Bases chemically modified with a cleavable group and methods of selecting functional nucleic acids comprising same

Abstract

The present invention concerns the field of modified nucleic acids, and in particular aptamer, ribozyme or deoxyribozyme functional nucleic acids, and methods for selecting (SELEX) such modified nucleic acids. The invention concerns nucleobases, nucleosides and nucleotides modified by a particular chemical group, and the uses thereof, in particular in methods for reverse transcription, amplification and selection of functional nucleic acids, regardless of the chemical group attached to the modified nucleic acids to be selected, provided that said group is cleavable via an SPICC reaction.

Description

[0001] DESCRIPTION

[0002] TITLE: Chemically modified bases with a cleavable group and methods for selecting functional nucleic acids comprising them

[0003] FIELD OF INVENTION

[0004] The present invention relates to the field of modified nucleic acids, and in particular to functional nucleic acids of the aptamer, ribozyme, or deoxyribozyme type, as well as selection methods (SELEX) for such modified nucleic acids. It concerns nucleobases, nucleosides, and nucleotides modified by a specific chemical group, and their uses, particularly in reverse transcription, amplification, and selection methods for functional nucleic acids, regardless of the chemical group attached to the modified nucleic acids to be selected, provided that it is cleavable by sterically strained iminosydnone-cycloalkyne cycloaddition (SPICC).

[0005] STATE OF THE ART

[0006] Nucleic acids (e.g., DNA or RNA) can serve as carriers of genetic information. Their primary structure, resulting from the chaining of nucleotides, can also fold to adopt secondary structures that can interact with each other to form tertiary structures. These structures can allow them to have numerous activities, such as specific interactions with molecules (small chemical compounds, peptides, proteins, etc.) or catalytic activities (e.g., ligation). Consequently, nucleic acid sequences can simultaneously contain a phenotype (activity) and a genotype (information) that allows this phenotype to be replicated. They are the only known polymers on Earth to possess this property.Molecular evolution strategies, also called directed molecular evolution, in vitro selection, or SELEX (for "Systematic Evolution of Ligands by Exponential Enrichment"), can identify nucleic acid structures with a chosen activity without prior knowledge of them. These strategies have been used to identify, from libraries of billions of oligonucleotide sequences, sequences capable of exhibiting catalytic activities (often called ribozymes or DNAzymes) or binding activities with a target molecule (often called aptamers). The advantage of these techniques is that they allow the application, at the molecular level, of the principles of evolution described by Charles Darwin.The principle of these techniques is based on the synthesis of a population of oligonucleotides called "candidates," which contain a random sequence flanked by two constant sequences essential to the different stages of enzymatic amplification. This random population (generally 10. 13 at 10 15The collection of different sequences is then subjected to selection pressure. The selection criterion can be catalytic activity or affinity for a chosen target. Candidates meeting the selection criteria are separated from the others and then amplified using polymerases (e.g., by PCR and / or in vitro transcription) for use in a new selection cycle. The polymerases used during the amplification step can generate mutations that are an integral part of the Darwinian evolution of the sequence population. Indeed, these mutations generate diversity in the selected sequences and can lead to the creation of sequences that respond better to selection pressure, even though they were not present in the original library.Repeating selection, amplification, and mutation steps allows the population to evolve in a Darwinian manner by progressively amplifying sequences with the best properties for the desired function. These sequences can then be identified by sequencing.

[0007] Even though molecular evolution technologies for nucleic acids are very efficient, natural nucleic acid sequences can only be formed from four nucleotides: deoxyadenosine, deoxyguanosine, deoxycytosine, and deoxythymidine for DNA, and adenosine, guanosine, cytosine, and uridine for RNA. However, these nucleotides cover a limited range of possible chemical interactions, which restricts the potential for molecular interaction and catalytic activity. For example, unlike proteins, nucleic acids do not contain sulfur atoms, positively charged groups, or hydrophobic groups. To overcome this limitation, chemical modifications have been introduced into nucleic acids to improve their existing properties or to impart entirely new ones.These chemical modifications can be introduced by chemical synthesis once a nucleic acid sequence has been identified through a molecular evolution process. However, care must be taken to ensure that these subsequent modifications do not disrupt the nucleic acid structure that confers the desired property. Experience to date shows that the consequences of chemical modifications on nucleic acid structure are difficult to predict. Therefore, it is necessary to synthesize and then test numerous sequences with chemical modifications at different positions, which is time-consuming and expensive. A more advantageous approach is to incorporate a chemical modification during the molecular evolution process in order to select chemically modified nucleic acid sequences that meet a specific selection criterion.This field of research is part of "xenobiology" or "synthetic biology".

[0008] Several methods have been proposed for the selection and molecular evolution of chemically modified nucleic acids. Early strategies relied on the use of chemically modified triphosphate nucleotides that could still be read and incorporated by polymerases. This approach has been used for the incorporation of modified nucleotides onto the ribose, the phosphodiester bond, or the nucleic base. Examples include 5-(l-pentynyl)-2'-deoxyuridine triphosphate, 2'-aminopyrimidine (2'-NH2Py) triphosphate, and 2'-fluoropyrimidine ((2'-F-Py)) triphosphate. For instance, the American company SomaLogic uses several modified nucleotides, such as deoxyuridines or deoxycytosines, chemically modified at the 5' position by a benzyl, naphthyl, indole, or isobutyl group (US10221207B2, US10239908B2, US10316321B2, US2021 / 0171951A1).This company markets "Somamers" (aptamers containing these chemical modifications) and has demonstrated that these modifications allow for the production of ligands with better affinities compared to chemically unmodified aptamers, notably with slower dissociation rates ("slow off rate").

[0009] However, a significant limitation currently restricts this xenobiology to the use of nucleotides that are only slightly modified chemically. Indeed, these chemical modifications must be compatible with the nucleic acid replication process, which uses polymerases. But polymerases are highly specific enzymes that have been selected over millions of years to function very specifically with natural nucleotides. Therefore, they cannot tolerate significant chemical modifications.

[0010] Thus, chemically modified triphosphate nucleotides are often incorporated with reduced polymerization yields. Furthermore, sequences incorporating these chemical modifications often have difficulty being used as templates for the synthesis of a complementary strand, as the ability of polymerases to read bases is even more affected by the chemical modifications than their ability to incorporate modified bases once the template sequence has been read.

[0011] To address these issues, mutations have been introduced into polymerases. For example, the Y639F mutation in T7 RNA polymerase is known to incorporate 2'-F-Py triphosphate more efficiently. Some laboratories have even evolved polymerases using molecular evolution techniques to identify polymerases capable of incorporating certain non-canonical nucleotides. For instance, Philip Holliger's laboratory at the MRC in Cambridge selected polymerases capable of incorporating several types of chemically modified nucleotides, called XNAs (xeno-nucleic acids). This group was able to use their polymerases to perform directed molecular evolution of sequences containing four chemically modified bases (EP2074211, US9228179B2). However, molecular evolution of polymerases is a very complex technology to implement, and each modified polymerase can only incorporate one specific type of modification.

[0012] Another approach, called "Click-SELEX," was developed by Günter Mayer's laboratory at the University of Bonn (EP3201353). In this case, 5-ethynyl-2'-deoxyuridine (EdU) is used to replace deoxythymidine in DNA. The advantage is that EdU can be incorporated by DNA polymerases with the same yield as deoxythymidine. The EdU incorporated into the DNA can then be covalently conjugated to various chemical groups via a copper(I)-catalyzed azide-alkyne cycloaddition bio-orthogonal chemistry reaction (CuAAC). This two-step modification overcomes the difficulties encountered by polymerases when incorporating chemically modified triphosphate nucleotides. A similar strategy was developed by the American company AM Biotechnologies LLC using oligonucleotide libraries grafted onto beads (US9988623B2).However, in both approaches, the sequences that have incorporated chemical modifications must still be able to be used as a template for the synthesis of a complementary strand, which again considerably limits the chemical modifications that can be used.

[0013] Another approach, called SELMA (“Selection with Modified Aptamers”), was developed by Isaac Krauss’s laboratory at Brandeis University by combining click conjugation with strand displacement (WO2015 / 084846, WO2018 / 152470, US10125162B2). In this method, a DNA strand is chemically modified by click chemistry while remaining covalently bound to a double-stranded DNA template, allowing its replication. Thus, at each iteration, the unmodified double-stranded DNA template can be replicated and amplified, then used to generate a new chemically modified strand. This has made it possible to select chemically modified RNAs without the need for reverse transcription and to select aptamers modified by complex sugars that would have been impossible to amplify enzymatically.However, this technique is very complicated to implement, and the folding of chemically modified sequences can be affected by attachment to the double-stranded DNA template. Thus, once detached from the double-stranded DNA template, the specificity of the selected sequences can be altered.

[0014] Therefore, there is still a need to develop new molecular evolution methods that allow for chemical modification of DNA or RNA and are simpler to implement.

[0015] SUMMARY OF THE INVENTION

[0016] To overcome the aforementioned problems, the inventors of the present invention have developed a novel molecular evolution method coupled with bio-orthogonal chemical reactions that enables the Darwinian evolution of a population of nucleic acid polymers conjugated to a wide variety of chemical groups without hindering replication. This method can be applied to identify high-affinity ligands that can be used, for example, in diagnostics, purification, the design of new drugs, or for the selection of new catalysts or the improvement of existing catalysts such as (deoxy)ribozymes.

[0017] This method allows the selection of sequences containing chemical modifications of any type without concern for polymerase compatibility and without requiring them to be attached to an unmodified DNA template. As a result, the chemically modified sequences can fold freely to adopt their three-dimensional conformation and then be selected by molecular evolution.

[0018] This method is based on:

[0019] (1) the generation of a library of nucleic acid sequences which contain non-canonical nucleotides modified by cleavable chemical groups;

[0020] (2) the selection of modified sequences that fulfill a selection criterion (e.g., binding to a target or catalyst);

[0021] (3) the cleavage of chemical groups to produce nucleic acid sequences that can serve as a template for the synthesis of a complementary strand by a polymerase;

[0022] (4) the amplification of these sequences; and

[0023] (5) the generation of a new library of nucleic acid sequences containing non-canonical nucleotides modified by cleavable chemical groups as in (1), and then the repetition of steps (2) to (5) to allow enrichment by Darwinian-type molecular evolution of chemically modified sequences that meet the selection criterion. The method further includes, at least in the last cycle, sequencing analysis of a portion of the library after step (4). When this step is performed in one or more intermediate cycles, it serves to measure the enrichment in nucleic acids of interest.

[0024] The advantage of this method is that it is possible to add chemical groups of all types in step (1), even groups that could inhibit sequence replication by polymerases or their reading when incorporated into a template strand, because these groups will be cleaved in step (3) in order to generate readable and amplifiable sequences.

[0025] The present invention thus relates first to a modified nucleobase, namely a nucleobase in which a hydrogen atom is replaced by Ro or a nitrogen atom is replaced by CRo where Ro is a substitution group of formula -A0-X1-A1-Y1-R1 or -A0-X1-A1-Y2 in which:

[0026] - Ao represents a single bond or a Ci-10-alkanediyl chain possibly preceded and / or interrupted and / or followed and / or replaced by one or more motifs chosen from the group , in which the wavy line indicates the point of connection with Ao and the dotted line indicates the point of connection with Ai;

[0027] - Ai represents a Ci-20-alkanediyl chain possibly preceded and / or interrupted and / or followed and / or replaced by one or more motifs chosen from the group consisting of -OC-, -C(R8)=C(R9)-, -O-, -S-, -NR10-, -C(O)-, -C(S)-, and -C(NRn)-; - Yi represents a group , in which X2 represents -NH-

[0028] CO-, -O-CO-, -SO2-, or -P(O)(Ci-6-alkyl)-, the -NH-CO- and -O-CO- groups being linked to Ai by the NH or O motif respectively and to the rest of the Yi group by the CO motif, R31 represents H or a halogen (I, Br, Cl or F), the dotted line indicates the point of bonding with Ai and the wavy line indicates the point of bonding with Ri;

[0029] - - Ri represents a group of formula A2-R30, in which A2 is a single bond or a Ci-20-alkanediyl chain possibly preceded and / or interrupted and / or followed and / or replaced by one or more motifs selected from the group consisting of an arylene, a C3-12- divalent carbocycle, -C=C-, -C(Ri2)=C(Ri3)-, -O-, -S-, -NR14-, -C(O)-, -C(S)-, and -C(NRis)-, and R30 is a Ci-20-alkanediyl, aryl, Cs-n-carbocyclyl, heteroaryl, or heterocycle group, said group possibly being substituted by one or more groups selected from ORIÔ, SR17, SOR18, SO2R19, OSO2R20, SO3R21, NR22R23, CO2R24, CONR25R26, NH-(C=NH)-NH2, N + R27R28R29Gf, NH-(C=NH2 + G2')-NH2, CO2 G3 + , SO3 G4 +, a metal complex derivative, an amino acid derivative, a cyanine derivative, a rhodamine derivative, a BODIPY derivative, a fluorophore group, and a photobridging group, or R30 is a group selected from ORIÔ, SR17, SOR18, SO2R19, OSO2R20, SO3R21, NR22R23, CO2R24, CONR25R26, NH-(C=NH)- NH2, N + R27R28R29Gf, NH-(C=NH2 + G2)-NH2, CO2G3 + , SO3G4 + , a metal complex derivative, an amino acid derivative, a cyanine derivative, a rhodamine derivative, a BODIPY derivative, a fluorophore group, and a photobridging group;

[0030] - R2 to R29 each represent, independently of each other, H or Ci-6-alkyl;

[0031] - Gf and G2 represent, independently of each other, a negatively charged counter-ion;

[0032] - G3 and G4 + represent, independently of each other, a positively charged counterion; and

[0033] - Y2 represents in which the dotted line indicates the point of connection with

[0034] Ai.

[0035] The present invention has as its second object a modified nucleoside consisting of a modified nucleobase according to the invention linked to the anomeric carbon atom in position 1' of a pentose residue, advantageously chosen from deoxyribose residues, ribose and ribose derivatives.

