Process for labeling a ribonucleic acid, including optional signal amplification, and labeled RNA fragments which are obtained by this process

The present invention relates to a new method for labeling a synthetic or natural ribonucleic acid (RNA).

By synthetic RNA, it is necessary to understand RNA obtained by a technique perfected by man, for example an amplification technique (PCR followed by transcription) or transcriptional amplification (TMA). By natural RNA, it is necessary to understand RNA obtained by extraction of a cell, for example messenger, ribosomal, transfer RNA.

The state of the art shows that numerous methods exist for labeling nucleotides, oligonucleotides or nucleic acids; oligonucleotides and nucleic acids will be designated by the term polynucleotides. With regard to the oligonucleotides, the labeling can be carried out either during the synthesis, or by incorporation of at least one labeled nucleotide.

A first method consists in fixing the marker on the base, whether the latter is natural or modified. A second method proposes to fix the marker on the sugar, again whether it is natural or modified. A third method relates to the attachment of the marker to the phosphate.

Labeling on the base has been used in particular in the approach of labeling nucleic acids by incorporating directly labeled nucleotides.

The labeling on sugar is often used in the case of nucleic acid probes prepared by chemical synthesis.

Labeling on phosphate has also been used to introduce functionalized arms and markers during the chemical synthesis of polynucleotides.

In fact, a person skilled in the art, who must label a nucleotide, or a nucleotide analogue or a nucleic acid, is inclined to effect this fixation on the base or on the sugar which offers him more convenience and alternative. This is what emerges from the study of numerous documents, such as EP-A-0.329.198, EP-A-0.302.175, EP-A-0.097.373, EP-A-0.063.879 , US-A-5,449,767, US-A-5,328,824, WO-A-93 / 16O94, DE-A-3.910.151, EP-A-0.567.841 for the base or EP-A-0.286.898 for the sugar .

However, these techniques have certain drawbacks, the two main ones being steric discomfort and the influence generated by the presence of a marker.

In the case of a labeling carried out on the base, the steric hindrance is due to the encroachment of the marker on the space where a neighboring base is present, whether this neighboring base is carried by an adjacent nucleotide of the same strand or by the complementary strand. It is also fairly obvious that the presence of the marker on the base can hinder efficiency and specificity, during enzymatic incorporation, and can influence the quality of the hydrogen bonds between the two complementary strands, which can harm the hybridization.

In the case of labeling on sugar, steric discomfort is due to the encroachment of the marker on the space where an adjacent sugar is present carried by the same strand. This presence of the marker can separate two adjacent bases carried by the same strand and subsequently prevent good hybridization with the complementary strand, since the hydrogen bonds are not optimal between the strands.

Attaching the label to the phosphate is a more complex technique than the technique of functionalizing the base or sugar.

However, in certain documents, phosphate marking techniques have been proposed. This is for example the case with document EP-A-0.280.058, which describes the labeling of a nucleotide by fixing the marker on the phosphate, the latter being fixed on the sugar in position 2 'and / or 5' when the nucleotide is a deoxyribonucleotide, and in position 2 ', 3' and / or 5 'when the nucleotide is a ribonucleotide. It also describes a polynucleotide or oligonucleotide which comprises at least one nucleotide labeled as described above; this nucleotide is incorporated into the polynucleotide or the oligonucleotide during the synthesis.

However, the labeling proposed by document EP-A-0.280.058 does not make it possible to obtain uniform labeling of the nucleic acids. Indeed, the incorporation of the labeled nucleotides into the polynucleotides cannot be controlled, it entirely depends on the composition of polynucleotides which will be synthesized. Thus some polynucleotides may contain many labeled nucleotides while others may not contain any at all. The intensity of the signal emitted by these nucleic acids will therefore not be uniform, which risks making interpretation of the results difficult during their detection.

In this case, the labeling is a biological labeling by incorporation, where the position of the labeled nucleotides is not controlled.

Document US-A-5,317,098 relates to nucleic acids which are labeled at their 5 ′ end. This fixation uses imidazole and a link arm. There is no associated fragmentation. In addition, phosphate is reported, so there is use of kinase.

However and logically, there will be a phosphate at each free end of the nucleic acid, which induces at least one additional step. This marking is not associated with fragmentation.

In addition, with regard to the two previous documents, the labeling is carried out on large nucleic acids. Indeed, no fragmentation phase, also called cleavage phase, has been described before the labeling steps. Thus, when these target nucleic acids are hybridized to capture probes, the duplexes formed after hybridization are not stable. This is also the case when the polynucleotides are used as detection probes. The reasons may be due to steric hindrance or lack of specificity between the polynucleotide, which has been synthesized, and its target which is not necessarily of the same size. There will therefore be a quantitative and qualitative loss of the signal.

Steric hindrance may be due not only to the length of the nucleic acid, but also to the existence or preservation of secondary structures. Fragmentation makes it possible to destroy these structures and thus to optimize hybridization. This steric hindrance plays a particularly important role in the case of hybridization on surfaces containing high density capture probes, for example the DNA chips developed by the company Affymetrix ("Accessing Genetic Information with High-Density DNA arrays ", M. Shee et al., Science, 274, 610-614." Light-generated oligonucleotide arrays for rapid DNA sequence analysis ", A. Caviani Pease et al., Proc. Natl. Acad. Sci. USA, 1994 , 91, 5022-5026). In this technology, the capture probes are generally of reduced size, around twenty nucleotides.

