdaddsd saadassaad
SSaSsdSaad garden SSsSsdad ddSddssaSs daadadsass sdaddSaaSd sdssdaddsd ddsddsdsd ssdsSSsads asSdaadsad daaaaadsds sdssSddSa
SdadSasdas aasdsSeeae
СсдСдссаСс ааадааадас дааадссСаС
SddsaaaSSS sasssdsSsa sdaddsdaas saaddasSas sdsadSsdSs daadsssada ddaaddsdad sdsaSdddad C Food DDA dG aSSasSsddS sSdsasdasd SasaSdasaa sadsadaSad ddddssaSdd aaddasaasd sdssaSsSda SSdassdada dSddadsSdS sddsSddsds ddsdadSSsa ddsddSdssd ssdaSddsds saasSdddsd saaaesdssa Sdddsdsdss daddasdSSS aadsaaSsa aadadsadad SssaSddssa aSsasasadS sSsdassSdd daddSSssad sSsdSddsSs daddSddssa dssadssaaa
SaaSaaaSda sSdaaaaaas aSaadsdssd
Sddssaddds adsddssdds aSaasaSsas aSdaadssss sdsaasssad adaaaassds
Sdassaada
SSddSaSSdd aaaaddedda asSaSSssad
SdaadaassS adaSSaadsd adsadSSssSS
SddsssaSda
Ссддсадда addСдсса с сдадасссс адаСсасда ссСсСасс ссдасдСдс сДсСдСдсссддсддсдсдсааа addadссса дсСдсддсддсдсдсдсдсдддддддддддддддддддддддддддддддддддддддддддддstickanned/emax
Sdsdsaaaad sasddSsdas dassaadaasd ssdsdsdaSass
Sdadsadsdd dassSaSass dsdadadSSd daasdasdss
Sddsadsdas dSSdSSSSSS daSdsSddds sadsdaassS sdsdsdsds ssdsddsdaaa ssdddaasd ssssdsdsdsddsdsdsadaa
SdaSdadsas saaddsass
Sdasbaa
120
180
240
300
360
420
480
540
600
660
720
780
940
900
960
1020
1080
1140
1200
1260
1320
1380
1440
1500
1560
1617
Amino acid sequence of the phage protelomerase UR882 from Schlr (8E0 10N N0: 13)
MZZEZSCHCHUI'S BIGGEST EUCTUOUKEA TE1AZTUK UMTMAKAUT Ε0ΝΜΚ.4Η3Ε £ ЬАНКОЬКЗ! ΚϋΝΟΕΑΓΕϋΙ KZMKONEUM ΙΝΥΗΕΙΜΑΟν UEYTKKTKT USZOZTUZGZ EEZSOAKKK SSAyUZETUT ΙΥΊΊ, νΟΞΟΙ, ν ΙΝΚΚΕΛΚΤΙ, Ν OTAKOGGSZO ΕΚνΡΚϋΒΚΑΙ ΗΕΟΙΕΤβΚΆΥ Κ0ΓΚν0Υ3ΕΡ EORUNKRSKR ΕΚΙΑΕϋΡΕΑΝ 1T05YTKE S5SKKU1K0U KSRKKAOKRK b '/ ANOUOPE !! JEIAIAJUESE
1TKZEKTKI ΤΚΑΑΤΚΓΚΤΚ LKOOKKKKRA ΟΟΙΕΚΙιΑΚΚΗ ΡΟΥΑΕΟΙΛΑΙ SELF-GAME. YNETRZAOK AKAEEEAAE ТЕ TEKTATUO KАЬСТСАТАТ СЕ СЕККККККККККΐΝΙΐΝΙΐΝΙΐΝΙ А РЕ РЕ РЕ РЕООСАСАСАСАСАСАСАΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜΞΜΑΚΙΗΟΜ
120
180
240
300
360
420
480
538
- 26 021069
Table H
The nucleotide sequence of telomerase (ίβΙΝ) and the secondary immune repressor (cA1 of the bacteriophage N15 from Euchenigma soya (5EO N N0: 14) 'Bd'a'b 120 cba £ bccdd £ bbd £ d £$ cc £ bbb £ bbc bbc £ bb a bcdb1; cbcb baaa baaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 240 asdddddaas sddda ^ dads aaddBaaaaa bcdddbd ^ b. daSaasasd sbddaad 300 add-onadds aaSdaDdss baaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. ^ baa!: yes1aa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. 