[0036] The present invention has as its third object a modified nucleotide consisting of a nucleoside modified according to the invention in which the OH group in position 5' of the pentose residue is replaced by an -O-(Z3i) group q i-(Z32) q 2-Z33-H or a salt thereof for which:

[0037] - q1 and q2 independently represent 0 or 1, and

[0038] - Z31, Z32 and Z33 represent, independently of each other, -P(O)(OH)-O-, -P(O)(SH)- O-, -P(O)(CH3)-O-, -P(S)(OH)-O-, -P(S)(SH)-O-, OR -P(S)(CH3)-O- (the phosphorus atom (P) of groups Z31, Z32 and Z33 being bonded to an oxygen atom of the group preceding it in the formula -O-(Z3i) q i-(Z32) q 2-Z33-H).

[0039] The present invention relates, as a fourth object, to a modified nucleic acid comprising at least one nucleotide modified according to the invention (hereinafter referred to as the modified nucleic acid). The nucleic acid will more particularly be a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA).

[0040] The present invention has as its fifth object a method for reverse transcription into DNA of an RNA modified according to the invention in which Ro is a substitution group of formula - A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined in this description, comprising contacting the modified RNA with a reverse transcriptase in the presence of a primer and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under appropriate conditions.

[0041] The present invention has as its sixth object a method for amplifying a nucleic acid of the type RNA or DNA modified according to the invention in which Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined in this description, comprising the following steps: a) when the nucleic acid to be amplified is an RNA modified according to the invention in which Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined in this description, the reverse transcription of the modified RNA into DNA by contacting it with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under appropriate conditions;b) contacting the DNA obtained in step a) or the DNA modified according to the invention in which Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined in this description, with a DNA polymerase in the presence of a sense primer capable of hybridizing to the 5' portion of one of the two DNA strands, an antisense primer capable of hybridizing to the 5' portion of the other DNA strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase when the nucleic acid to be amplified is an RNA modified according to the invention, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives; and c) amplification of the DNA by polymerase chain reaction comprising the following substeps:;

[0042] (i) a DNA denaturation step enabling the separation of the two DNA strands, during which the mixture obtained in step b) is advantageously heated to a temperature of 90°C to 100°C;

[0043] (ii) a step of pairing the sense and antisense primers to the separated DNA strands, during which the mixture obtained in substep (i) is advantageously heated to a temperature of 45°C to 72°C, advantageously from 45°C to 65°C;

[0044] (iii) an elongation step of a complementary strand of each DNA strand comprising a primer by DNA polymerase, during which the mixture obtained in substep ii) is advantageously heated to a temperature of 65°C to 80°C; (d) optionally, N iterations of step c), N being an integer greater than 1, in which for each iteration n (l <n<N-l), la sous-étape (i) de l’itération n+1 est mise en œuvre sur le mélange obtenu à la sous-étape (iii) de l’itération n au lieu du mélange obtenu à l’étape b), et e) lorsque l’acide nucléique à amplifier est un ARN, la transcription des ADN amplifiés à l’étape c) et éventuellement à l’étape d) en ARN avec une ARN polymérase en présence d’un mélange de ribonucléotides comprenant trois groupements phosphate ou dérivé de phosphate dans des conditions appropriées.

[0045] The present invention relates as a seventh object to a method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a library of natural or modified DNA according to the invention in which Ro is replaced by a group of the formula -Ao-Xn, where Ao is as defined in this description and Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR=CH2, RÔ and R7 being as defined in this description, the sequence of which comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0046] - the sequence of the region at the 5' end is known and identical for all DNA,

[0047] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0048] - the sequence of the central region is random; b) systematically when the library comprises natural DNA and optionally when the library comprises DNA modified according to the invention as defined in step a), amplification by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives of which one deoxyribonucleotide comprises a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group where Ao is as defined in this description and where Xn represents - C=CH, -N3, -C(O)Re, -O-NH2, -NH-NH2, -SH or -CR7=CH2, RÔ and R7 being as defined in this description, c) then,when Xn is -C=CH, -N3, -SH or -C(R7)=CH2, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R7)-AI-YI-RI, OR SH-A1-Y1-R1 respectively; when Xn is -CO-RÔ, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1; and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri; d) selection of modified DNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri having the function of interest; e) cleavage of the Yi group to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y2; f) optionally, N iterations of steps a) to e), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l),The library provided in step a) of iteration n+1 corresponds to the mixture of modified DNA obtained in step e) of iteration n; and g) the sequencing of the DNA obtained at the end of step d) or, where present, at the end of step f). The present invention relates as an eighth object to a method for selecting modified functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:,

[0049] - the sequence of the region at the 5' end is known and identical for all DNA,

[0050] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0051] - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives of which one ribonucleotide comprises a nucleobase of which a hydrogen atom is replaced by an -Ao-Xn group or of which a nitrogen atom is replaced by a C-Ao-Xn group where Ao is as defined in this description and where Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR?=CH2, RÔ and R7 being as defined in this description, under appropriate conditions; d) when Xn is -C=CH, -N3, -SH or -C(R?)=CH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R?)-AI-YI-Ri, or SH-A1-Y1-R1 respectively; when Xn is -CO-Re, the coupling reaction of the RNAs obtained in step c) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1;and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula Re-CO-Ai-Yi-Rl, to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1; e) the selection of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1 having the function of interest; f) the cleavage of the Y1 group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2; g) the reverse transcription of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 into DNA by contacting a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under appropriate conditions;h) Optionally, N iterations of steps a) to g), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’acides nucléiques obtenu à la sous-étape (iii) de l’étape g) de l’itération n ; et i) le séquençage des ADN obtenus à la fin de l’étape g) ou, lorsqu’elle est présente, à la fin de l’étape h).;

[0052] The present invention relates as a ninth object to a method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0053] - the sequence of the region at the 5' end is known and identical for all DNA,

[0054] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0055] - the sequence of the central region is random;b) amplification of the DNAs from the library provided in step a) by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives, one of which comprises a nucleobase in which a hydrogen atom is replaced by a Ro group or a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of the formula -Ao-Xi-Ai-Yi-Ri, where Ao, Xi, Ai, Yi, and Ri are as defined in this description, to give modified DNAs bearing a substitution group of the formula -A0-X1-A1-Y1-R1; c) selection of modified DNA bearing a substitution group of formula -A0-X1-A1-Y1-Ri having the function of interest;d) cleavage of the Yi group to give modified DNAs bearing a substitution group of the formula -A0-X1-A1-Y2; e) optionally, N iterations of steps a) to d), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à la sous-étape (iii) de l’étape d) de l’itération n ; et f) le séquençage des ADN obtenus à la fin de l’étape d) ou, lorsqu’elle est présente, à la fin de l’étape e).;

[0056] The tenth object of the present invention is a method for selecting modified functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0057] - the sequence of the region at the 5' end is known and identical for all DNA,

[0058] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0059] - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives, one of which is a ribonucleotide comprising a nucleobase in which a hydrogen atom is replaced by a Ro group or in which a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -A0-X1-A1-Y1-R1, in which Ao, Xi, Ai, Yi and Ri are as defined in this description under conditions suitable to give RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1; d) selection of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri having the function of interest; e) cleavage of the Yi group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2;f) reverse transcription of modified RNAs bearing a substitution group of the formula -A0-X1-A1-Y2 into DNA by contact with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under suitable conditions; g) optionally, N iterations of steps a) to f), N being an integer greater than or equal to 1, wherein for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à l’étape f) de l’itération n ; et h) le séquençage des ADN obtenus à la fin de l’étape f) ou, lorsqu’elle est présente, à la fin de l’étape g).;

[0060] The present invention relates as its eleventh object a method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a library of natural or modified DNA according to the invention in which Ro is replaced by a group of formula -Ao-Xn, where Ao is as defined in this description and Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR7=CH2, RÔ and R7 being as defined in this description, the sequence of which comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0061] - the sequence of the region at the 5' end is known and identical for all DNA,

[0062] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0063] - the sequence of the central region is random; b) systematically when the library comprises natural DNA and optionally when the library comprises DNA modified according to the invention as defined in step a), amplification by polymerase chain reaction of the DNAs of the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives of which one deoxyribonucleotide comprises a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group where Ao is as defined in this description and where Xn represents - C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR7=CH2, RÔ and R7 being as defined in this description, c) then,when Xn is -C=CH, -N3, -SH or -C(R7)=CH2, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R7)-AI-YI-RI, OR SH-A1-Y1-R1 respectively; when Xn is -CO-RÔ, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1; and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri; e) cleavage of the Yi group to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y2; d) selection of modified DNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; f) optionally, N iterations of steps a) to e), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l),the library provided in step a) of iteration n+1 corresponds to the mixture of modified DNA obtained in step e) of iteration n; and g) the sequencing of the DNA obtained at the end of step d) or, where present, at the end of step f).,

[0064] The present invention relates, as its twelfth object, to a method for selecting functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0065] - the sequence of the region at the 5' end is known and identical for all DNA,

[0066] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0067] - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives of which one ribonucleotide comprises a nucleobase of which a hydrogen atom is replaced by an -Ao-Xn group or of which a nitrogen atom is replaced by a C-Ao-X11 group where Ao is as defined in this description and where Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR?=CH2, RÔ and R7 being as defined in this description, under appropriate conditions; d) when Xn is -C=CH, -N3, -SH or -C(R?)=CH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R?)-AI-YI-Ri, or SH-A1-Y1-R1 respectively; when Xn is -CO-Re, the coupling reaction of the RNAs obtained in step c) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1;and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1; e) the cleavage of the Yi group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2; f) the selection of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; g) the reverse transcription of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 into DNA by contacting a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivative under appropriate conditions;h) Optionally, N iterations of steps a) to g), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’acides nucléiques obtenu à la sous-étape (iii) de l’étape g) de l’itération n ; et g) le séquençage des ADN obtenus à la fin de l’étape g) ou, lorsqu’elle est présente, à la fin de l’étape h).;

[0068] The present invention relates as its thirteenth object a method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0069] - the sequence of the region at the 5' end is known and identical for all DNA,

[0070] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0071] - the sequence of the central region is random;b) amplification of the DNAs from the library provided in step a) by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives, one of which comprises a nucleobase in which a hydrogen atom is replaced by a Ro group or a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -A0-X1-Ai-Yi-Ri, in which Ao, Xi, Ai, Yi, and Ri are as defined in this description, to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1, d) the cleavage of the Yi group to give modified DNAs carrying a substitution group of the formula -A0-X1-A1-Y2;c) the selection of modified DNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; e) optionally, N iterations of steps a) to d), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à la sous-étape (iii) de l’étape d) de l’itération n ; et f) le séquençage des ADN obtenus à la fin de l’étape d) ou, lorsqu’elle est présente, à la fin de l’étape e).;

[0072] The present invention relates, as its fourteenth object, to a method for selecting functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0073] - the sequence of the region at the 5' end is known and identical for all DNA,

[0074] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0075] - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives, one of which is a ribonucleotide comprising a nucleobase in which a hydrogen atom is replaced by a Ro group or in which a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -A0-X1-A1-Y1-R1, in which Ao, Xi, Ai, Yi and Ri are as defined in this description, under conditions suitable to give RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1; e) the cleavage of the Yi group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2; d) the selection of modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest;f) reverse transcription of modified RNA bearing a substitution group of the formula -A0-X1-A1-Y2 into DNA by contact with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under suitable conditions; g) optionally, N iterations of steps a) to f), N being an integer greater than or equal to 1, wherein for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à l’étape f) de l’itération n ; et h) le séquençage des ADN obtenus à la fin de l’étape f) ou, lorsqu’elle est présente, à la fin de l’étape g).;

[0076] DETAILED DESCRIPTION OF THE INVENTION

[0077] The present invention is described in detail below.

[0078] Definitions

[0079] For the purposes of this invention, "nucleobase", "nitrogenous base", "nucleic base" or "nucleotide base" means a heterocyclic organic compound containing one or more nitrogen atoms and having basic properties, advantageously selected from the nucleobases of the pyrimidine family (including in particular cytosine, thymine and uracil) and of the purine family (including in particular adenine and guanine).

[0080] A nucleobase can be, in particular, a naturally occurring nucleobase present in natural nucleic acids, that is, adenine, guanine, thymine, uracil, or cytosine, preferably adenine, guanine, uracil, or cytosine. The naturally occurring nucleobases of nucleic acids (adenine, guanine, thymine, uracil, and cytosine) correspond to the formulas shown in Table 1.

[0081] [Table 1]

[0082]

[0083] For the purposes of this invention, "nucleoside" means a glycosylamine consisting of a nucleobase linked to the anomeric carbon atom of a pentose residue by a glycosidic bond from the nitrogen atom NI of a pyrimidine or the N9 atom of a purine.

[0084] In natural nucleosides, the pentose residue is generally a residue of ribose (then called a "ribonucleoside") or of deoxyribose (then called a "deoxyribonucleoside").

[0085] The natural nucleoside is then: in which Nu represents a nucleobase as defined above linked via an NH group.

[0086] A natural nucleoside can therefore be an adenosine, a deoxyadenosine, a guanosine, a deoxyguanosine, a ribothymidine, a thymidine, a uridine, a deoxyuridine, a cytidine or a deoxy cytidine.

[0087] However, modified pentose residues may also be present in a nucleoside as defined in the present invention. In particular, a nucleoside according to the invention may comprise a ribose derivative in which the OH group at the 2' position is replaced by another group, advantageously by a group selected from methoxy (or -OMe of formula -OCH3), methoxymethoxy (or -OMOE of formula -O-CH2-OCH3), methoxyethoxy (or -OMOE of formula -O-(CH2)2-OCH3), fluoro (F), amino (NH2), and azido (N3). Such a nucleoside is also referred to as a "ribonucleoside," a term encompassing both nucleosides comprising a ribose residue and those comprising a ribose derivative residue with a substitution of the OH group at the 2' position by another group (in particular those described above).