With regard to the fragmentation of nucleic acids, numerous methods are described in the state of the art.

First, the fragmentation can be enzymatic, that is to say that the fragmentation of the nucleic acids can be carried out by nucleases (DNases or RNases). Small fragments are then generated with 3'-OH, 5'-OH, 3'-phosphate, 5'-phosphate ends.

Second, the fragmentation can be chemical. For example, in the case of DNA, depurination or depyrimidination of DNA can be carried out, which is then fragmented in the presence of a base by a mechanism called "β-elimination". DNA fragmentation can be achieved by mechanisms of oxidation, alkylation, addition of free radicals among others. To fragment the RNA, metal cations are often used, often associated with organic molecules used as chemical catalysts, for example imidazole. This fragmentation is preferably carried out in an alkaline medium and generates fragments with 3'-phosphate ends.

However, these fragments are not intended to facilitate or allow labeling.

The document WO-A-88/04300 proposes a method which allows the fragmentation and the labeling of RNA, the fragmentation being carried out via RNA having enzymatic properties, the ribozymes. This fragmentation by ribozymes releases for each cut an end (5 ') HO-nucleic acid and an end (3') HO-PO₂-nucleic acid. Labeling, which is only radioactive, is then carried out via an incorporation enzyme (kinase) which allows the incorporation of an added radioactive phosphate, originating from a γ-GTP molecule. This fixing is only carried out at the 5 'end. In addition, fragmentation is carried out only by ribozymes, which implies that there is specificity between these and the target nucleic acids to be cut. The phosphate then acts as a marker.

Our invention allows a marker to be fixed at the level of the only phosphate, of a nucleic acid fragment, released during the cleavage. There is no specificity, the fragmentation can be carried out on any type of nucleic acid and this at random. In fact, our method allows for example the preparation of detection probe. Finally, the phosphate is only a link arm between the nucleic acid and the marker.

No fragmentation process before marking in one or two stages has been described in the prior art.

The present invention therefore provides a method which overcomes the aforementioned drawbacks. Indeed, it makes it possible to obtain a uniform marking of the RNA fragments, as soon as the fragmentation is complete. In addition, the fragmentation makes it possible to obtain fragments of an optimal size for possible hybridization. The hybridization being of better quality, the revelation of this hybridization will be faster and more efficient.

The term “marking” is intended to mean the attachment of a marker capable of directly or indirectly generating a detectable signal. A non-exhaustive list of these markers follows:<ul id="ul0001" list-style="bullet" compact="compact"><li>the enzymes which produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, α-galactosidase, glucose-6-phosphate dehydrogenase,</li><li>chromophores such as fluorescent, luminescent, dye compounds,</li><li>groups with electron density detectable by electron microscopy or by their electrical property such as conductivity, amperometry, voltammetry, impedance,</li><li>detectable groups, for example whose molecules are large enough to induce detectable changes in their physical and / or chemical characteristics, this detection can be carried out by optical methods such as diffraction, surface plasmon resonance, surface variation , contact angle variation or physical methods like atomic force spectroscopy, tunnel effect,</li><li>radioactive molecules like the ³²P, the ³⁵S or the ¹²⁵I.</li></ul>

Indirect systems can also be used, such as for example ligands capable of reacting with an anti-ligand. The ligand / anti-ligand pairs are well known to those skilled in the art, which is the case for example of the following couples: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complementary to the polynucleotide. In this case, it is the ligand which carries the binding agent. The anti-ligand can be detectable directly by the markers described in the preceding paragraph or be itself detectable by a ligand / anti-ligand.

These indirect detection systems can lead, under certain conditions, to an amplification of the signal. This signal amplification technique is well known to those skilled in the art, and reference may be made to the prior patent applications FR98 / 10084 or WO-A-95/08000 of the applicant or to article J. Histochem. Cytochem. 45: 481-491, 1997.

To this end, the present invention relates to a method for labeling a synthetic or natural ribonucleic acid (RNA), characterized in that it consists:<ul id="ul0002" list-style="dash" compact="compact"><li>to fragment RNA, and</li><li>to be labeled at the terminal phosphate located at the 3 'and / or 5' end of each fragment of said RNA, said terminal phosphate having been released during fragmentation.</li></ul>

According to a preferred mode of operation, the labeling of the 3 'end of each fragment of the RNA is carried out for each fragment with the exception of the fragment constituting the 3' and / or 5 'end of the RNA of departure, and / or the labeling of the 5 ′ end of each fragment of the RNA is carried out for each fragment with the exception of the fragment constituting the 5 ′ end of the starting RNA.

According to a first embodiment, the fragmentation and the marking is carried out in one step.

According to a second embodiment, the fragmentation and the marking is carried out in two stages.

Whatever the embodiment, the labeling of the 3 ′ or 5 ′ end of an RNA fragment is carried out by fixation, on the phosphate in position 2 ′, in position 3 ′ or in position 2 ′ - Cyclic 3'-monophosphate, with respect to ribose, of a reactive function carried by a marker or which can be associated subsequently with at least one marker. When there are at least two markers, this is a signal amplification technique.