540 aCssg-sb ^ a sadsdaada £ ba1cb1ca £ dds £$ bc £ a £ dss ± asdds ± aa £ aY: sdss 600 d adsaaaaey adsaadaea aaaaaaaa 720 assada ^ e ^^^ Dads ds sadSda' aaasadda da''ddaad dadsdssdd 780 as1a sYa £ £ £ 1 AMA: a £ bc saasaadds sdsdSTd adaadaas ^ a sassadsHsa addsaassa £ 840 £ Duddy C1: d assaSsSds ad_aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa! and ££ dasass 960 saasaasa dsadsas adaa * ^ 1: daa1: AA1: ss dsdas1; £ A and: adY: 1AA 1020 asassdis ddaaddsa ssSddss SdsSsdds dsddasa dddsdaadaa 1080 dadada aad £ 1sad dddaa1 £ d d ssdMsadd aaadaasd ££ a ^ 'ch 1140 sadddsaads aaaaaasds Ssdaada £ ^ a aaadsdaas sadaasda 1aSass1a £ 1200 dsdaadsaaa and asd £ £ ± daaPaaa sadaeSded £ £ x Ts x cb £ £ £ d dsas 1260 asdada dd Lq £ ££ aaa ddaaddaa eddadaas aaddsdad aasddsadda 1320 1: aah dsa £ £ THadsaaaa dsa ^ baass cMddddbaa a £ ca £ £ b £ bc ddsdadass 1380 e! sd1d1Ya aaadaads sdsdsa asdsSsdsa sdsSadad and dMs ^ ^ ss 1440 dsdgsdass asddddaaa aasdsdasd addadd ^ b ^ c C saddad aSYuSsddas 1500 asdasda ^ yes daasasssad with dsas £ £ £ and a adsadsaa ds'ddssaas sSssadaa 1560 cc ^ ddsdass £ DAAD £ Sddd dadaaaasa ssadds1dd £ ddsSsSdsad aaasddasd 1620 adaaa * gcc gcc adds £££ adadddasd sddsd1ssd bc ^ ssaHdaa assdaads 1680 adsddgdda dsaddasssa sadsaaaaa aasseasad sassssdd dssaaa 1740 gadsssdas dadaSads sddassSdd AD1 ££ dssds £ ££ dadsa etc. dddsad * sd 1800 ££ ddsdadaa sdddsadSdd SADC ^ DAAD adadaezes SdsaaSsd's sdssSdaSd 1860 Aadal £ SSD £ dadassas dasdaassdd adadads ssaadasdas dadsddad Aada 1920 £ £ yeah dadssdas dadddddsd dsdadaass aassdaadad daadddssad 1980 aadaasasa dssaasds with £ aaaasssd iS1.saadss £ dsaaaaaa aasddddasd 2040 daasd ^ asaa yes ^ adad): ^! daaasasda'd daaadsessaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaha SasssaSsss sSdsaaddda 2220 sddaaddaSS addsddaaas SdsadsSdsa asSasddasa SsdssdSsss dasSdsaddd 2280 asSSssssds dSaaadsddd x £ 1AA & CCc dddsSddssa asssSa1 £ SS SsSdsaaSsd 2340 sSddsdaSdS SadSSSsdSd daSadsd ^ SS ssadsSSSSs aasddssads SsaaaaSdSd 2400 eSddsadsas sSsSssadS SssdSaSsaa SaSsddSdaS sddsadeSsS ssasaadasa 2460 asssddsd assdssasda asasasds dsadsadss ssdsdad asasdsad 2520 dsseadads sd (b £ c £ dsa dssd'Xaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ddsdsddsdd adadssass 2640 adasdssds aasssa £ DSD Oasdd acb ^ daaaasd dsadsd s, 1, 2700 asadds ssdead Ec £ £ £ £ to £££ e DSE dssad unseat ^ ^ ddEsased DSD cosh £ ££ hell dssaddss 2760 dasaaaadsa Hassdssd b ^ ssdda adsddsada asssd 'B'dcdcsseas £ £ 2,820 dsddaassds cdds! D £ cd Csscdcdc cdcdcdcdcdcdcdcxcdcdcd 2880 dc adadcsse ^ ccdccccdcccdccdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdcdccdc sa a'Sb ^ 'Sesa's a'aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa sadssdssasa aassasddd sassassdas 3060 aadaassass sda-dadddd dds ^^ 1: ss <: d aaadadaaad asdadadad ssi * bsa ^ 1: ds 3120 dss £ ssssddd a £$$ sarsed s £ sadaaadddddd ddddddd dsSkssSdsa aazsadsd ddssssa aa £ 3240 sGddadaNad dsdddads adadsseaea adsdsIsaa ssdsadsad DSD ££ sssa 3300 a1sdsssa dsaddsssd ddsdaas daaSsSdd £ £ eadsda with asssds1 yes 3360 ssdsdagasd ddsdasada asdaddasaa aasddsHdds daasddsda sdads ^ sHs 3420 dssddada dsaaddd daaaddsdd DDA £ and £ ddde "SIdgss dgdsddasda 3480 sadsdsaaa bj daaSd aasadda' dsass ab ^ c ^ d ^ d ^ aadd dsassas 3540 Sadad 1dad aassa £ ^ and £ dddddd hell sssadasadd d £ s1: Saas seddasaad 3600 eadaaassdd sssaassdaa dLddssssa sHdadssas saaaa £$ garden daa1: kindergarten 3660 a1: b1; aasas saaaaaaasa kaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa; £ yes £ sdddd 3720 daddsssdds dsadaG dsdsadsdd ddGaassa ssdssaaads adaasdsasd 3780 saa £ aa £££ addddasa assssd dassadsas d ^ dsasadd GVD ^^ a ^ al 3840 1Hds £ L £ al dasdaSsa daassdas adSLadda dssddaa s1La ££ dad assdsadssa 3900 cc £ ada & db dssaaas sssa ^ asdds sasdaa'dad ssasddaas 3960 ddaa hell ad
Amino acid sequence of telomerase of bacteriophage N15 from ExCepsyla coh (Zeo Yu N0: 15)
M5KUK1SёY ΙίΤΐ, νΝΕνΕΑΙ OASOPROZOLK ΤΚΚΙΚΑΑΑΑΕ ΪΚΝΑΙ, ΡΝΟΚΚ КГЙСКСЬ £ К? 60 ΙΤΑΝΤΓΝΑΥΜ 3ΚΑΚΚΕΕΌΟΚ NN5G0KM1M KZEKURUZ BEZZNZMR ΤΑΝΙΒΦΗΜ33 120 05KKO1MR ΙΑΕΕΣ3ϊΐνΚΙ 05KS50AK1A KYKKUROIZ GAEZOYZO ΜΚΕΗΚΟΥΊ, ΥΚ 180 GOOSZAE ENOKUNNE νΕΥΗΣΟΙδΡΑ ΕΕΤ3Ι00ΕΗΑ OUEEKKOU νΫΙΟΥΡΤΥΜ £ 240 δΙΥϋΙΙΝΝΡΑ TRZNTKZS ΜΑΡΙΑΡΑ1ΑΑ U5SEKMHE1M Pj SEGAUZSK UTUNRZSOAK 300 ΚΚδΕϋΚδνΤΕ T1UTSEAK GUETEKZ SZAAZRROEU UKSUSKORTN 5ΕΝΘΚΙΝΑΙΙ, 360 ΑΚΑΓΝΡίίνΚδ ΓΕΌΟΟΚΕνΥΚ 05ΕΑΙΥΑΕΙΑ UEMGGNUORE ΗΚΝνΟΕΟνΓΓ ΜΕΙΕΘΗΟϋΕΝ 420 ΤΟΙ * ΗΥΚζ> ΓΚΙ, ΑΝΡΞΕΤΜΚΡΕ TSOEYTKUA OKOOEMRb GAESOASUK ΗΕΤνΚΟΙΛ / ΕΏ 480 ΟΡΞΑΚΙΓΝ3Τ EAEKGZRTM ΙΞΚΥΙ, ΕΓΑΑΟ Α1ΘΰΓν5ΕΝ6 0H £ K1ETRA TURLEOZUE 540 ΤΙΟΕΡϋϋΕδΰ ϋΟΕΙιΟΕϋΕΙΕ OESSSEERT EEESREEN ^ P TAKRUGKRA ΚΝΝΟΟΘΤΥΚΙ 600 ΕΡΕΥΟΟΚΗΥΑ NZSRAO5RMA AMEZAIETUU 5631
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Contents13
23 sheets
Sheet 1 Sheet 2 Sheet 3 Sheet 4 Sheet 5 Sheet 6 Sheet 7 Sheet 8 Sheet 9 Sheet 10 Sheet 11 Sheet 12 Sheet 13 Sheet 14 Sheet 15 Sheet 16 Sheet 17 Sheet 18 Sheet 19 Sheet 20 Sheet 21 Sheet 22 Sheet 23
Every citation, both ways
| Document | Relation | Office | Cited during |
|---|---|---|---|
| WO2017060650A1 | Cited by | World Intellectual Property Organization (WIPO) | Applicant |
| RU2616279C1 | Cited by | Russian Federation | Search report |