[0088] For the purposes of this invention, "nucleotide" means a nucleoside as defined above in which the OH group at the 5' position is replaced by one to three phosphate or phosphate-derived groups. In particular, a nucleotide as defined in this invention may be a nucleoside as defined above in which the OH group at the 5' position is replaced by an -O-(Z3i) group. q i-(Z32)q2-Z33-H or a salt thereof for which:

[0089] - q1 and q2 independently represent 0 or 1, and

[0090] - Z31, Z32 and Z33 represent, independently of each other, -P(O)(OH)-O- (phosphate group allowing the generation of nucleic acids with phosphodiester type bonds), -P(O)(SH)-O- (group allowing the generation of nucleic acids with phosphorothiolate type bonds), -P(O)(CH3)-O- (group allowing the generation of nucleic acids with phosphonate type bonds), -P(S)(OH)-O- (group allowing the generation of nucleic acids with phosphorothioate type bonds), -P(S)(SH)-O- (group allowing the generation of nucleic acids with phosphorodithioate type bonds), or -P(S)(CH3)-O- (group allowing the generation of nucleic acids with methyl phosphorothioate type bonds).

[0091] When a nucleotide contains a deoxyribonucleoside, it is called a "deoxyribonucleotide." When a nucleotide contains a ribonucleoside (containing a ribose residue or a ribose derivative whose OH group at the 2' position is replaced by another group (including those described above)), it is called a "ribonucleotide." When a nucleotide contains only one phosphate or derivative group (q1=q2=0), it is called a mono-(phosphate or derivative) nucleotide (abbreviated here as "MPN"). When a nucleotide contains two phosphate or derivative groups (q1 or q2=1, the other is zero), it is called a di-(phosphate or derivative) nucleotide (abbreviated here as "NDP"). When the nucleotide includes three phosphate or derivative groups (ql=2=l), it is called a triphosphate or derivative nucleotide (abbreviated here as "NTP").For amplification by polymerase chain reaction, tri-(phosphate or derivative) deoxynucleotides (abbreviated here as "dNTP") are used, which are added by extension of a primer by DNA polymerase.

[0092] For the purposes of this invention, "nucleic acid" or "polynucleotide" means a polymer of any size whose basic unit or monomer is a nucleotide, the nucleotides being covalently linked to one another. The type of covalent bond between two consecutive nucleotides of a nucleic acid depends on the type of phosphate or derivative group attached to the 3' pentose residue of the nucleotide. When it is a phosphate group, the bond is a phosphodiester bond. When it is a phosphate derivative group, it may be a phosphorothiolate, phosphonate, phosphorothioate, phosphorodithioate, or methyl phosphorothioate bond. When the pentose residue is a ribose or a ribose derivative (including those described herein), the nucleic acid is referred to in the context of the invention as a ribonucleic acid (RNA), regardless of the type of bond between the different ribonucleotides.When the pentose residue is a deoxyribose, the nucleic acid is a deoxyribonucleic acid (DNA), regardless of the type of bond between the different deoxyribonucleotides.

[0093] By "oligonucleotide" we mean a short polynucleotide, usually single-stranded, usually synthetic, whose length generally does not exceed 310 nucleotides.

[0094] Nucleic acids are divided into "coding nucleic acids," whose function is to code for a peptide or protein of interest, and "non-coding nucleic acids," whose function is not to code for a peptide or protein of interest. Examples of "non-coding nucleic acids" include ribosomal RNA (rRNA) and transfer RNA (tRNA), which are not coding but participate in the coding process of peptides and proteins, and "functional nucleic acids," whose function does not contribute to the coding process of peptides and proteins.Among the functional nucleic acids, we can notably mention microRNAs (or "miRNAs", involved in the post-transcriptional regulation of mRNAs), small interfering RNAs (or "siRNAs", produced after exposure to a foreign RNA and 100% complementary to the target mRNAs), "specific ligand nucleic acids" (capable of binding specifically to a ligand of interest) such as aptamers and "catalytic nucleic acids" (which have a catalytic function) such as ribozymes and deoxyribozymes.

[0095] An "aptamer" is a single-stranded nucleic acid, which can be DNA or RNA as defined here, with a three-dimensional (3D) structure that allows it to interact specifically, and most often with high affinity, with a target molecule. Aptamers are generally composed of 60 to 150 nucleotides and include a central region of variable size (usually between 20 and 90 nucleotides) and variable sequence flanked by two constant sequences of approximately 14 to 50 nucleotides each. These constant sequences are necessary in the process called "SELEX" (an acronym for "systematic evolution of ligands by exponential enrichment") or "directed molecular evolution," used to select aptamers that bind specifically to a target molecule of interest.

[0096] By “ribozyme” we mean an RNA as defined here, which has the enzymatic property of catalyzing one or more specific chemical reactions.

[0097] By "deoxyribozyme" we mean a single-stranded DNA as defined here, which has the property of catalyzing one or more specific biochemical reactions.

[0098] Ribozymes and deoxyribozymes are typically composed of 90 to 310 nucleotides and include a central region of variable size (usually between 30 and 250 nucleotides) and variable sequence flanked by two constant sequences of about 14 to 50 nucleotides each, which are required in the "directed molecular evolution" process used to select ribozymes or deoxyribozymes with catalytic activity of interest.

[0099] The catalytic properties of ribozymes and deoxyribozymes are linked to the ability of RNA and DNA to fold into a well-defined, compact 3D structure, which, as in the case of proteins, allows the formation of cavities that create ligand-binding sites. Precisely oriented reactive groups then carry out the actual catalysis. The "SELEX" process (an acronym for "systematic evolution of ligands by exponential enrichment") or "directed molecular evolution" refers to a process that relies on the use of a library of candidate functional nucleic acids (particularly aptamers, ribozymes, or deoxyribozymes) (generally 10 13 at 10 15different sequences) and successive cycles of enrichment by amplification and selection. The candidate functional nucleic acids from the initial library are generally composed of 60 to 310 nucleotides and include a central region of variable size (generally between 20 and 200 nucleotides) and variable and random sequence framed by two constant sequences of about 14 to 50 nucleotides each, indispensable to the different amplification steps of each enrichment cycle.

[0100] By "DNA polymerase" we mean an enzyme that catalyzes the synthesis of new DNA strands from an existing template strand in the presence of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives during DNA replication.

[0101] By "RNA polymerase" we mean an enzyme that catalyzes the synthesis of RNA from a template DNA strand in the presence of ribonucleotides comprising three phosphate groups or phosphate derivatives during transcription.

[0102] By “reverse transcriptase” or “reverse transcriptase”, we mean an enzyme capable, under appropriate conditions, of synthesizing a strand of DNA from a template RNA strand in the presence of a primer and dNTPs.

[0103] A "primer" is a single-stranded oligonucleotide or fragment of DNA or RNA that hybridizes to a template nucleic acid strand such that the 3' end of the primer can serve as a polymerization and extension site using a DNA or RNA polymerase under appropriate conditions (buffer / pH, temperature, presence of NTPs). The primer can consist of any combination of nucleotides and can be of any suitable length, for example, approximately 10 to 60 nucleotides, 12 to 50 nucleotides, 15 to 30 nucleotides, and 15 to 40 nucleotides. Preferably, the primer length is between 15 and 30 nucleotides.

[0104] For the purposes of this invention, "Ci-6-alkyl" means a monovalent saturated hydrocarbon chain, linear or branched, comprising 1 to 6, preferably 1 to 4, carbon atoms. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl groups. For the purposes of this invention, "Ci-x-alkanediyl" means a divalent saturated hydrocarbon chain, linear or branched, preferably linear, comprising 1 to x carbon atoms. Preferably, it is a -(CH2) chain. n - with n = 1 to x.

[0105] For the purposes of this invention, "aryl" means a monovalent aromatic hydrocarbon group, preferably comprising 6 to 10 carbon atoms, and including one or more fused rings, such as a phenyl or naphthyl group. Advantageously, this refers to phenyl.

[0106] For the purposes of this invention, "arylene" means a divalent aromatic hydrocarbon group, preferably comprising 6 to 10 carbon atoms, and including one or more fused rings, such as a phenylene or naphthylene group. Advantageously, this refers to phenylene.

[0107] For the purposes of this invention, "Cs-n-carbocyclyl" means a monocyclic or polycyclic hydrocarbon system, saturated or unsaturated but non-aromatic, preferably saturated, and monovalent, comprising from 3 to 12 carbon atoms. The polycyclic system comprises at least 2, in particular 2 or 3, fused or bridged rings. Each ring of the monocyclic or polycyclic system advantageously comprises 3 to 8, in particular 4 to 7, especially 5 or 6, carbon atoms. Examples include an adamantyl, cyclohexyl, cyclopentyl, cyclopropyl, or cyclohexenyl group. Preferably, it is a C3-8-cycloalkyl group.

[0108] For the purposes of this invention, "C3-12-divalent carbocycle" means a monocyclic or polycyclic hydrocarbon system, saturated or unsaturated but non-aromatic, preferably saturated, and divalent, comprising from 3 to 12 carbon atoms. The polycyclic system comprises at least 2, in particular 2 or 3, fused or bridged rings. Each ring of the monocyclic or polycyclic system advantageously comprises 3 to 8, in particular 4 to 7, especially 5 or 6, carbon atoms. Examples include a divalent adamantane group, divalent cyclohexane, divalent cyclopentane, divalent cyclopropane, and divalent cyclohexylene. Preferably, it is a C3-8-cycloalkanediyl group.

[0109] For the purposes of this invention, "C3-8-cycloalkyl" means a saturated, monovalent, monocyclic hydrocarbon system comprising 3 to 8, in particular 4 to 7, especially 5 or 6, carbon atoms. It may, in particular, be a Cs-6-cycloalkanediyl group comprising 5 or 6 carbon atoms. Examples include a cyclohexyl, cyclopentyl, or cyclopropyl group.

[0110] For the purposes of this invention, "C3-8-cycloalkanediyl" means a saturated, divalent, monocyclic hydrocarbon system comprising 3 to 8, in particular 4 to 7, especially 5 or 6, carbon atoms. Examples include cyclohexanediyl, cyclopentanediyl, and cyclopropanediyl groups.

[0111] For the purposes of this invention, "heteroaryl" means an aromatic group comprising one or more, in particular 1, 2, or 3, fused hydrocarbon rings, wherein one or more carbon atoms, advantageously 1, 2, 3, or 4, are each replaced by a heteroatom, such as, for example, a sulfur, nitrogen, or oxygen atom. Preferably, it is a heteroaryl comprising 1, 2, or 3 fused rings, of which 1, 2, or 3 carbon atoms have been replaced by a heteroatom selected from oxygen and nitrogen, preferably nitrogen. Preferably, each ring of the heteroaryl comprises 5 or 6 members. Preferably, the heteroaryl comprises 5 to 15 intracyclic atoms (i.e., excluding hydrogen atoms).Examples of heteroaryl groups are furyl, thienyl, pyrrolyl, pyridyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolyl, quinoxalyl, indolyl, carbazolyl, etc.

[0112] For the purposes of this invention, "heterocycle" means a saturated or unsaturated but non-aromatic hydrocarbon cyclic system comprising one or more, in particular 1, 2, 3 or 4, hydrocarbon rings joined and / or bridged and / or linked together by a spiro bond, in which one or more carbon atoms, advantageously 1, 2, 3 or 4 and even more advantageously 1 or 2, are each replaced by a heteroatom such as, for example, a sulfur, nitrogen or oxygen atom. Preferably, it is a saturated or unsaturated but non-aromatic hydrocarbon cyclic system comprising 1, 2, or 3 bonded and / or bridged hydrocarbon rings, in which 1, 2, or 3 carbon atoms are each replaced by a heteroatom selected from oxygen and nitrogen, particularly nitrogen. Preferably, each ring of the heterocycle comprises 3, 4, 5, 6, or 7 members, particularly 5 or 6 members. Preferably, the heteroaryl comprises 5 to 15 intracyclic atoms (i.e., excluding hydrogen atoms).This may include, in particular, a pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl group, etc.

[0113] For the purposes of this invention, a "metal complex" is defined as a metal cation surrounded by, or complexed with, one or more anionic or neutral ligands. Examples include iron-porphyrin, copper-porphyrin, or ruthenium-bipyridine (bpy) complexes.