The fragmentation and / or labeling of the 3 ′ or 5 ′ end of an RNA fragment is carried out by fixation, on the phosphate in the 2 ′ position, in the 3 ′ position or in the 2 ′ -3 ′ position. cyclic monophosphate, with respect to ribose, of a nucleophilic, electrophilic, halide function carried by a marker or which can subsequently be associated with a marker.

The RNA is fragmented by enzymatic, chemical or physical means.

The enzymatic fragmentation of RNA is carried out by nucleases.

The chemical fragmentation of the RNA is carried out by metal cations associated or not with a chemical catalyst.

In this case, the metal cations are Mg ions⁺⁺, Mn⁺⁺, Cu⁺⁺, Co⁺⁺ and / or Zn⁺⁺, and the chemical catalyst consists of imidazole, a substituted analogue, for example N-methyl-imidazole, or any chemical molecule having an affinity for RNA and carrying an imidazole nucleus or a substituted analogue.

The physical fragmentation of RNA is carried out by sonication or by radiation.

In all cases, the marking of the 3 ′ or 5 ′ end of an RNA fragment is carried out by fixing, on the phosphate connected to the 2 ′ position, to the 3 ′ position or to the position Cyclic ribose 2'-3'-monophosphate, of an RX molecule, where R is constituted by the marker and X is the binding agent between the marker and the RNA, such as a hydroxyl, amine or hydrazine group , alkoxylamine, alkyl halide, phenyl-methyl halide, iodoacetamide, maleimide.

The present invention also consists of an RNA fragment obtained by the method, according to the characteristics described above, which is characterized in that the RNA fragment comprises, on the one hand, a single nucleotide labeled at the level of the terminal phosphate located at the 3 'or 5' end of the RNA fragment, said terminal phosphate having been released during fragmentation, and on the other hand, at least one other nucleotide, the base of which (purine: adenine / guanine or pyrimidine: uracyl / cytosine) is identical to that of the labeled nucleotide.

This RNA fragment comprises from 10 to 100 nucleotides, preferably from 30 to 70 and preferably from 40 to 60 nucleotides.

According to a preferred embodiment, the RNA fragment comprises at least one thiophosphate nucleotide.

In addition, the labeled nucleotide is a thiophosphate nucleotide.

The invention relates to the use of an RNA fragment, as defined above, as a probe for detecting RNA and / or DNA or an RNA fragment and / or DNA.

The invention finally relates to the use of an RNA fragment, as defined above, as a labeled target capable of binding to a capture probe.

The attached figures show different modes of synthesis of RNA fragments labeled according to the invention, as well as the fragments thus obtained. They represent particular embodiments and cannot be considered as limiting the scope of the present invention.

FIG. 1 represents a schematic view of the chemical fragmentation of an RNA, in the presence of Mn cations⁺⁺ and imidazole.

FIG. 2 represents a schematic view of the fragmentation and labeling of an RNA, with a marker carrying a nucleophilic function.

FIG. 3 represents a marker which can be used with the method exposed in FIG. 2, this marker being constituted by fluorescein-cadaverine.

FIG. 4 represents a marker which can be used with the method exposed in FIG. 2, this marker being constituted by fluorescein-hydrazide.

FIG. 5 represents a halogenated marker usable with the method according to the invention.

FIG. 6 represents a schematic view of the 3 ′ end of an RNA fragment obtained by a method according to the present invention, where the marker is fixed on a natural phosphate, Z being a coupling arm, also called spacer arm , as defined in a previous patent application of the plaintiff filed on 1^er August 1997 under number PCT / FR97 / 01445. This definition of Z is also true for Figures 7 and 8.

FIG. 7 represents a schematic view of the 3 ′ end of an RNA fragment obtained by a method according to the present invention, where the label is fixed on a thiophosphate.

Finally, FIG. 8 represents a schematic view of the 5 ′ end of an RNA fragment obtained by a method according to the present invention, where the label is fixed on a natural phosphate.

The process described in the figures can be in one step, as is the case in FIG. 2 where the fragmentation and the labeling are carried out jointly by a labeled nucleophile.

The process can also be carried out in two stages. A first fragmentation step according to FIG. 1, by the action of imidazole for example. Finally, a second marking step using a marker, shown without limitation in Figures 3 to 5; such labeling is carried out on the released phosphate and is exposed in FIGS. 6 to 8.

Example 1 Preparation of the Amplicon RNAs and Transcribed RNAs

A - Preparation of natural amplicons:

Natural amplicons (single-stranded RNA) are prepared using the TMA (Transcription-Mediated Amplification) amplification technique developed by Gen Probe (San Diego, CA). The amplified RNA strand corresponds to the 16S sequence of the ribosomes of<i>Mycobacterium tuberculosis</i> (ATCC -27294). The 16 S RNA was cloned into a plasmid and transformed into bacteria from the TA Cloning Dual Promoter kit (Ref: K2050-01- Invitrogen, Groningen, Netherlands). After culture in LB medium (described for example in Maniatis) and extraction of bacterial DNA by alkaline lysis, the strand was transcribed by the kit "Ampli Scribe T7 Transcription" (Ref .: AS 2607 - Epicenter Technologies (Madison, WI)). The number of copies per microliter was determined by measuring the absorbance at 260 nm.