| WO2025190971A1 | Cited by | World Intellectual Property Organization (WIPO) | Applicant |
| FR3160186A1 | Cited by | France | Applicant |
| WO0104280A2 | Cites | World Intellectual Property Organization (WIPO) | Search report |
| WO2004028562A2 | Cites | World Intellectual Property Organization (WIPO) | Search report |
| MARDANOV A.V. ET AL.: "Functional characterization of the repA replication gene of linear plasmid prophage N15" RESEARCH IN MICROBIOLOGY, ELSEVIER, AMSTERDAM, NL LNKD-DOI: 10.1016/J.RESMIC.2005.06.008, vol. 157, no. 2, 1 March 2006 (2006-03-01), pages 176-183, XP025101129 ISSN: 0923-2508 [retrieved on 2006-03-01] page 176 - page 182 | Non-patent | – | Search report |
| RAVIN N.V. ET AL.: "The protelomerase of the phage-plasmid N15 Is responsible for its maintenance in linear form" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB LNKD-DOI: 10.1006/JMBI.2001.5019, vol. 312, no. 5, 5 October 2001 (2001-10-05), pages 899-906, XP004490133 ISSN: 0022-2836, page 899 - page 905 | Non-patent | – | Search report |
| HUANG W.M. ET AL.: "Protelomerase Uses a Topoi somerase IB/Y-Recombinase Type Mechanism to Generate DNA Hairpin Ends" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB LNKD-DOI: 10.1016/J.JMB.2004.01.012, vol. 337, no. 1, 12 March 2004 (2004-03-12), pages 77-92, XP004491619 ISSN: 0022-2836, page 77 - page 86 | Non-patent | – | Search report |
| JOCHEN HEINRICH ET AL.: "Linear closed mini DNA generated by the prokaryotic cleaving-joining enzyme TelN is functional in mammalian cells" J. MOL. MED., vol. 80, 2002, pages 648-654, XP002580374, page 648 - page 653 | Non-patent | – | Search report |
| JAN DENEKE ET AL.: "THE PROTELOMERASE OF TEMPERATE ESCHERICHIA COLI PHAGE N15 HAS CLEAVING-JOINING ACTIVITY" PNAS, vol. 97, no. 14, 5 July 2000 (2000-07-05) pages 7721-7726, XP002580375 page 7721 - page 7725; figure 3 | Non-patent | – | Search report |
| RAVIN V. ET AL.: "Genomic sequence and analysis of the atypical temperate bacteriophage N15" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB LNKD-DOI: 10.1006/JMBI.2000. 3731, vol. 299, no. l, 26 May 2000 (2000-05-26), pages 53-73, XP004470889 ISSN: 0022-2836 the whole document | Non-patent | – | Search report |
44 members in 20 offices
Priority claims2
| Document | Office | Kind | Date |
|---|---|---|---|
| 0901593 | United Kingdom | A | |
| 2010000165 | United Kingdom | W |
Members44
| Document | Office | Kind | |
|---|---|---|---|
| GB0901593D0 | United Kingdom | D0 | |
| CA2751130A1 | Canada | A1 | |