[0114] For the purposes of this invention, "metal complex derivative" means a metal complex as defined above linked to the rest of the molecule by one of its ligands. For the purposes of this invention, "amino acid" means all residues of naturally occurring amino acids (e.g., Alanine (Ala), Arginine (Arg), Asparagine (Asn), Aspartic Acid (Asp), Cysteine ​​(Cys), Glutamine (Gin), Glutamic Acid (Glu), Glycine (Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr), and Valine (Val)) in the D or L form, as well as non-natural amino acids (e.g., P-alanine, I-allylglycine, tert-leucine, 3-aminoadipic acid, 2-aminobenzoic acid, 3- aminobenzoic acid, 4-aminobenzoic acid, 2-aminobutanoic acid, 4-amino-l-carboxymethyl piperidine, 1-amino-1-cyclobutanecarboxylic acid,4-Aminocyclohexaneacetic acid, 1-Amino-1-cyclohexanecarboxylic acid, (17,2A)-2-Aminocyclohexanecarboxylic acid, (1A,25)-2-Aminocyclohexanecarboxylic acid, (15,2A)-2-Aminocyclohexanecarboxylic acid, (15,2S)-2-Aminocyclohexanecarboxylic acid, 3-Aminocyclohexanecarboxylic acid, 4-Aminocyclohexanecarboxylic acid, (1A,2R)-2-Aminocyclopentanecarboxylic acid, (1R,2S)-2-Aminocyclopentanecarboxylic acid, 1-Amino-1-cyclopropanecarboxylic acid, 4-(2-Aminoethoxy)benzoic acid 3-aminomethylbenzoic acid, 4-aminomethylbenzoic acid, 2-aminobutanoic acid, 4-aminobutanoic acid, 6-aminohexanoic acid, 1-aminoindane-1-carboxylic acid, 4-aminomethylphenylacetic acid, 4-aminophenylacetic acid, 3-amino-2-naphthoic acid, 4-aminophenylbutanoic acid, 4-amino-5-(3-indolyl)pentanoic acid, (4A,55)-4-amino-5-methylheptanoic acid,(R)-4-amino-5-methylhexanoic acid, (R)-4-amino-6-methylthiohexanoic acid, (5)-4-aminopentanoic acid, (R)-4-amino-5-phenylpentanoic acid, 4-aminophenylpropionic acid, (R)-4-aminopimeric acid, (4A,5A)-4-amino-5-hydroxyhexanoic acid, (R)-4-amino-5-hydroxypentanoic acid, (R)-4-amino-5-( / ?-hydroxyphenyl)pentanoic acid, 8-aminooctanoic acid, (25,4A)-4-aminopyrrolidine-2-carboxylic acid, (2S,4S)-4-aminopyrrolidine-2-carboxylic acid, azetidine-2-carboxylic acid (25,4A)-4-benzyl-pyrrolidine-2-carboxylic acid, (S)-4,8-diaminooctanoic acid, tert-butylglycine, γ-carboxyglutamate, P-cyclohexylalanine, citrulline, 2,3-diaminopropionic acid, hippuric acid, homocyclohexylalanine, moleucine, homophenylalanine, 4-hydroxyproline, indoline-2-carboxylic acid, isonipecotic acid, α-methylalanine, nicopetic acid, norleucine, norvaline,octahydroindole-2-carboxylic acid, ornithine, penicillamine, phenylglycine, 4-phenylpyrrolidine-2-carboxylic acid, pipecolic acid, propargylglycine, 3-pyridinylalanine, 4-pyridinylalanine, 1-pyrrolidine-3-carboxylic acid, sarcosine, statins, tetrahydroisoquinoline-1-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, tranexamic acid).

[0115] For the purposes of this invention, "amino acid derivative" means an amino acid as defined above linked to the rest of the molecule by its COOH, NH2 function or possibly by another functional group present on the amino acid (e.g. SH, NH, OH function, etc.).

[0116] For the purposes of this invention, "cyanine" means a molecule comprising two nitrogen atoms separated by a polymethine chain, one of the nitrogen atoms being in an iminium form. The polymethine chain is a conjugated unsaturated hydrocarbon chain that may advantageously comprise 3, 5, or 7 carbon atoms. Examples include Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, etc. cyanine.

[0117] For the purposes of this invention, "cyanine derivative" means a cyanine as defined above linked to the rest of the molecule by one of its atoms.

[0118] For the purposes of this invention, "rhodamine" means a molecule comprising a fluorone or imino-3H-xanthene motif. Examples include tetramethylrhodamine, rhodamine 6G, rhodamine B, rhodamine 123, etc.

[0119] For the purposes of this invention, "rhodamine derivative" means a rhodamine as defined above linked to the rest of the molecule by one of its atoms.

[0120] For the purposes of this invention, "BODIPY" means a molecule comprising a boron-dipyrromethene motif, and more particularly a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene motif. Examples include BODIPY, 3,5-dimethylBODIPY, 3,5-dimethyl-8-phenylBODIPY, etc.

[0121] For the purposes of this invention, "BODIPY derivative" means a BODIPY as defined above linked to the rest of the molecule by one of its atoms.

[0122] For the purposes of this invention, "fluorophore group" means a chemical group capable of emitting fluorescence light after excitation.

[0123] For the purposes of this invention, "photo-bridging group" means a group capable of covalently binding to the target under appropriate irradiation, such as a benzophenone linked to the rest of the molecule by a carbon atom.

[0124] The "salts" according to the present invention include, in particular:

[0125] (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as formic acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and the like, and

[0126] (2) base addition salts formed when an acid proton present in the parent compound is either replaced by a metal ion, for example an alkali metal ion (e.g. Na, K), an alkaline earth metal ion (e.g. Ca), a zinc ion, a silver ion or an aluminium ion; or coordinated with an organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like; or with an inorganic base such as aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide and the like.

[0127] modified nucleobase

[0128] The modified nucleobase according to the invention is a nucleobase (hereinafter referred to as the unmodified nucleobase) in which a hydrogen atom is replaced by Ro or a nitrogen atom is replaced by CRo, where Ro is a substitution group of formula -A0-X1-A1-Y1-R1 or -A0-X1-A1-Y2, in particular a substitution group of formula -A0-X1-A1-Y2 in which Y2 preferably represents

[0129] The unmodified nucleobase can therefore be:

[0130] (a) a pyrimidine, advantageously a uracil or a cytosine; or

[0131] (b) a purine, advantageously an adenine or a guanine.

[0132] Thus, a modified nucleobase according to the invention is advantageously derived:

[0133] (a) of a pyrimidine, advantageously of a uracil or a cytosine; or

[0134] (b) of a purine, advantageously of an adenine or a guanine.

[0135] When the unmodified nucleobase is a pyrimidine, the modified nucleobase will most likely be a uracil or cytosine whose hydrogen atom at position 5 is replaced by the Ro substitution group. When the unmodified nucleobase is a purine, the modified nucleobase will most likely be an adenine or a guanine, whose nitrogen atom at position 7 of the purine ring is replaced by CRo.

[0136] Thus, a modified nucleobase according to the invention is advantageously a modified nucleobase in which:

[0137] (a) in the case of a nucleobase derived from a pyrimidine, the nucleobase is modified by replacing the hydrogen atom attached to the carbon in position 5 of the pyrimidine ring with Ro; or

[0138] (b) in the case of a nucleobase derived from a purine, the nucleobase is modified by replacing the nitrogen atom located at position 7 of the purine ring with CRo.

[0139] Ao represents a single bond or a Ci-io-alkanediyl chain possibly preceded and / or interrupted and / or followed and / or replaced by one or more motifs chosen from the group consisting of -C=C-, -C(R2)=C(R3)-, -O-, -S-, -NR4-, -C(O)-, -C(S)-, and -C(NR5)-.

[0140] Advantageously, Ao represents a single bond or a Ci-io-alkanediyl chain possibly preceded and / or interrupted by one or more motifs selected from the group consisting of -C=C- and -O-, preferably -C=C-. Advantageously, Ao represents a single bond; a Ci-io-alkanediyl chain possibly preceded and / or interrupted by one or more -C=C- motifs; or a -(O) chain. n i(CH2CH2O) n 2(CH2CH2) n3- with ni = 0 or 1, n3 = 0 or 1 and n2 representing an integer from 1 to 5 with the condition that n2+n3 does not exceed 5. Preferably, Ao represents a single bond or a Ci-io-alkanediyl chain possibly preceded and / or interrupted by one or more -C=C- motifs, such as -C=C- (CH2)4-, the last atom being bonded to Xi.

[0141] According to one embodiment, Ao is chosen from the group consisting of -C=C-, and -C=C-(CH2)4- C=C-, , in which the wavy line indicates the point of connection with Ao and the dotted line indicates the point of connection with Ai.

[0142] Preferably, Xi represents a group chosen from the group consisting of

[0143] Ai represents a Ci-20-alkanediyl chain, in particular Ci-10-alkanediyl, possibly preceded and / or interrupted and / or followed and / or replaced by one or more motifs chosen from the group consisting of -C=C-, -C(Rs)=C(R9)-, -O-, -S-, -NR10-, -C(O)-, -C(S)-, and -C(NRn)-.

[0144] Preferably, Ai represents -(CH2) m o- with mO representing an integer from 1 to 20, in particular from 1 to 10; or a string -(O) m i(CH2CH2O) m 2(CH2CH2)m3- with ml = 0 or 1, m3 = 0 or 1 and m2 representing an integer from 1 to 10 with the condition that m2+m3 does not exceed 10. Preferably, Ai represents -(CH2) m o- with mO representing an integer from 1 to 20, in particular from 1 to 10, such as 3.

[0145] Yi represents a group , in which X2 represents -NH-CO-

[0146] , -O-CO-, -SO2-, or -P(O)(Ci-6-alkyl)-, the -NH-CO- and -O-CO- groups being linked to Ai by the NH or O motif respectively and to the rest of the Yi group by the CO motif, R31 represents H or a halogen (I, Br, Cl or F) and preferably H, the dotted line indicates the bonding point with Ai and the wavy line indicates the bonding point with Ri; and

[0147] Y2 represents 2, in which the dotted line indicates the point of bonding with Ai; for which X2 represents -NH-CO-, -O-CO-, -SO2-, or -P(O)(Ci-6-alkyl)-, the -NH-CO- and -O-CO- groups being bonded to Ai by the NH or O motif respectively and to the rest of the Yi group by the CO motif.

[0148] Preferably, X2 represents -NH-CO- or -O-CO-, preferably -NH-CO-,

[0149] Preferably, Yi represents preferably, Yi represents

[0150] According to a particular embodiment:

[0151] - Ao represents a single bond or a Ci-io-alkanediyl chain possibly preceded and / or interrupted by one or more motifs chosen from the group consisting of -C=C- and -O; preferably, a single bond; a Ci-io-alkanediyl chain possibly preceded and / or interrupted by one or more -C=C- motifs; or a -(O) chain n i(CH2CH2O) n 2(CH2CH2) n 3- with ni = 0 or 1, n3 = 0 or 1 and n2 representing an integer from 1 to 5 with the condition that n2+n3 does not exceed ; preferably, a single bond or a Ci-io-alkanediyl chain possibly preceded and / or interrupted by one or more -C=C- motifs, such as -C=C- (CH2)4-, the last atom being bonded to Xi;

[0152] - Ai represents -(CH2) m o- with mO representing an integer from 1 to 20, in particular from 1 to 10; or a string -(O) m i(CH2CH2O) m2(CH2CH2)m3- with ml = 0 or 1, m3 = 0 or 1 and m2 representing an integer from 1 to 10 with the condition that m2+m3 does not exceed 10; preferably -(CH2) m o- with mO representing an integer from 1 to 20, in particular from 1 to 10, such as 3; - Yi represents

[0153] - Y2 represents

[0154] Ri represents a group of formula A2-R30, in which:

[0155] - A2 is a single bond or a Ci-20-alkanediyl chain possibly preceded and / or interrupted and / or followed and / or replaced by one or more motifs selected from the group consisting of an arylene, a divalent Cs-n-carbocycle, -OC-, -C(Ri2)=C(Ri3)-, -O-, -S-, -NR14-, -C(O)-, -C(S)-, and -C(NRI5)-, and

[0156] - R30 is a Ci-20-alkanediyl, aryl, Cs-n-carbocyclyl, heteroaryl, or heterocyclic group, said group being optionally substituted by one or more groups selected from OR16, SR17, SOR18, SO2R19, OSO2R20, SO3R21, NR22R23, CO2R24, CONR25R26, NH-(C=NH)-NH2, N + R27R28R29G , NH-(C=NH2 + G2)-NH2, CO2G3 + , SO3G4 + , a metal complex derivative, an amino acid derivative, a cyanine derivative, a rhodamine derivative, a BODIPY derivative, a fluorophore group, and a photobridging group, or R30 is a group selected from OR10, SR17, SOR18, SO2R19, OSO2R20, SO3R21, NR22R23, CO2R24, CONR25R26, NH-(C=NH)-NH2, N + R27R28R29G , NH-(C=NH2 + G2)-NH2, CO2G3 + , SO3G4 + , a metal complex derivative, an amino acid derivative, a cyanine derivative, a rhodamine derivative, a BODIPY derivative, a fluorophore group, and a photobridging group;

[0157] - R2 to R29 each represent, independently of each other, H or Ci-6-alkyl;

[0158] - Gf and G2 represent, independently of each other, a negatively charged counterion; and

[0159] - G3 and G4 + independently represent a positively charged counterion. Advantageously, A2 is a single bond or a Ci-20-alkanediyl chain optionally preceded and / or interrupted and / or followed and / or replaced by one or more motifs selected from the group consisting of an arylene, a divalent C3-i2-carbocycle, -OC-, or -O-. Preferably, A2 is a single bond; a Ci-20-alkanediyl chain, in particular Ci-6-alkanediyl, optionally preceded and / or interrupted and / or followed and / or replaced by one or more motifs selected from the group consisting of an arylene, a Cs-6-cycloalkanediyl, or -OC-; or a -(O) P I(CH2CH2O) P 2(CH2CH2) P3- with p1 = 0 or 1, p3 = 0 or 1, and p2 representing an integer from 1 to 10, with the condition that p2 + p3 does not exceed 10. Advantageously, A2 represents a single bond; or a Ci-20-alkanediyl chain, in particular Ci-6-alkanediyl, optionally preceded and / or interrupted and / or followed and / or replaced by one or more motifs selected from the group consisting of an arylene, a Cs-6-cycloalkanediyl, or -OC-, such as a phenylene (-C0U-) OR a bis-phenylene (-C0U-C0U-). Preferably, the C1-20-alkanediyl chain, in particular Ci-6-alkanediyl, is at least preceded by an arylene group such as phenylene (-C0EU-) OR bis-phenylene (-C0EU-C0EU-).

[0160] Advantageously, R30 is a Ci-20-alkanediyl, aryl, C3-12-carbocyclyl, heteroaryl, or heterocyclic group, said group being optionally substituted by one or more groups selected from OR10, SR17, SOR18, SO2R19, OSO2R20, SO3R21, NR22R23, CO2R24, CONR25R26, NH-(C=NH)-NH2, N +R27R28R29G , NH-(C=NH2 + G2)-NH2, CO2G3 + , and SO3G4 + In particular, R30 is a Ci-20-alkanediyl, aryl, C3-12-carbocyclyl, heteroaryl, or heterocyclic group, said group being optionally substituted by one or more groups selected from ORIÔ, NR22R23, CO2R24, CONR25R26, NH-(C=NH)-NH2, N R27R28R29G1', NH- (C=NH2 + G2)-NH2, CO2 G3 + , and SO3 "G4. In particular, R30 is a Ci-20-alkanediyl, aryl, C3-i2-carbocyclyl, heteroaryl, or heterocyclic group. In particular, R30 is a heteroaryl or heterocyclic group, specifically heteroaryl.