B - Preparation of the RNAs transcribed from DNA obtained by TMA:

It should first of all be noted that the TMA amplification product contains, in addition to the RNAs, DNA amplicons, in a proportion of substantially 90% of RNA for 10% of DNA. These DNA amplicons were also used to produce single stranded RNA by transcription.<i>in vitro.</i>

Firstly, in a first step, amplicons were obtained with the Mycobacterium Tuberculosis Direct (MTD) kit from Gen Probe (Ref: 1001E), in the same way as previously according to the protocol described in step 1-A ci -above.

In a second step, transcription was carried out on a DNA matrix obtained by TMA of the first step, from a 16S RNA matrix of <i>Mycobacterium tuberculosis.</i>

The transcription was carried out from 5 μl of TMA products using the MEGAscript T7 kit (AMBION, Austin, TX, Ref: 1334). The reaction took place for one hour at 37 ° C.

C - Preparation of the RNAs transcribed from DNA obtained by PCR:

From a bacterial isolate (ATCC-27294), cultivated on Lowenstein-Jensen medium, one or two colonies (3-5 mm in diameter equivalent to 10⁸ bacteria) were collected using a spatula and resuspended in 250 µl of sterile water in a 1.5 ml Eppendorf tube. The nucleic acids were extracted from the cellular material of the bacterial suspension by vigorous shaking by means of a vortex in the presence of glass beads. Such an extraction is well described in patent applications FR97 / 12164 of September 23, 1997 and FR98 / 09583 of July 23, 1998 filed by the applicant.

A 5 µl aliquot of the lysate was added directly to the PCR reaction. It is also possible to add 20 ng of plasmid DNA directly to the PCR reaction.

The hypervariable 16S region was amplified by PCR using genus-specific primers <i>Mycobacterium</i> (positions 213-236 and 394-415 on the reference reference sequence <i>M. tuberculosis,</i> M20940, Genbank), the amplicon size being 202 base pairs (bp). The primers also contain T3 or T7 bacteriophage promoter sequences at their 5 'end. In the following representation of the primers, the promoter sequence is in lowercase and bold characters, while the sequence of mycobacteria is in capital letters:<img file="EP1260596A2_D0001.tif" />

The PCR was carried out in a reaction volume of 100 μl containing 50 mM KCl, 10 mM Tris, pH = 8.3, 1.5 mM MgCl₂, 0.001% (m / v) gelatin, 5% (v / v) DMSO, 0.5 µM of each primer, 200 µM of each of the four deoxynucleotide triphosphates, and 1.5 units of Taq polymerase (AmpliTaq, Perkin -Elmer, Norwalk, CT). The PCR reaction was carried out in a Perkin-Elmer 2400 thermocycler (Norwalk, CT) with an initial denaturation step at 94 ° C of 5 minutes (min), and 35 cycles of 45 seconds (s) at 94 ° C, 30 s at 60 ° C, 30 s at 72 ° C, followed by 10 min at 72 ° C after the last cycle. The PCR reaction products were analyzed by agarose gel electrophoresis.

Amplicons containing promoters were used to produce single-stranded RNA by transcription <i>in vitro.</i> Each reaction with a volume of 20 µl contains approximately 50 ng of PCR products, 20 units of T3 or T7 polymerase (Promega, Madison, WI)), 40 mM Tris-Acetate buffer, pH = 8.1, 100 mM d Magnesium acetate [Mg (AcO)₂], 10 mM DTT, 1.25 mM from each nucleotide triphosphate (ATP, CTP, GTP, UTP). The reaction took place for one hour at 37 ° C.

D - Preparation of the transcribed RNAs containing thiophosphates from DNA obtained by PCR:

The amplicons obtained by PCR, according to the preceding step 1-C, containing the promoters were used to produce single-stranded RNA containing thiophosphates by transcription <i>in vitro.</i> Each reaction with a volume of 20 μl then contains approximately 50 ng of PCR products, 20 units of T3 or T7 polymerase (Promega, Madison, WI)), 40 mM of Tris-Acetate buffer, pH = 8.1, 100 mM Magnesium acetate [Mg (AcO)₂], 10 mM DTT, 1.25 mM of each kind of nucleotide triphosphates or thiophosphates (ATP-α-thiophosphate (ATP-α-S) or CTP-α-thiophosphate (CTP-α-S)) :<ul id="ul0003" list-style="dash" compact="compact"><li>ATP-α-S, CTP, GTP and UTP, or</li><li>ATP, CTP-α-S, GTP and UTP</li></ul> The reaction took place for one hour at 37 ° C.

The thiophosphate nucleotide used was present at 100% of the 1.25 mM, therefore replacing the corresponding natural nucleotide. In each case, the transcription product was analyzed by electrophoresis on 6% polyacrylamide gel in the presence of 7M urea. After staining with ethidium bromide, the size of the transcript is controlled and quantified with respect to a standard deposited on the gel.

The nucleotides ATP-α-S and CTP-α-S were obtained from N&amp;N Life Science Products (Boston, MA - USA).

Example 2 - Chemical fragmentation of RNA:

The chemical fragmentation of RNAs is often catalyzed by metal cations (Mn⁺⁺, Mg⁺⁺ ...) which, by binding to the phosphate group, neutralize the negative charge of oxygen and thus facilitate the nucleophilic attack on the phosphate by the hydroxyl in position 2 'of the ribose.

This nucleophilic attack can be reinforced by the presence of molecules which are donor and acceptor of protons, such as the imidazole nucleus (R. Breslow and R. Xu, Proc. Natl. Acad. Sci. USA, 90, 1201-1207, 1993 ), as shown in Figure 1.