| WO2010086626A1 | World Intellectual Property Organization (WIPO) | A1 | |
| AU2010209532A1 | Australia | A1 | |
| IL213930A0 | Israel | A0 | |
| IL213930D0 | Israel | D0 | |
| MX2011007937A | Mexico | A | |
| SG173102A1 | Singapore | A1 | |
| KR20110107846A | Republic of Korea | A | |
| EP2391731A1 | European Patent Office (EPO) | A1 | |
| CN102301010A | China | A | |
| EA201101141A1 | Eurasian Patent Organization (EAPO) | A1 | |
| JP2012516147A | Japan | A | |
| HK1159693A | Hong Kong, China | A | |
| HK1159693A1 | Hong Kong, China | A1 | |
| US2012282283A1 | United States of America | A1 | |
| EP2391731B1 | European Patent Office (EPO) | B1 | |
| NZ594004A | New Zealand | A | |
| ZA201105013B | South Africa | B | |
| ES2400890T3 | Spain | T3 | |
| DK2391731T3 | Denmark | T3 | |
| EP2612925A1 | European Patent Office (EPO) | A1 | |
| IL213930A | Israel | A | |
| EA021069B1This record | Eurasian Patent Organization (EAPO) | B1 | |
| AU2010209532B2 | Australia | B2 | |
| CN102301010B | China | B | |
| US9109250B2 | United States of America | B2 | |
| CN104911177A | China | A | |
| US2015329902A1 | United States of America | A1 | |
| JP2016047049A | Japan | A | |
| EP2612925B1 | European Patent Office (EPO) | B1 | |
| EP3150722A1 | European Patent Office (EPO) | A1 | |
| CA2751130C | Canada | C | |
| KR101926662B1 | Republic of Korea | B1 | |
| JP6454243B2 | Japan | B2 | |
| BRPI1005683A2 | Brazil | A2 | |
| US2019203282A1 | United States of America | A1 | |
| EP3150722B1 | European Patent Office (EPO) | B1 | |
| BRPI1005683B1 | Brazil | B1 | |
| LT3150722T | Lithuania | T | |
| DK3150722T3 | Denmark | T3 | |
| ES2749629T3 | Spain | T3 | |
| US11384388B2 | United States of America | B2 | |
| US2022372565A1 | United States of America | A1 |
1 legal event, as the office reported them to INPADOC
Events
| Event | Code | |
|---|---|---|
| Lapse of a eurasian patent due to non-payment of renewal fees within the time limit in the following designated state(s)LapsedMM4A | MM4A |
Numbers
- Publication
- 021069
- Application
- 201101141
Titles2
- English
- PRODUCTION OF CLOSED LINEAR DNA
- Russian
- ПОЛУЧЕНИЕ ЗАКРЫТОЙ ЛИНЕЙНОЙ ДНК
Classification
- CPC, 24
- C12N9/0069
- C12Q1/6846
- C12Q1/6853
- C12Q1/6844
- C12Y113/12007
- A61P31/04
- A61P31/10
- A61P31/12
- A61P31/14
- A61P31/16
- A61P31/18
- A61P31/20
- A61P31/22
- A61P33/00
- A61P35/00
- A61P37/00
- A61P37/04
- A61P37/08
- A61P39/02
- A61K39/00
- A61K31/711
- C12N15/11
- A61K48/00
- A61K2039/53
- IPC, 2
- C12Q1 68
- C12N15 11