[0161] R30 could be, for example dotted line indicating the attachment point to A2.

[0162] According to a preferred embodiment:

[0163] - Ao represents a single bond or a Ci-10-alkanediyl chain possibly preceded and / or interrupted by one or more motifs chosen from the group consisting of -OC- and -O; preferably, a single bond; a Ci-10-alkanediyl chain possibly preceded and / or interrupted by one or more -OC- motifs; or a -(O) chain n i(CH2CH2O) n 2(CH2CH2) n 3- with ni = 0 or 1, n3 = 0 or 1 and n2 representing an integer from 1 to 5 with the condition that n2+n3 does not exceed ; preferably, a single bond or a Ci-io-alkanediyl chain possibly preceded and / or interrupted by one or more -C=C- motifs;

[0164] - Ai represents -(CH2) m o- with mO representing an integer from 1 to 20, in particular from 1 to 10; or a string -(O) m i(CH2CH2O) m2(CH2CH2)m3- with ml = 0 or 1, m3 = 0 or 1 and m2 representing an integer from 1 to 10 with the condition that m2+m3 does not exceed 10; preferably -(CH2) m o- with mO representing an integer from 1 to 20, in particular from 1 to 10;

[0165] - Yi represents

[0166] - Y2 represents

[0167] - R30 is a Ci-20-alkanediyl, aryl, Cs-n-carbocyclyl, heteroaryl, or heterocyclic group; in particular heteroaryl or heterocyclic, especially heteroaryl; for example the dotted line designates the point of attachment to A2.

[0168] A modified nucleobase according to the present invention can be prepared by:

[0169] (1) Coupling reaction between: a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group, where Ao is as defined above and Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR=CH2, and a molecule of formula X12-A1-Y1-R1, where Ai, Yi and Ri are as defined above and X represents:

[0170] -N3 when Xn represents -C=CH,

[0171] -C=CH when Xn represents -N3,

[0172] -O-NH2 or -NH-NH2 when Xn represents -C(O)RÔ,

[0173] -C(O)RÔ when Xn represents -O-NH2 or -NH-NH2,

[0174] -CR?=CH2 when Xn represents -SH, or

[0175] -SH when Xn represents -CR?=CH2, to give a modified nucleobase according to the invention in which a hydrogen atom is replaced by Ro' or a nitrogen atom is replaced by CRo' where Ro' is a substitution group of formula -A0-X1-A1-Y1-R1,

[0176] (2) then, if necessary, cleavage of the Yi group of the modified nucleobase obtained in step (1) to give a modified nucleobase according to the invention in which a hydrogen atom is replaced by Ro” or a nitrogen atom is replaced by CRo” where Ro” is a substitution group of formula -A0-X1-A1-Y2.

[0177] Step (1):

[0178] Coupling in step (1) between a molecule carrying a group Xn and a molecule carrying a group Xn allows the formation of a group Xi according to Table 2 below.

[0179] [Table 2]

[0180]

[0181] The type A coupling reaction is a Huisgen azide-alkyne cycloaddition (1,3-dipolar cycloaddition), also known as copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). This reaction is typically catalyzed by a copper(I) catalyst such as CuBr or Cul. The copper(I) catalyst can also be formed in situ by the reduction of a copper(II)-containing substance, and in particular by the reduction of a copper(II) salt such as CuSCU in the presence of a reducing agent such as ascorbic acid or a salt thereof. The reaction can also be carried out in the presence of a stabilizing ligand such as tris(3-hydroxypropyltriazolylmethyl)amine (THPTA). This reaction can be carried out in various solvents such as alcohols (e.g., / c / 7-butanol), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone, water, or a mixture thereof.

[0182] Type B coupling reactions involve oxime ligation, while type C coupling reactions involve hydrazone ligation. These reactions can be carried out at room temperature in neutral or slightly acidic media. They can also be performed in an organic solvent or in aqueous solution.

[0183] The D-type coupling reaction is a thiol-ene reaction forming a thioether adduct. This reaction can be carried out between a thiol and an alkene in two different ways. The first method involves placing the two reactants in the presence of a base, preferably in organic solvents such as dichloromethane (DCM) or tetrahydrofuran (THF), or in water, and proceeds via a Michael addition when the alkene used is electron-deficient. The second method involves placing the two reactants in the presence of a chemical initiator (such as azobisisobutyronitrile (AIBN)) or a photoinitiator (such as 2,2-dimethoxy-2-phenylacetophenone (DMP A)) which will generate a radical that allows the radical addition reaction to the alkene. These reactions can occur at room temperature, for example in mixtures of organoaqueous solvents (DMSO / H2O for example) or in totally aqueous media.

[0184] A nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group may be commercially available. Examples include:

[0185] The molecule with the formula X12-A1-Y1-R1 can be prepared by coupling reactions well known to those skilled in the art. For example, the Yi group with R31 = H can be formed by reacting a molecule bearing an aminoacetonitrile group with a nitrite such as α-butyl nitrite. Coupling reactions then allow substitution of each end to introduce the terminal groups X12 and Ri. The Yi group with R31 = halogen can be obtained in the same way with an additional halogenation step in the presence of a halogenating agent such as 7V-chlorosuccinimide (if the desired halogen is chlorine), 7V-bromosuccinimide (if the desired halogen is bromine), or 7V-iodosuccinimide (if the desired halogen is iodine).

[0186] Step (2):

[0187] The cleavage reaction can be carried out by a SPICC (strain-promoted iminosydnone-cycloalkyne cycloaddition) reaction, namely by reaction with a compound comprising a cyclic alkyne, possibly substituted, preferably comprising a cyclooctyne such as cyclooctyne, DBCO or DIBO (dibenzocyclooctyne), DBCO-NH2 or DBCO-amine (dibenzocyclooctyne-amine), m-dPEGn-DBCO, DBCO-PEG4-COOH or DBCO-PEG4-acid (dibenzoazacyclooctyne-PEG4-acid), DBCO-PEG5-COOH or DBCO-PEG5-acid (dibenzoazacyclooctyne-PEG5-acid), or (1R,8S,9S)-bicyclo[6.1.0]nonyne (BCN). m-dPEGn-DBCO corresponds to the formula below:

[0188] This cleavage reaction can be carried out, for example, in a solvent such as water, DMSO, DMF or a mixture of these.

[0189] In the process mentioned above, additional steps of protection and / or deprotection of various chemical groups may be necessary, which a person skilled in the art will be able to determine.

[0190] Modified nucleoside

[0191] A modified nucleoside according to the invention consists of a modified nucleobase as defined above bonded to the anomeric carbon atom at position 1' of a pentose residue. The pentose residue is advantageously selected from deoxyribose and ribose residues or ribose derivatives, particularly ribose derivatives in which the OH group at position 2' has been replaced by another group, notably selected from O-methyl (or O-O-methoxyethyl, O-(CH2)2-OCH3), O-methoxyethyl (or O-(CH2)2-OCH3), fluoro (F), amino (NH2), and azido (N3) groups. The modified nucleoside according to the present invention can be prepared by:

[0192] (3) coupling reaction between: a modified nucleoside consisting of a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group, Ao and Xn being as defined above, the nucleobase being bonded to the anomeric carbon atom at position 1' of a pentose residue, advantageously chosen from ribose and deoxyribose, and a molecule of formula X12-A1-Y1-R1, in which Ai, Yi, Ri, and Xn are as defined above, to give a modified nucleoside according to the invention in which the modified nucleobase is a nucleobase in which a hydrogen atom is replaced by Ro' or a nitrogen atom is replaced by CRo' where Ro' is a substitution group of formula -A0-X1-A1-Y1-R1,

[0193] (4) then, if necessary, cleavage of the Yi group of the modified nucleoside obtained in step (3) to give a modified nucleoside according to the invention having the modified nucleobase a nucleobase in which a hydrogen atom is replaced by Ro” or a nitrogen atom is replaced by CRo” where Ro” is a substitution group of formula -A0-X1-A1-Y2.

[0194] Step (3) can be carried out under the same conditions as step (1) above.

[0195] Step (4) can be carried out under the same conditions as step (2) above.

[0196] The modified nucleoside, consisting of a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group, may be commercially available. Examples include:

[0197] in which Zi = H, OH, -methoxy, -methoxymethoxy (O-CH2-OCH3), -methoxyethoxy (O-5 (CH2)2-OCH3), fluoro (F), or amino (NH2) and Z2 = H or (CH2)4C=CH. In the process mentioned above, additional steps for protecting and / or deprotecting various chemical groups may be necessary, which a person skilled in the art will be able to determine.

[0198] Modified nucleotide

[0199] A modified nucleotide according to the invention consists of a modified nucleoside as defined above in which the OH group at position 5' of the pentose residue is replaced by an -O- (Z3i) group q i-(Z32)q2-Z33-H or a salt thereof for which:

[0200] - q1 and q2 independently represent 0 or 1, and

[0201] - Z31, Z32, and Z33 represent, independently of each other, a phosphate group -P(O)(OH)-O- or a phosphate derivative. By "phosphate derivative" is meant a phosphate group in which one or more oxygen atoms are replaced by a sulfur atom, and / or in which the OH group is replaced by a methyl group. A phosphate group derivative is most advantageously chosen from -P(O)(SH)-O-, -P(O)(CH3)-O-, -P(S)(OH)-O-, -P(S)(SH)-O-, and -P(S)(CH3)-O-,

[0202] It can be a deoxyribonucleotide (in which the nucleoside is a deoxyribonucleoside as defined above) or a ribonucleotide (in which the nucleoside is a ribonucleoside as defined above), advantageously a deoxyribonucleotide.

[0203] Preferably, ql = q2 = 1, because reverse transcriptases and DNA polymerases are capable of incorporating deoxynucleotides comprising three phosphate groups or phosphate derivatives.

[0204] Preferably, Z31, Z32 and Z33 are identical, and preferably represent -P(O)(OH)-O-,

[0205] The modified nucleotide according to the present invention can be prepared by:

[0206] (5) coupling reaction between: a modified nucleotide consisting of a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group, Ao and Xn being as defined above, the nucleobase being bonded to the anomeric carbon atom in position 1' of a pentose residue, advantageously chosen from ribose and deoxyribose, the OH group in position 5' of the pentose residue being replaced by an -O-(Z3i) group qi-(Z32)q2-Z33-H or a salt thereof, and a molecule of formula X12-A1-Y1-R1, in which Ai, Yi, Ri, and X12 are as defined above, to give a modified nucleotide according to the invention having the modified nucleobase a nucleobase in which a hydrogen atom is replaced by Ro' or a nitrogen atom is replaced by CRo' where Ro' is a substitution group of formula -A0-X1-A1-Y1-R1,

[0207] (6) then, if necessary, cleavage of the Yi group of the modified nucleotide obtained in step (5) to give a modified nucleotide according to the invention having the modified nucleobase a nucleobase in which a hydrogen atom is replaced by Ro” or a nitrogen atom is replaced by CRo” where Ro” is a substitution group of formula -A0-X1-A1-Y2.

[0208] Step (5) can be carried out under the same conditions as step (1) above. Step (6) can be carried out under the same conditions as step (2) above.

[0209] The modified nucleotide consisting of a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group, Ao and Xn being as defined above, the nucleobase being linked to the anomeric carbon atom in position 1' of a pentose residue, advantageously chosen from ribose and deoxyribose, the OH group in position 5' of the pentose residue being replaced by an -O-(Z3i) group q i-(Z32)q2-Z33-H or a salt thereof, may be commercially available. For example:

[0210] in which Zi = H, OH, -methoxy, -methoxymethoxy (O-CH2-OCH3), -methoxyethoxy (O-

[0211] 5 (CH2)2-OCH3), fluoro (F), or amino (NH2); Z2= H or (CH2)4C=CH; and Z3= -(P(O)(OH)-O)3-

[0212] H or a salt thereof. In the process mentioned above, additional steps for protecting and / or deprotecting various chemical groups may be necessary, which a person skilled in the art will be able to determine.

[0213] Modified nucleic acid

[0214] The modified nucleic acid according to the invention comprises at least one nucleotide modified according to the invention. In other words, at least one of the monomers constituting the nucleic acid is a nucleotide modified according to the invention.

[0215] A modified nucleic acid according to the invention may comprise nucleotides including several types of nucleobases, in particular selected from adenine, guanine, thymine, uracil, or cytosine, preferably from adenine, guanine, uracil, or cytosine. Advantageously, a modified nucleic acid according to the invention comprises nucleotides including at least two different nucleobases, preferably at least three different nucleobases, and even more preferably, a modified nucleic acid according to the invention comprises four different nucleotides including the four nucleobases adenine, guanine, uracil, and cytosine, respectively.

[0216] Advantageously, at least 50%, preferably at least 60%, or even at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even 100% of the nucleotides of the modified nucleic acid comprising the same nucleobase (advantageously chosen from adenine, guanine, uracil or cytosine, preferably uracil) are nucleotides modified according to the invention.

[0217] When modified nucleotides (comprising a nucleobase modified according to the invention in which a hydrogen atom is replaced by Ro or a nitrogen atom is replaced by CRo, where Ro is a substitution group of the formula -A0-X1-A1-Y1-R1 or -A0-X1-A1-Y2, where A0, Xi, Ai, Yi, Y2, and Ri are as defined above) can be directly incorporated by a DNA or RNA polymerase, then up to 100% of the nucleotides in the modified nucleic acid comprising the same nucleobase can be nucleotides modified according to the invention. It is sufficient to amplify a template strand with a DNA or RNA polymerase in the presence of a mixture of tri-(phosphate or derivative) nucleotides in which 100% of the nucleotides comprising the same nucleobase are nucleotides modified according to the invention.