Fragmentation can be carried out at different temperatures and under different conditions. According to the description which follows, two different types of fragmenting have been carried out.

There may be fragmentation at 60 ° C. In this case, the 16S RNA transcripts of<i>Mycobacterium tuberculosis,</i> hereinafter called <i>Mycobacteria</i>, which contain 330 nucleotides (approximately 66 pmol), are incubated in an aqueous solution of imidazole (30mM) and manganese chloride (30mM) at 60 ° C for 30 min. The total volume of the fragmentation solution is 100 µl. The fragmented RNA is then analyzed on polyacrylamide gel (6X, urea 7M) and stained with ethidium bromide.

There can also be fragmentation at 95 ° C. So, the 16S RNA transcripts of 330 nucleotides (66 pmoles)<i>Mycobacteria</i> are incubated in an aqueous solution of magnesium chloride (30mM) at 95 ° C for 30 min. The total volume of the fragmentation solution is 100 µl. The fragmented RNA is then analyzed on polyacrylamide gel (6X, urea 7M) and stained with ethidium bromide.

In both types of fragmentation, gel analysis shows the disappearance of the starting product and the appearance of several shorter fragments, the largest population of which is between 20 and 50 nucleotides in size.

These two protocols were used to introduce a fluorescent label on the RNA chain during its fragmentation.

Example 3 - Marking during fragmentation by introduction of markers carrying a nucleophilic function:

A more nucleophilic function than hydroxyl, in the 2 'position of ribose, can attack the neutralized phosphate and thus allow the fragmentation of the RNA chain by generating fragments linked to this function, via the phosphate group. According to Figure 2, this function can be linked to a marker to generate labeled RNA fragments.

Fluorescein-cadaverine, exposed in FIG. 3, carrying an amine function and fluorescein-hydrazide, detailed in FIG. 4, carrying a hydrazide function, were used to label a 16S RNA (330 nucleotides) of <i>Mycobacteria</i>, during fragmentation at 65 ° C. Such RNAs were obtained according to Example 1B

Fluorescein cadaverine and fluorescein hydrazide were solubilized in DMF to a final concentration of 7.5 mM. They come from Molecular Probes (Eugene, OR, USA).

To the target of 16S RNA (66 pmol) in solution in the fragmentation buffer at 65 ° C (Example 1), 1 μl of the marker solution (7.5 mM in DMF) was added. After incubation at 65 ° C for 30 minutes, each reaction product was hybridized, detected and analyzed on a DNA chip (Affymetrix, Santa Clara, CA USA) according to the protocol provided by the manufacturer. These chips are designed for the identification of a particular region, and in this case of the region 213-415 of the sequence M20940 "Genbank" of the 16S RNA of <i>Mycobacterium tuberculosis.</i> In the case of cadaverine fluorescein or hydrazide fluorescein, the sequence was found to be 66%. This indicates that in each case the marker was introduced during the fragmentation thus generating fluorescent and detectable fragments. Such an identification is well described in the article by A. Troesch et al, J. Clin Microbiol., 37 (1), p. 49-55, 1999.

Example 4 Marking During Fragmentation Via a Marker Carrying a Methyl Halide:

It is known that a monophosphate can be substituted by a marker carrying a phenyl-methyl halide group (See T. Furuta et al., J. Chem. Soc. Perkin Trans. 1 3139- 3142, 1993). 5-bromo-fluorescein, represented in FIG. 5, belongs to this category of halogenated markers.

Knowing that the fragmentation reaction releases cyclic 3'-monophosphate ends, in equilibrium with the open forms, the fragmentation step can be used to attach a marker to the phosphate groups at 3 'of the RNA fragments.

A - Labeling of the RNA amplicons obtained by TMA amplification:

16S RNA amplicons (330 nucleotides) of <i>Mycobacteria</i> were prepared by TMA amplification, as described in Example 1-A. 5-bromo-fluorescein was obtained from Molecular Probes (Eugene, OR, USA). Manganese chloride was obtained from Sigma and imidazole from Aldrich.

A - 1. Marking of 50 μl of TMA: protocol using 6 mM of imidazole and 60 mM of manganese chloride:

To 165 μl of RNase free water (Sigma), in a polypropylene tube of 5 ml, the dimensions of which are 12 mm in diameter and 75 mm in length, 15 μl of an imidazole solution (0, 1 M), 15 μl of a solution of manganese chloride (1 M), 2.5 μl of 5-bromofluorescein (100 mM) in DMSO then 50 μl of TMA products. The mixture is homogenized by vortexing and incubated at 60 ° C for 30 min.

After incubation, the solution was used without any purification for hybridization and detection on a DNA chip. (Affymetrix, Santa Clara, CA USA), also called a biochip. The protocol used for this hybridization step is that provided by the manufacturer. These chips are designed for the identification of the 213-415 region of the M20940 “Genbank” sequence of the 16S RNA of<i>Mycobacterium tuberculosis.</i> The results are given in table 1 below: <tables id="tabl0001" num="0001"><table frame="all"><title>Table 1:</title><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col3" align="center">Results of labeling of RNA fragments labeled without purification</entry></row><row><entry namest="col1" nameend="col1" align="center">Score obtained</entry><entry namest="col2" nameend="col2" align="center">Median Intensity (Rfu)</entry><entry namest="col3" nameend="col3" align="center">Background noise (Rfu)</entry></row></thead><tbody valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" align="center">91,2 %</entry><entry namest="col2" nameend="col2" align="center">1727</entry><entry namest="col3" nameend="col3" align="center">834</entry></row></tbody></tgroup></table></tables>

The percentage of identification or score obtained corresponds, in identification by biochip technology, to a percentage of analysis compared to the reference sequence. The sequence is identified at 91.2% and the median intensity is 1727 Rfu. This indicates that in each case the marker was introduced during the fragmentation thus generating fluorescent and detectable fragments. It is also important to note that the labeling reaction product was hybridized directly on the chip without any prior purification. This result is very interesting and shows that unpurified transcribed RNAs can be efficiently labeled by this fragmentation protocol using imidazole and manganese chloride.

In a second step, we attempted the labeling of 50 μl then 100 μl (total volume) of a TMA reaction described in example 1-A, using the labeling protocol described above and a purification step before hybridization on the DNA chip.

After incubation at 65 ° C, the two labeling reaction products containing 50 μl and 100 μl of TMA were treated with butanol-1 to extract the excess of 5-bromofluorescein. This extraction was carried out with twice 1 ml of butanol-1 saturated with water. Such an extraction, like those which will follow, is well known from the state of the art. Additional information can in particular be found in the document Sambrook, Fritsch &amp; Maniatis, Molecular Cloning (Second Edition, 1.46) Cold Spring Harbor Laboratory Press, 1989. The results are given in table 2 below. These measurements are made after hybridization and reading on the biochip.<tables id="tabl0002" num="0002"><table frame="all"><title>Table 2:</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col4" align="center">Results of labeling of the RNA fragments labeled with purification</entry></row><row><entry namest="col1" nameend="col1" align="center">TMA volume</entry><entry namest="col2" nameend="col2" align="center">Score obtained</entry><entry namest="col3" nameend="col3" align="center">Median Intensity (Rfu)</entry><entry namest="col4" nameend="col4" align="center">Background noise (Rfu)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="center">50 µl</entry><entry namest="col2" nameend="col2" align="center">95,9 %</entry><entry namest="col3" nameend="col3" align="center">6867</entry><entry namest="col4" nameend="col4" align="center">169</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="center">100 µl</entry><entry namest="col2" nameend="col2" align="center">96,7%</entry><entry namest="col3" nameend="col3" align="center">5775</entry><entry namest="col4" nameend="col4" align="center">275</entry></row></tbody></tgroup></table></tables>

The percentage of identification or score obtained is close to 100% and the intensities obtained are very high. This shows that a purification step before hybridization can improve the identification percentages, the labeling intensities and reduce the background noise. Indeed, possible additional post-hybridization washing cycles are no longer necessary.

A - 2. Labeling of 50 μl of TMA: protocol using 30 mM of imidazole and 30 mM of manganese chloride

To 112 µl of RNase free water (Sigma) in a 5 ml polypropylene tube, 75 µl of an imidazole solution (0.1 M), 7.5 µl of a manganese chloride solution are added. (1 M), 2.5 μl 5-bromofluorescein (100 mM) in DMSO then 50 μl of TMA products. The mixture is homogenized by shaking with a vortex and incubated at 60 ° C for 30 min.

After incubation, the two labeling reaction products on 50 μl of TMA products were treated with butanol-1 to extract the excess of 5-bromofluorescein. These measurements are made after hybridization and reading on the biochip. This extraction was carried out with twice 1 ml of butanol-1 saturated with water. The results are given in Table 3 below.<tables id="tabl0003" num="0003"><table frame="all"><title>Table 3:</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col4" align="center">Results of labeling of the RNA fragments labeled with purification</entry></row><row><entry namest="col1" nameend="col1" align="center">TMA volume</entry><entry namest="col2" nameend="col2" align="center">Score obtained</entry><entry namest="col3" nameend="col3" align="center">Median Intensity (Rfu)</entry><entry namest="col4" nameend="col4" align="center">Background noise (Rfu)</entry></row></thead><tbody valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" align="center">50 µl</entry><entry namest="col2" nameend="col2" align="center">92,7 %</entry><entry namest="col3" nameend="col3" align="center">444</entry><entry namest="col4" nameend="col4" align="center">131</entry></row></tbody></tgroup></table></tables>

The amplified sequence is identified at 92.7% and the median intensity is 444 Rfu. These results show that the protocol using 30 mM of imidazole and 30 mM of MnCl₂ produces labeled amplicon fragments. However, the intensity level is lower than that obtained with the imidazole / MnCl protocol₂ = 6 mM / 60 mM.

This strategy can be optimized. The high concentration of metal salt is certainly very important for the labeling reaction. Other metals or other buffers can be used to carry out this labeling reaction during fragmentation.

B - Labeling of the transcribed RNAs obtained by TMA post-amplification transcription:

16S transcribed RNA targets (330 nucleotides) of mycobacteria were prepared by transcription, as described in Example 1-B.

To 165 µl of RNase free water (Sigma) in a 5 ml polypropylene tube, 15 µl of an imidazole solution (0.1 M) is added, 15 µl of a manganese chloride solution (1 M), 2.5 μl 5-bromofluorescein (100 mM) in DMSO then 50 μl of transcription product. The mixture is homogenized by shaking with a vortex and incubated at 60 ° C for 30 min.