[0218] When the modified nucleotides cannot be directly incorporated by a DNA or RNA polymerase (because the chemical modification is too large), the nucleic acids modified according to the invention can be obtained by incorporating nucleotides comprising the same nucleobase in which a hydrogen atom is replaced by an -Ao-Xu group or in which a nitrogen atom is replaced by a C-Ao-Xn group, where Ao is as defined above and Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH, or -CR=CH2 (R6 and R7 being as defined above), and then carrying out a coupling reaction (in particular under the conditions described above for step (2)) with a molecule of formula X12-A1-Y1-R1, in which Ai, Yi, and Ri are as defined above and X12 represents: -N3 when Xn represents -C=CH, -C=CH when Xn represents -N3,

[0219] -O-NH2 or -NH-NH2 when Xn represents -C(O)RÔ,

[0220] -C(O)RÔ when Xn represents -O-NH2 or -NH-NH2,

[0221] -CR?=CH2 when Xn represents -SH, or

[0222] -SH when Xn represents -CR?=CH2.

[0223] In this case, the coupling is never total. Nevertheless, at least 50%, preferably at least 60%, or even at least 70% of the nucleotides of the nucleic acid comprising the same nucleobase (advantageously chosen from adenine, guanine, uracil or cytosine, preferably uracil) are nucleotides modified according to the invention.

[0224] The modified nucleic acid according to the invention is preferably a functional nucleic acid, advantageously chosen from specific ligand nucleic acids (i.e., capable of binding specifically to a ligand of interest), such as aptamers, and catalytic nucleic acids, such as ribozymes and deoxyribozymes. Advantageously, the functional nucleic acid modified according to the invention is chosen from aptamers, ribozymes, and deoxyribozymes. More advantageously, it is an aptamer.

[0225] The modified nucleic acid according to the invention can be a ribonucleic acid (RNA, as defined above, preferably whose pentose residue is a ribose) or a deoxyribonucleic acid (DNA).

[0226] The modified nucleic acid according to the invention may in particular be an aptamer of DNA or RNA.

[0227] Reverse transcription process

[0228] The inventors demonstrated that a reverse transcriptase was capable of reading a modified RNA template comprising at least one modified ribonucleotide in which a hydrogen atom of the nucleobase is replaced by Ro or a nitrogen atom of the nucleobase is replaced by CRo where Ro is a substitution group of formula -A0-X1-A1-Y2, where Ro, Ao, Xi, Ai and Y2 are as defined above.The present invention therefore also relates to a method of reverse transcription into DNA of an RNA modified according to the invention in which Ro is a substitution group of formula - -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined above (i.e. a modified RNA comprising at least one modified nucleotide in which a hydrogen atom of the nucleobase is replaced by Ro or a nitrogen atom of the nucleobase is replaced by CRo where Ro is a substitution group of formula A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined above), comprising contacting the RNA modified according to the invention with a reverse transcriptase in the presence of a primer and a mixture of deoxynucleotides comprising three phosphate groups or phosphate derivatives as described above under appropriate conditions.

[0229] In this process, any type of deoxynucleotide comprising three phosphate groups or phosphate derivatives as described above can be used, including deoxynucleotides comprising three phosphate groups or phosphate derivatives possessing a natural nucleobase (thus obtaining natural DNA) or deoxynucleotides modified according to the invention as described above (thus obtaining DNA according to the invention). Furthermore, any type of reverse transcriptase can be used. Many reverse transcriptases (RTs) are commercially available, and any of them can be used, under the conditions recommended by the manufacturer.One can use, in particular, a reverse transcriptase chosen from the avian myeloblastosis virus reverse transcriptase (abbreviated as "AMV RT", sequence SEQ ID NO: 18, see Table 3 below) or a sequence with at least 90% identity with SEQ ID NO: 18, the Moloney murine leukemia virus reverse transcriptase (abbreviated as "MML-V RT", sequence SEQ ID NO: 19, see Table 3 below, or a sequence with at least 90% identity with SEQ ID NO: 19), or derivatives of MML-V RT. Examples of MML-V RT derivatives include:

[0230] - Superscript II including the D524G, E562Q, and D583N mutations compared to the wild-type sequence of MML-V RT,

[0231] - Superscript III including the mutations H204R, T306K, F309N, V223H, D524G, E562Q compared to the wild-type sequence of MML-V RT, and D583N and

[0232] - Superscript IV corresponding to SEQ ID NO:4 of US9663770B2 and including the mutations P51L, S67R, E69K, T197A, H204R, E302K, F309N, W313F, T330P, L435G, N454K, D524G, D583N, H594Q, D653N, and L671P compared to the wild-type sequence of MML-V RT.

[0233] Other variants of MML-V RT that may be used in the context of the present invention have been described, including in US5244797A (see amino acid sequence in Figure 6), US7056716B2 (see SEQ ID NO:6 in Figure 8 of this patent), W02007022045A2 (see SEQ ID NO:20 to 35 of this application, in particular SEQ ID NO:35 of this application), US7595179B2 (see SEQ ID NO:2 of this patent), W02009125006A2 (see SEQ ID NO:26 to 128 of this application), US9580698B1 (see SEQ ID NO:1 of this patent), and US20170159032A1 (see SEQ ID NO:1 to 18 of this application).

[0234] [Table 3]

[0235] Other reverse transcriptases known to those skilled in the art can also be used. The buffer used for reverse transcription can be chosen by those skilled in the art depending on the chosen reverse transcriptase. For example, the reverse transcription buffer might include:

[0236] - 10 to 100 mM Tris-HCl, pH 8.3, in particular;

[0237] - 10 to 150 mM KCl;

[0238] - 0 to 10 mM MgCh, - 0 to 0.05 M DTT; and

[0239] - O to lOmM MnCh.

[0240] The following stamp can be used, in particular:

[0241] - 50 mM Tris-HCl, pH 8.3;

[0242] - 75 mm KC1;

[0243] 3 mM MgCh), and

[0244] - 0.02 M DTT.

[0245] The reverse transcription conditions can also be chosen by a person skilled in the art depending on the reverse transcriptase chosen. For example, suitable reverse transcription conditions may be the following: 30 minutes at 1:30 (advantageously 45 minutes at 1:11 5, 50 minutes at 1:11 0, 55 minutes at 1:05, in particular about 1:00) at a temperature of + / - 2 degrees relative to the optimal temperature of the reverse transcriptase (optimal temperature indicated by the supplier, for example, for Superscript IV, a temperature between 50 and 65°C, between 52 and 65°C, between 53 and 57°C, or between 54 and 56°C, such as about 55°C).

[0246] RNA / DNA amplification method

[0247] The inventors also demonstrated that a DNA polymerase was capable of reading a modified DNA template comprising at least one modified deoxyribonucleotide in which a hydrogen atom of the nucleobase is replaced by Ro or a nitrogen atom of the nucleobase is replaced by CRo where Ro is a substitution group of formula or -A0-X1-A1-Y2, where Ro, Ao, Xi, Ai and Y2 are as defined above.

[0248] The present invention therefore also relates to a method for amplifying a modified RNA or DNA according to the invention in which Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined above (i.e., a modified RNA or DNA comprising at least one modified nucleotide in which a hydrogen atom of the nucleobase is replaced by Ro or a nitrogen atom of the nucleobase is replaced by CRo where Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined above), comprising the following steps: a) when the nucleic acid to be amplified is a modified RNA in which Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined in this description,the reverse transcription of modified TRNA into DNA by contacting it with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under appropriate conditions; b) contacting the DNA obtained in step a) or the modified DNA according to the invention in which Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined in this description, with a DNA polymerase in the presence of a sense primer capable of hybridizing to the 5' portion of one of the two DNA strands, and an antisense primer capable of hybridizing to the 5' portion of the other DNA strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase when the nucleic acid to be amplified is an RNA according to the invention,and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives; and c) DNA amplification by polymerase chain reaction comprising the following substeps:,

[0249] (i) a DNA denaturation step enabling the separation of the two DNA strands, during which the mixture obtained in step b) is advantageously heated to a temperature of 90°C to 100°C;

[0250] (ii) a step of pairing the sense and antisense primers to the separated DNA strands, during which the mixture obtained in substep (i) is advantageously heated to a temperature of 45°C to 72°C, advantageously from 45°C to 65°C;

[0251] (iii) an elongation step of a complementary strand of each DNA strand comprising a primer by DNA polymerase, during which the mixture obtained in substep ii) is advantageously heated to a temperature of 65°C to 80°C; (d) optionally, N iterations of step c), N being an integer greater than 1, in which for each iteration n (l <n<N-l), la sous-étape (i) de l’itération n+1 est mise en œuvre sur le mélange obtenu à la sous-étape (iii) de l’itération n au lieu du mélange obtenu à l’étape b), et e) lorsque l’acide nucléique à amplifier est un ARN, la transcription des ADN amplifiés à l’étape c) et éventuellement à l’étape d) en ARN avec une ARN polymérase en présence d’un mélange de ribonucléotides comprenant trois groupements phosphate ou dérivé de phosphate dans des conditions appropriées.

[0252] Thus, when the nucleic acid to be amplified is DNA modified according to the invention in which Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined above, the amplification process according to the invention comprises the following steps b), c) and optionally d) (steps a) and e) are not necessary since the nucleic acid to be amplified is not an RNA): b) contacting the modified DNA to be amplified with a DNA polymerase in the presence of a sense primer capable of hybridizing to the 5' portion of one of the two DNA strands, an antisense primer capable of hybridizing to the 5' portion of the other DNA strand, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives; and c) DNA amplification by polymerase chain reaction comprising the following substeps:

[0253] (i) a DNA denaturation step enabling the separation of the two DNA strands, during which the mixture obtained in step b) is advantageously heated to a temperature of 90°C to 100°C;

[0254] (ii) a step of pairing the sense and antisense primers to the separated DNA strands, during which the mixture obtained in substep (i) is advantageously heated to a temperature of 45°C to 72°C, advantageously from 45°C to 65°C;

[0255] (iii) an elongation step of a complementary strand of each DNA strand comprising a primer by DNA polymerase, during which the mixture obtained in substep ii) is advantageously heated to a temperature of 65°C to 80°C; and (d) optionally, N iterations of step c), N being an integer greater than 1, in which for each iteration n (l <n<N-l), la sous-étape (i) de l’itération n+1 est mise en œuvre sur le mélange obtenu à la sous-étape (iii) de l’itération n au lieu du mélange obtenu à l’étape b).

[0256] When the nucleic acid to be amplified is an RNA modified according to the invention in which Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined above, the amplification process according to the invention comprises the following steps a) to c), optionally d), and step e): a) reverse transcription of the modified RNA to be amplified into DNA by contacting it with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under appropriate conditions;b) contacting the DNA obtained in step a) with a DNA polymerase in the presence of a sense primer capable of hybridizing to the 5' portion of one of the two DNA strands, an antisense primer capable of hybridizing to the 5' portion of the other DNA strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives; and c) amplification of the DNA by polymerase chain reaction comprising the following substeps:;

[0257] (i) a DNA denaturation step enabling the separation of the two DNA strands, during which the mixture obtained in step b) is advantageously heated to a temperature of 90°C to 100°C;

[0258] (ii) a step of pairing the sense and antisense primers to the separated DNA strands, during which the mixture obtained in substep (i) is advantageously heated to a temperature of 45°C to 72°C, advantageously from 45°C to 65°C;

[0259] (iii) an elongation step of a complementary strand of each DNA strand comprising a primer by DNA polymerase, during which the mixture obtained in substep ii) is advantageously heated to a temperature of 65°C to 80°C; (d) optionally, N iterations of step c), N being an integer greater than 1, in which for each iteration n (l <n<N-l), la sous-étape (i) de l’itération n+1 est mise en œuvre sur le mélange obtenu à la sous-étape (iii) de l’itération n au lieu du mélange obtenu à l’étape b), et e) la transcription des ADN amplifiés à l’étape c) et éventuellement à l’étape d) en ARN avec une ARN polymérase en présence d’un mélange de ribonucléotides comprenant trois groupements phosphate ou dérivé de phosphate dans des conditions appropriées.

[0260] Step d) is optional, but preferably present, and advantageously comprises 2 to 35 iterations, more advantageously 5 to 35 iterations, 10 to 35 iterations, 15 to 35 iterations, preferably 20 to 30 iterations, for example about 25 iterations.

[0261] Deoxyribonucleotide mixtures comprising three phosphate groups or phosphate derivatives (step b)) or ribonucleotide mixtures comprising three phosphate groups or phosphate derivatives (step e)) include deoxyribonucleotides or ribonucleotides comprising the 4 different types of bases useful for amplification (A, T or U, G, and C) or transcription (A, U, G, and C).

[0262] In one embodiment, particularly when the modified nucleic acid to be amplified is DNA, one of the deoxyribonucleotides comprising three phosphate groups or a phosphate derivative (advantageously all or part of the deoxyribonucleotides comprising three phosphate groups comprising the same nucleobase) of the mixture added in step b) may comprise a nucleobase in which a hydrogen atom is replaced by a Ro or -Ao-Xn group or in which a nitrogen atom is replaced by a CRo or C-Ao-Xn group, where Ro, Ao, and Xn are as defined previously (in particular, Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH, or -CR=CH2). In this embodiment, the resulting DNA is DNA modified according to the invention. However, all deoxyribonucleotides comprising three phosphate groups or phosphate derivatives may alternatively include natural nucleobases. In this embodiment, the resulting DNA is natural DNA.

[0263] In an alternative or combined embodiment, particularly when the modified nucleic acid to be amplified is RNA, one of the ribonucleotides comprising three phosphate groups or a phosphate derivative (advantageously all or part of the ribonucleotides comprising three phosphate groups comprising the same nucleobase) of the mixture used for transcription in step e) may comprise a nucleobase in which a hydrogen atom is replaced by a Ro or -Ao-Xn group or in which a nitrogen atom is replaced by a CRo or C-Ao-Xn group, where Ro, Ao, and Xn are as defined above (in particular, Xn represents -C=CH, -N3, -C(O)RO, -O-NH2, -NH-NH2, -SH, or -CR7=CH2). In this embodiment, the resulting RNA is RNA modified according to the invention. However, all ribonucleotides comprising three phosphate groups or phosphate derivatives may alternatively include natural nucleobases.In this embodiment, the RNA obtained is natural RNA.

[0264] In the amplification methods according to the invention, the optional back-transcription step can be carried out according to any embodiment described in the section above concerning back-transcription methods according to the invention.

[0265] In the amplification processes according to the invention, the sense and antisense primers added for amplification are each complementary to one of the DNA strands to be amplified and each allows the extension by DNA polymerase of a strand complementary to the strand to which the primer hybridizes. Their sequences are chosen according to the sequence of the DNA to be amplified.

[0266] In the amplification processes according to the invention, any type of DNA polymerase may be used under the conditions recommended by the manufacturer. DNA polymerases have been categorized into seven families based on phylogenetic analysis and the similarity of their nucleic acid sequences: families A, B, C, X, Y, and RT. The DNA polymerase used in the amplification processes according to the invention is advantageously selected from among the thermostable DNA polymerases of archaea in family B, as well as their optimized mutants. In particular, the DNA polymerase may be selected from those of the genera Pyrococciis, Thermococciis, Thermotoga, Pyrobaculum, and Ignicoccus, and their mutants, especially those of the genera Pyrococcus and Thermococcus.

[0267] The DNA polymerase can notably be chosen from among the thermostable DNA polymerases of the B family archaea described in Table 4 below, or a DNA polymerase with at least 90%, advantageously at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identity with the sequences described in Table 4. These polymerases are close to each other and some of them (KOD DNA polymerase from Thermococcus kodakarensis, PWO DNA polymerase from Pyrococcus woesei and DeepVent (exo-) DNA polymerase from Pyrococcus species GB-D) are thermostable polymerases known to have better compatibility with chemically modified nucleotides.

[0268] [Table 4]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274] Advantageously, the DNA polymerase used includes the DNA polymerase Deep Vent (SEQ ID NO: 1 or a sequence with at least 90%, advantageously at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identity with SEQ ID NO: 1), alone or in combination with another DNA polymerase. In particular, a combination of Deep Vent (SEQ ID NO: 1 or a sequence with at least 90%, advantageously at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identity with SEQ ID NO: 1) with Taq polymerase (SEQ ID NO: 17 or a sequence with at least 90%, advantageously at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identity with SEQ ID NO: 17) may be used, such a combination being commercially available under the name "One Taq" from New England Biolabs (NEB; ref M0481S).

[0275] The buffer used for amplification advantageously includes, particularly when using:

[0276] - a Tris-SCU buffer at a concentration of 5 to 200 mM, advantageously 50 to 150 mM, more advantageously 60 to 100 mM, 70 to 90 mM, in particular about 80 mM;

[0277] - of (NH4)2SU4 at a concentration of 1 to 100 mM, advantageously of 5 to 60 mM, of 10 to 40 mM, of 15 to 30 mM, in particular about 20 mM;

[0278] - Mg ions 2+ advantageously in the form of MgSCU at a concentration of 1 to 10 mM, advantageously from 1 to 5 mM, from 1.5 to 3 mM, in particular about 2 mM;

[0279] - glycerol at a concentration of 0 to 20% (volume / volume abbreviated as "v / v"), advantageously from 1 to 10% (v / v), from 2.5 to 7.5% (v / v), in particular about 5% (v / v),

[0280] - DMSO (dimethyl sulfoxide) at a concentration of 0 to 20% (v / v), advantageously 1 to 10% (v / v), 2.5 to 7.5% (v / v), in particular about 5% (v / v),

[0281] - of octylphenoxy poly(ethyleneoxy)ethanol (IGEPAL® CA-630) at a concentration of 0 to 1% (v / v), advantageously of 0.01 to 0.1% (v / v), of 0.04 to 0.08% (v / v), in particular of about 0.06% (v / v),

[0282] - polysorbate 20 at a concentration of 0 to 1% (v / v), advantageously 0.01 to 0.1% (v / v), 0.025 to 0.075% (v / v), in particular about 0.05% (v / v),

[0283] - a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives as defined above at a concentration of 0.1 to 3 mM, advantageously 0.1 to 1 mM, 0.1 to 0.5 mM, 0.1 to 0.3 mM, in particular about 0.2 mM, and

[0284] - 20 to 30 units / mL, advantageously 23 to 27 units / mL, in particular about 25 units / mL of DNA polymerase.

[0285] The amplification conditions in steps c) and d) of the amplification process according to the invention are preferably as follows:

[0286] - at step c) (1 er amplification cycle):

[0287] Step (c) advantageously comprising a substep (iii) of long elongation, of at least 15 minutes, advantageously at least 20 minutes, at least 25 minutes, or even at least 30 minutes. The elongation step may advantageously last between 15 and 90 minutes, between 20 and 75 minutes, or between 30 and 60 minutes, and in particular about 30 minutes, about 45 minutes or about 1 hour.

[0288] The substeps (i) of denaturation and (ii) of hybridization are of usual duration, i.e. between 2 and 3 minutes, advantageously between 2 and 2.5 minutes, for example about 2 minutes and 15 seconds for the initial substep (i) of denaturation and between 15 and 45 seconds, advantageously between 20 and 40 seconds, for example about 30 seconds for the substep (ii) of hybridization.

[0289] Advantageously, the more precise conditions of step c) are as follows:

[0290] (i) a DNA denaturation step allowing the two DNA strands to be separated, wherein the mixture obtained in step b) is heated to a temperature of 90°C to 100°C for 10 to 30 seconds, preferably to a temperature of 93°C to 97°C for 10 to 30 seconds, in particular to a temperature of about 95°C for about 15 seconds;

[0291] (ii) a primer-to-separated DNA strands pairing step, during which the mixture obtained in substep (i) is advantageously heated to a temperature of 45°C to 72°C for 15 to 45 seconds, preferably to a temperature of 55°C to 60°C for 10 to 30 seconds, in particular to a temperature of about 57°C for about 15 seconds;

[0292] (iii) an elongation step of a complementary strand of each DNA strand comprising a primer by DNA polymerase, during which the mixture obtained in substep ii) is advantageously heated at a temperature of 65°C to 80°C for 15 to 90 minutes, preferably at a temperature of 65°C to 72°C for 20 to 60 minutes, in particular at a temperature of about 68°C for about 30 or 45 or 60 minutes;

[0293] - at step d) optional but preferably present with 2 to 35 iterations, advantageously 5 to 35 iterations, 10 to 35 iterations, 15 to 35 iterations, 20 to 30 iterations, in particular about 25 iterations, the conditions are the usual ones, and advantageously the following:

[0294] (i) a DNA denaturation step allowing the two DNA strands to be separated, wherein the mixture obtained in step b) is heated to a temperature of 90°C to 100°C for 10 to 30 seconds, preferably to a temperature of 93°C to 97°C for 10 to 30 seconds, in particular to a temperature of about 95°C for about 15 seconds;

[0295] (ii) a primer-to-separated DNA strands pairing step, during which the mixture obtained in substep (i) is advantageously heated to a temperature of 45°C to 72°C for 15 to 45 seconds, preferably to a temperature of 55°C to 60°C for 10 to 30 seconds, in particular to a temperature of about 57°C for about 15 seconds;

[0296] (iii) an elongation step of a complementary strand of each DNA strand comprising a primer by DNA polymerase, during which the mixture obtained in substep ii) is advantageously heated to a temperature of 65°C to 80°C for 45 to 75 seconds, preferably to a temperature of 65°C to 72°C for 55 to 65 seconds, in particular to a temperature of about 68°C for about 60 seconds.

[0297] In the amplification processes according to the invention, the transcription of the DNA amplified in step c) and possibly in step d) into RNA is carried out with an RNA polymerase. Many RNA polymerases are commercially available, and any of them can be used, under the conditions recommended by the manufacturer. In particular, an RNA polymerase can be used, chosen from among the T7 RNA polymerase comprising the amino acid sequence SEQ ID NO:20 or a sequence with at least 90% identity with SEQ ID NO:20, or the sp6 RNA polymerase comprising the amino acid sequence SEQ ID NO:21 or a sequence with at least 90% identity with SEQ ID NO:21 (see Table 5 below).

[0298] [Table 5]

[0299] The buffer used for transcription can be chosen by a person skilled in the art depending on the RNA polymerase selected. For example, the transcription buffer might include:

[0300] - 10 to 100mM HEPES, - 3 to 20mM MgC12, - 0 to 50mM NaCl,

[0301] - 0 to 10m Spermidine,

[0302] - 1 to 100m DTT, and

[0303] - 0 to 10% triton XI 00.

[0304] For example, the following can be used as a transcription buffer:

[0305] - 40mm HEPES,

[0306] - 12 mM MgC12,

[0307] - 10M NaCl

[0308] - 2mM Spermidine,

[0309] - lOmM DTT, and

[0310] - 0.1% triton X100.

[0311] Transcription conditions can also be chosen by a person skilled in the art depending on the RNA polymerase chosen. For example, appropriate transcription conditions may be the following: 1h to 12h, advantageously 1h to 10h, 1h to 5h, 1h to 3h, 1h30 to 2h30, or 1h45 to 2h5, especially about 2h, at a temperature of 35 to 39 °C, advantageously 36 to 38 °C, especially about 37 °C.

[0312] Advantageously, DNase A is then added and the reaction is further incubated for 10 to 30 minutes, advantageously for 15 to 25 minutes, especially about 20 minutes, at a temperature of 35 to 39 °C, advantageously 36 to 38 °C, especially about 37 °C.

[0313] Method for selecting functional nucleic acids

[0314] The nucleobases, nucleosides and nucleotides according to the invention were primarily developed to implement a SELEX-type process with nucleotides chemically modified by numerous chemical groups of varying formula and size, despite the constraints imposed by the very limited ability of reverse transcriptases and DNA polymerases and RNA polymerases to read and incorporate modified nucleotides.

[0315] Indeed, thanks to the possibility of cleaving the chemical modification after selection and before a new enrichment cycle, it is now possible to select functional nucleic acids (including aptamers, ribozymes and deoxyribozymes) with any type of chemical modification using a SELEX-type process, provided that it includes a chemically cleavable group with a satisfactory cleavage yield, and without effect on the nucleic acid itself (unlike the case of ultraviolet photocleavage, which at low ultraviolet doses may not have a sufficient yield and which at high ultraviolet doses is likely to damage the nucleic acid).

[0316] If the ability of DNA polymerases and RNA polymerases to incorporate nucleotides with chemical modifications is limited, their ability to read a strand of nucleic acid comprising nucleotides with chemical modifications is even more limited.

[0317] Therefore, there may be cases where the chemical modification allows a DNA polymerase or RNA polymerase to incorporate the modified nucleotide but not to read it.

[0318] Depending on whether the chemical modification of interest allows a DNA polymerase or RNA polymerase to incorporate the modified nucleotide, the SELEX selection process may vary slightly. The SELEX selection process also varies depending on whether the functional nucleic acid to be selected is DNA or RNA.

[0319] A first SELEX-type selection process according to the invention is applicable regardless of the chemical modification.

[0320] When the modified functional nucleic acid to be selected is DNA, this is a method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a library of natural or modified DNA according to the invention in which Ro is replaced by a group of formula -Ao-Xn, where Ao is as defined in this description and Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR=CH2, RÔ and R7 being as defined in this description, the sequence of which comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0321] - the sequence of the region at the 5' end is known and identical for all DNA,

[0322] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0323] - the sequence of the central region is random; b) when the library comprises natural DNA or optionally when the library comprises DNA modified according to the invention as defined in step a), amplification by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate or phosphate derivative groups, one of which comprises a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-ni group

[0324] Ao-Xn where Ao is as defined in this description and where Xn represents -C=CH, -N3, - C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR?=CH2, RÔ and R7 being as defined in this description, c) then, when Xn is -C=CH, -N3, -SH or -C(R?)=CH2, the coupling reaction of the DNAs obtained in step b) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R?)- A1-Y1-R1, or SH-A1-Y1-R1 respectively; when Xn is -CO-Re, the coupling reaction of the DNAs obtained in step b) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1- Y1-R1; and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the DNAs obtained in step b) with a molecule of formula Re-CO-Ai-Yi-Ri, to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri; d) the selection of the modified DNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri having the function of interest;e) cleavage of the Yi group to give modified DNAs bearing a substitution group of the formula -A0-X1-A1-Y2; f) optionally, N iterations of steps a) to e), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN modifiés obtenu à l’étape e) de l’itération n ; et g) le séquençage des ADN obtenus à la fin de l’étape e) ou, lorsqu’elle est présente, à la fin de l’étape f).;

[0325] In this process, the chemical modification of the deoxyribonucleotide, consisting of a nucleobase with a chemically altered hydrogen or nitrogen atom, added for the amplification step, is a minor intermediate chemical modification that always allows amplification by a DNA polymerase. A coupling reaction then allows the final chemical modification of interest, with a chemically cleavable group, to be cleaved. After the selection step, the chemical modification of interest is chemically cleaved back to another minor chemical modification that always allows the DNA polymerase to read the resulting DNA.

[0326] When the modified functional nucleic acid to be selected is an RNA, a similarly applicable method regardless of the chemical modification but adapted to RNAs is a method for selecting modified functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0327] - the sequence of the region at the 5' end is known and identical for all DNA,

[0328] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0329] - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives of which one ribonucleotide comprises a nucleobase of which a hydrogen atom is replaced by an -Ao-Xn group or of which a nitrogen atom is replaced by a C-Ao-Xn group where Ao is as defined in this description and where Xn represents -C=CH, -N3, -C(O)RO, -O-NH2, -NH-NH2, -SH or -CR=CH2 under appropriate conditions; d) when Xn is -C=CH, -N3, -SH or -C(R?)=CH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R?)-AI-YI-Ri, or SH-A1-Y1-R1 respectively; when Xn is -CO-RÔ, the coupling reaction of the RNAs obtained in step c) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1;and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1; e) the selection of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1 having the function of interest; f) the cleavage of the Y1 group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2; g) the reverse transcription of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 into DNA by contacting a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under appropriate conditions;h) Optionally, N iterations of steps a) to g), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’acides nucléiques obtenu à la sous-étape (iii) de l’étape g) de l’itération n ; et g) le séquençage des ADN obtenus à la fin de l’étape g) ou, lorsqu’elle est présente, à la fin de l’étape h).;

[0330] In some cases, the chemical modification of interest may be incorporated by a DNA polymerase or an RNA polymerase but not be read by a reverse transcriptase or by a DNA polymerase.