After incubation, the solution was treated as described in Example 3 (A - 1.) to remove the excess of 5-bromofluorescein. The hybridization and the detection were then carried out on the DNA chip (Affymetrix, Santa Clara, CA USA), designed for the identification of the region 213-415 of the sequence M20940 "Genbank" of the 16S RNA of<i>Mycobacterium tuberculosis.</i> The results are given in Table 4 below. <tables id="tabl0004" num="0004"><table frame="all"><title>Table 4:</title><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col3" align="center">Results of labeling of the RNAs obtained by TMA post-amplification transcription</entry></row><row><entry namest="col1" nameend="col1" align="center">Score obtained</entry><entry namest="col2" nameend="col2" align="center">Median Intensity (Rfu)</entry><entry namest="col3" nameend="col3" align="center">Background noise (Rfu)</entry></row></thead><tbody valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" align="center">97,6 %</entry><entry namest="col2" nameend="col2" align="center">1426</entry><entry namest="col3" nameend="col3" align="center">147</entry></row></tbody></tgroup></table></tables>

The sequence is identified at 97.6% and the median intensity is 1426 Rfu. This result shows that the labeling strategy during fragmentation used to label RNA produced by post-TMA transcription is effective. These transcribed RNA targets are used in the labeling reaction without any purification.

C - Labeling of the transcribed RNAs containing thiophosphates post-PCR amplification:

16S RNA targets (330 nucleotides) of mycobacteria were produced by post-PCR transcription reactions where 100% of adenine tri-phosphate (ATP) was replaced by ATP-α-thio (two manipulations were performed) or else 100% of cytidine tri-phosphate (CTP) has been replaced by CTP-α-thio (manipulation has been carried out), as indicated in Example 1-D.

These RNAs were labeled using the protocol described above in the presence of imidazole and manganese chloride. After incubation at 65 ° C for 30 min, the reaction product was hybridized, detected and analyzed on a DNA chip (Affymetrix, Santa Clara, CA USA) according to the protocol provided by the manufacturer. The results are given in Table 5 below.<tables id="tabl0005" num="0005"><table frame="all"><title>Table 5:</title><tgroup cols="3" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col3" align="center">Results of labeling of RNAs containing thiophosphates obtained by post-amplification PCR transcription</entry></row><row><entry namest="col1" nameend="col1" align="center">Transcribed</entry><entry namest="col2" nameend="col2" align="center">Score obtained</entry><entry namest="col3" nameend="col3" align="center">Median Intensity (Rfu)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="center">ATP-α-S</entry><entry namest="col2" nameend="col2" align="center">97,1% 100%</entry><entry namest="col3" nameend="col3" align="center">1186 1290</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="center">CTP-α-S</entry><entry namest="col2" nameend="col2" align="center">93,6%</entry><entry namest="col3" nameend="col3" align="center">1027</entry></row></tbody></tgroup></table></tables>

The sequences are identified at 97.1 and 100% in the case of ATP-α-S and at 93.6% in the case of CTP-α-S. The median intensities are between 1000 and 1300 Rfu. This result shows that the labeling strategy during fragmentation used to label RNAs containing thio-phosphates is effective, and even as effective as non-sulfur containing nucleotides. These transcribed RNA targets are used in the labeling reaction without any purification.

Example 5 - Marking of natural RNA:

A bacterial suspension is obtained from a pure strain isolated on a solid medium. This suspension produced in sterile water is standardized to an index of 2 MacFarland (or approximately 3.10⁸ bacteria / ml) then is centrifuged in an Eppendorf tube for 1 min.

The RNA is extracted from the bacterial biomass as follows. The bacterial pellet is lysed by resuspension in 100 μl of lysis buffer (Tris.HCl 30 mM, EDTA 5 mM, 100 mM NaCl 2% SDS, proteinase K 5 mg / ml, pH 7.3 in the presence of 50 μl of beads of round glass 100 µm in diameter, made by VIA1 France). We vortex for 30 sec. Then incubate for 15 min at 37 ° C. 100 μl of saturated phenol are then added. It is subjected to a vortex for 30 s. The aqueous phase is recovered and then extracted with 100 μl of chloroform. It is again subjected to a vortex for 30 s. The aqueous phase is again recovered. Two ether extractions are carried out and this solvent is evaporated. There then remains approximately 50 μl of aqueous phase containing the total RNAs. Alternatively for this extraction, the Qiagen RNeasy kit can be used.

The labeling of the total RNAs of the bacterial biomass by the technique described in the present invention is carried out as follows by adding the reagents in the following order:<ul id="ul0004" list-style="dash" compact="compact"><li>all the RNA extracted (50 µl),</li><li>water (qs 100 µl),</li><li>6 µl imidazole 1 M (60 mM final),</li><li>6 µl MnCl₂ 1 M (60 mM final),</li><li>the solution is subjected to a vortex for 10 s,</li><li>2 µl of 50 mM 5-Bromo-fluorescein (1 mM final),</li><li>homogenized by removing several times using a pipette,</li><li>we submit again to a vortex for 1 s,</li><li>we centrifuge for 1 s to collect at the bottom of the tube, and</li><li>incubated for 30 minutes at 60 ° C.</li></ul>

To remove the excess free marker, the procedure is then as follows. From the 100 µl marked we add:<ul id="ul0005" list-style="dash" compact="compact"><li>40 µl salmon sperm DNA</li><li>100 µl final sodium acetate (610 mM) final pH 5.2</li><li>250 µl cold isopropanol (-20 ° C)</li></ul>

It is subjected to a vortex, then centrifuged for 1 min. The supernatant is removed. The pellet is then resuspended in the hybridization buffer (6 x SSPE, 5mM DTAB, 3M betaine, Triton 0.05%, 250 μg / ml of herring sperm DNA)

We visualize on a 1% agarose gel with ethidium bromide:<ul id="ul0006" list-style="bullet" compact="compact"><li>an aliquot of the preparation of total RNA before labeling according to the invention (A),</li><li>an aliquot of the preparation of total RNA after labeling according to the invention (B).</li></ul>

After visualization of ethidium bromide under ultraviolet (UV), we note:<ul id="ul0007" list-style="dash" compact="compact"><li>for the well With the presence of the characteristic bands of majority RNA, with from top to bottom: 23S, 16S then a diffuse band corresponding to the population of messenger RNA,</li><li>for well B, the presence at the bottom of the gel of a population corresponding to the total mixture of fragmented RNA.</li></ul>

After visualizing the fluorescein, we note:<ul id="ul0008" list-style="dash" compact="compact"><li>for well A: nothing at all,</li><li>for well B, the presence at the bottom of the gel of a population corresponding to the mixture of RNA fragmented and covalently labeled with fluorescein, which shows that the RNAs have been fragmented and labeled. This control was carried out for all the bacterial species described in the table below.</li></ul>

An aliquot of the RNAs thus labeled is hybridized on a solid glass support (biochip) where oligonucleotides are grafted by photolithography allowing the identification of these different species according to a process equivalent to that described in the article by A. Troesch et al, J. Clin Microbiol., 37 (1), p. 49-55, 1999. The results are reproduced in Table 6 below.<tables id="tabl0006" num="0006"><table frame="all"><title>Table 6:</title><tgroup cols="4" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row><entry namest="col1" nameend="col4" align="center">Results of labeling natural RNAs from different species</entry></row><row><entry namest="col1" nameend="col1" align="center"><b>Cash</b></entry><entry namest="col2" nameend="col2" align="center"><b>International strain reference</b></entry><entry namest="col3" nameend="col3" align="center"><b>Internal strain reference</b></entry><entry namest="col4" nameend="col4" align="center"><b>Score obtained</b></entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="center"><i>Escherichia coli</i></entry><entry namest="col2" nameend="col2" align="center">ATCC11775T</entry><entry namest="col3" nameend="col3" align="center">7308009</entry><entry namest="col4" nameend="col4" align="center">86,3%</entry></row><row><entry namest="col1" nameend="col1" align="center"><i>Streptococcus pneumoniae</i></entry><entry namest="col2" nameend="col2" align="center">NCTC7465T</entry><entry namest="col3" nameend="col3" align="center">7804060</entry><entry namest="col4" nameend="col4" align="center">87,2%</entry></row><row><entry namest="col1" nameend="col1" align="center"><i>Klebsiella pneumoniae</i></entry><entry namest="col2" nameend="col2" align="center">ATCC13883T</entry><entry namest="col3" nameend="col3" align="center">7308012</entry><entry namest="col4" nameend="col4" align="center">84,6%</entry></row><row><entry namest="col1" nameend="col1" align="center"><i>Pseudomonas aeruginosa</i></entry><entry namest="col2" nameend="col2" align="center">ATCC10145</entry><entry namest="col3" nameend="col3" align="center">7309001</entry><entry namest="col4" nameend="col4" align="center">79,1%</entry></row><row><entry namest="col1" nameend="col1" align="center"><i>Enterobacter cloacae</i></entry><entry namest="col2" nameend="col2" align="center">ATCC13047T</entry><entry namest="col3" nameend="col3" align="center">7308013</entry><entry namest="col4" nameend="col4" align="center">81%</entry></row><row><entry namest="col1" nameend="col1" align="center"><i>Streptococcus agalactiae</i></entry><entry namest="col2" nameend="col2" align="center">ATCC13813</entry><entry namest="col3" nameend="col3" align="center">7701031</entry><entry namest="col4" nameend="col4" align="center">89,1%</entry></row><row><entry namest="col1" nameend="col1" align="center"><i>Klebsiella oxytoca</i></entry><entry namest="col2" nameend="col2" align="center">ATCC13182T</entry><entry namest="col3" nameend="col3" align="center">9211047</entry><entry namest="col4" nameend="col4" align="center">85,5%</entry></row><row><entry namest="col1" nameend="col1" align="center"><i>Citrobacter freundii</i></entry><entry namest="col2" nameend="col2" align="center">ATCC29935T</entry><entry namest="col3" nameend="col3" align="center">9410068</entry><entry namest="col4" nameend="col4" align="center">81,5%</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="center"><i>Salmonella typhimurium</i></entry><entry namest="col2" nameend="col2" align="center">---</entry><entry namest="col3" nameend="col3" align="center">9810059</entry><entry namest="col4" nameend="col4" align="center">71,3%</entry></row></tbody></tgroup></table></tables>

The species with the highest score on the biochip is always the species sought, the score obtained is indicated in the right column.

For all the species, the identification is correct, showing that the process of the present invention is effective both on natural RNA and on RNA derived from an amplification technique.