[0331] In this case, when the chemical modification of interest can be incorporated by a DNA polymerase and the modified functional nucleic acid to be selected is DNA, a SELEX-type selection method according to the invention can be a method for selecting modified functional DNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region, and a 3' end region, characterized in that:

[0332] - the sequence of the region at the 5' end is known and identical for all DNA,

[0333] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0334] - the sequence of the central region is random;b) amplification of the DNAs from the library provided in step a) by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives, one of which comprises a nucleobase in which a hydrogen atom is replaced by a Ro group or a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of the formula -Ao-Xi-Ai-Yi-Ri, where Ao, Xi, Ai, Yi, and Ri are as defined in this description, to give modified DNAs bearing a substitution group of the formula -A0-X1-A1-Y1-R1; c) selection of modified DNA bearing a substitution group of formula -A0-X1-A1-Y1-Ri having the function of interest;d) cleavage of the Yi group to give modified DNAs bearing a substitution group of the formula -A0-X1-A1-Y2; e) optionally, N iterations of steps a) to d), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à la sous-étape (iii) de l’étape d) de l’itération n ; et f) le séquençage des ADN obtenus à la fin de l’étape d) ou, lorsqu’elle est présente, à la fin de l’étape e).;

[0335] When the chemical modification of interest can be incorporated by an RNA polymerase and the modified functional nucleic acid to be selected is an RNA, a SELEX-type selection method according to the invention can be a method for selecting modified functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region, and a 3' end region, characterized in that:

[0336] - the sequence of the region at the 5' end is known and identical for all DNA,

[0337] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0338] - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives, one of which is a ribonucleotide comprising a nucleobase in which a hydrogen atom is replaced by a Ro group or in which a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -A0-X1-A1-Y1-R1, in which Ao, Xi, Ai, Yi and Ri are as defined in this description under conditions suitable to give RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1; d) selection of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri having the function of interest; e) cleavage of the Yi group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2;f) reverse transcription of modified RNA bearing a substitution group of the formula -A0-X1-A1-Y2 into DNA by contact with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under suitable conditions; g) optionally, N iterations of steps a) to f), N being an integer greater than or equal to 1, wherein for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à l’étape f) de l’itération n ; et h) le séquençage des ADN obtenus à la fin de l’étape f) ou, lorsqu’elle est présente, à la fin de l’étape g).;

[0339] In certain cases, it may also be possible to select nucleic acids (DNA or RNA) modified according to the invention bearing a substitution group of the formula -A0-X1-A1-Y2. Nucleic acids modified according to the invention bearing a substitution group of the formula -A0-X1-A1-Y1-R1 may then serve as prodrugs, these not having the function of interest, but being able to be cleaved to give nucleic acids modified according to the invention bearing a substitution group of the formula -A0-X1-A1-Y2 having the function of interest.

[0340] When the nucleic acids modified according to the invention bearing a substitution group of formula -A0-X1-A1-Y2 are DNA, and regardless of the modifying chemical group of the prodrug, the present invention therefore also relates to a method for selecting functional modified DNA having a function of interest, comprising the following steps: a) providing a library of natural or modified DNA according to the invention in which Ro is replaced by a group of formula -Ao-Xn, where Ao is as defined in this description and Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR=CH2, RÔ and R7 being as defined in this description, the sequence of which comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0341] - the sequence of the region at the 5' end is known and identical for all DNA,

[0342] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0343] - the sequence of the central region is random; b) systematically when the library comprises natural DNA and optionally when the library comprises DNA modified according to the invention as defined in step a), amplification by polymerase chain reaction of the DNAs of the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives of which one deoxyribonucleotide comprises a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group where Ao is as defined in this description and where Xn represents - C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR7=CH2, RÔ and R7 being as defined in this description, c) then,when Xn is -C=CH, -N3, -SH or -C(R?)=CH2, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R?)-AI-YI-RI, OR SH-A1-Y1-R1 respectively; when Xn is -CO-RÔ, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1; and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri; e) cleavage of the Yi group to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y2; d) selection of modified DNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; f) optionally, N iterations of steps a) to e), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l),the library provided in step a) of iteration n+1 corresponds to the mixture of modified DNA obtained in step e) of iteration n; and g) the sequencing of the DNA obtained at the end of step d) or, where present, at the end of step f).,

[0344] When the nucleic acids modified according to the invention bearing a substitution group of formula -A0-X1-A1-Y2 are RNAs, and regardless of the modifying chemical group of the prodrug, the present invention has as its twelfth object a method for selecting modified functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that:

[0345] - the sequence of the region at the 5' end is known and identical for all DNA,

[0346] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0347] - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives of which one ribonucleotide comprises a nucleobase of which a hydrogen atom is replaced by an -Ao-Xn group or of which a nitrogen atom is replaced by a C-Ao-Xn group where Ao is as defined in this description and where Xn represents -C=CH, -N3, -C(O)RO, -O-NH2, -NH-NH2, -SH or -CR=CH2 under appropriate conditions; d) when Xn is -C=CH, -N3, -SH or -C(R?)=CH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R?)-AI-YI-Ri, or SH-A1-Y1-R1 respectively; when Xn is -CO-RÔ, the coupling reaction of the RNAs obtained in step c) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1;and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1; e) the cleavage of the Yi group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2; f) the selection of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; g) the reverse transcription of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 into DNA by contacting a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivative under appropriate conditions;h) Optionally, N iterations of steps a) to g), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’acides nucléiques obtenu à la sous-étape (iii) de l’étape g) de l’itération n ; et g) le séquençage des ADN obtenus à la fin de l’étape g) ou, lorsqu’elle est présente, à la fin de l’étape h).;

[0348] When the chemical modification of interest can be incorporated by a DNA polymerase and the modified functional nucleic acid to be selected is DNA, a SELEX-type selection method according to the invention can be a method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region, and a 3' end region, characterized in that:

[0349] - the sequence of the region at the 5' end is known and identical for all DNA,

[0350] - the sequence of the region at the 3' end is known and identical for all DNA, and

[0351] - the sequence of the central region is random;b) amplification of the DNAs from the library provided in step a) by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives, one of which comprises a nucleobase in which a hydrogen atom is replaced by a Ro group or a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -Ao-Xi-Ai-Yi-Ri, where Ao, Xi, Ai, Yi, and Ri are as defined in this description, to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1, d) the cleavage of the Yi group to give modified DNAs carrying a substitution group of the formula -A0-X1-A1-Y2;c) the selection of modified DNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; e) optionally, N iterations of steps a) to d), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à la sous-étape (iii) de l’étape d) de l’itération n ; et f) le séquençage des ADN obtenus à la fin de l’étape d) ou, lorsqu’elle est présente, à la fin de l’étape e).;

[0352] When the chemical modification of interest can be incorporated by an RNA polymerase and the modified functional nucleic acid to be selected is an RNA, a SELEX-type selection method according to the invention can be a method for selecting modified functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region, and a 3' end region, characterized in that:

[0353] - the sequence of the region at the 5' end is known and identical for all DNA,

[0354] - the sequence of the region at the 3' end is known and identical for all DNAs, and - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives, one of which is a ribonucleotide comprising a nucleobase in which a hydrogen atom is replaced by a Ro group or in which a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -A0-X1-A1-Y1-R1, in which Ao, Xi, Ai, Yi and Ri are as defined in this description, under conditions suitable to give RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1; e) the cleavage of the Yi group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2; d) the selection of modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest;f) reverse transcription of modified RNA bearing a substitution group of the formula -A0-X1-A1-Y2 into DNA by contact with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under suitable conditions; g) optionally, N iterations of steps a) to f), N being an integer greater than or equal to 1, wherein for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à l’étape f) de l’itération n ; et h) le séquençage des ADN obtenus à la fin de l’étape f) ou, lorsqu’elle est présente, à la fin de l’étape g).;

[0355] In any one of the selection methods according to the invention:

[0356] (1) where the process includes a reverse transcription step, the reverse transcriptase can be any reverse transcriptase as described previously in the section relating to reverse transcription processes according to the invention;

[0357] (2) where the process includes a back-transcription step, the buffer used for back-transcription may be any suitable buffer described above in the section concerning back-transcription processes according to the invention; (3) where the process includes a back-transcription step, this may be carried out under any suitable conditions described above in the section concerning back-transcription processes according to the invention;

[0358] (4) where the process includes a polymerase chain reaction amplification step, the DNA polymerase may be any DNA polymerase as previously described in the section relating to amplification processes according to the invention, in particular the DNA polymerase includes a thermostable DNA polymerase from the B family of archaea, advantageously a thermostable DNA polymerase from the GB-D strain of Pyrococcus comprising an amino acid sequence with at least 90% identity with SEQ ID NO:1, alone or in combination with a Taq polymerase comprising an amino acid sequence with at least 90% identity with SEQ ID NO:17);

[0359] (5) where the process includes a polymerase chain reaction amplification step, the buffer used for polymerase chain reaction amplification may be any suitable buffer described above in the section concerning amplification processes according to the invention;

[0360] (6) where the process includes a polymerase chain reaction amplification step, this step may be carried out under any suitable conditions described above in the section concerning amplification processes according to the invention; or

[0361] (7) where the process includes a transcription step, the RNA polymerase can be any RNA polymerase as described previously in the section relating to amplification processes according to the invention;

[0362] (8) where the process includes a transcription step, the buffer used for transcription may be any suitable buffer described above in the section concerning amplification processes according to the invention;

[0363] (9) where the process includes a transcription step, this can be carried out under any appropriate conditions described above in the section concerning amplification processes according to the invention;

[0364] (10) The sequencing step can be carried out:

[0365] - directly onto natural or modified DNA according to the invention bearing a substitution group of formula -A0-X1-A1-Y2 obtained in the previous step, or

[0366] - when the DNA obtained in the previous step is modified according to the invention and carries a substitution group of the formula -A0-X1-A1-Y2, after amplification of this DNA by polymerase chain reaction in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives whose nucleobase is natural, this being able to facilitate the sequencing step; or

[0367] (11) any combination of (1) to (10) above.

[0368] The selection methods according to the invention are suitable for selecting any type of functional nucleic acid. In particular, they can be used for selecting specific ligand nucleic acids (i.e., capable of binding specifically to a ligand of interest) such as aptamers, or for selecting catalytic nucleic acids such as ribozymes and deoxyribozymes.

[0369] DESCRIPTION OF THE FIGURES

[0370] Figure 1A. General procedure for the in vitro evolution of chemically modified DNA without the polymerase barrier. A. Left panel, schematic of the in vitro selection. A. Right panel, schematic of the chemical reactions involved in the process. The starting random library containing EdU is modified by CuAAC with a probe carrying an azide (triangle of the molecule to be grafted), an iminosydnone core (square of the molecule to be grafted), and a selected chemical modification (R group). The chemically conjugated library is incubated with a target. After several washes, the sequences that bind to the target are recovered, and part of the chemical modification is removed using a cyclooctyne that reacts with the iminosydnone via sterically constrained iminosydnone-cycloalkyne cycloaddition (SPICC).The oligonucleotide with the remaining triazole group (transferred probe square) and a terminal urea (transferred probe triangle), constituting the TdU DNA, is then amplified to EdU DNA by PCR to generate a suitable alkyne library for the next round of in vitro selection. B. General synthesis of the azide-C3-amino-iminosydnone-benzyl-R group. The first step involves introducing a nitrile group to para-iodoaniline, which allows the formation of the iminosydnone core in the second step. This step involves the in situ formation of an N-nitroso intermediate, immediately followed by cyclization in acidic medium. The third step is the conversion of the exocyclic nitrogen to introduce the azide function into the molecule. Finally, iodine allows Suzuki coupling to introduce any R group bearing a boronic acid function. C. Azurite-iminosydnone derivatives used in this study. Figure 2. High-throughput sequencing analysis of SELEX.Approximately 64,000 reads per cycle were analyzed. 2,229 sequences had a library frequency greater than 0.005% (50 copies per million) in at least one round. These sequences were extracted and grouped into families. The families were named according to their higher frequency in EdU-SELEX, Carb-SELEX, or Ind-SELEX (starting with E, C, or I, respectively). Top panels show the percentage of the library containing families with a frequency greater than 0.005%. Analysis from RI to R6 shows a high and drastic enrichment for EdU-SELEX and Carb-SELEX and a slight, gradual enrichment for Ind-SELEX. Middle panel shows the basic composition of the libraries at different rounds. Bottom panels show the frequencies of the predominantly amplified families.

[0371] Figure 3. Demonstration of the role of chemical modifications and specificity for Streptavidin using a mixture of aptamer libraries. The libraries from round R6 of each SELEX are pooled. The mixture is divided to be either unmodified (as in EdU SELEX) or modified by Carb and Ind (as in Carb-SELEX and Ind-SELEX, respectively). The same mixture in a different chemistry is then subjected to a series of SELEX processes with no beads or against M270 Streptavidin magnetic beads, Streptavidin agarose beads, and M270 Amine magnetic beads. High-throughput sequencing allowed quantification of the enrichment of the different families under the different conditions relative to the starting mixture.

[0372] Figure 4. Demonstration of the role of chemical modifications and specificity towards Streptavidin using an equimolar mixture of aptamers. An equimolar mixture of the 11 most enriched aptamer candidates in the EdU, Carb-dU, or Ind-dU chemistry, or a mixture of each chemistry, was subjected to a SELEX spin against Streptavidin M270 magnetic beads. High-throughput sequencing allowed quantification of the enrichment of the different families under the various conditions relative to the initial pool.

[0373] Figure 5. Demonstration of the key role of Carb-modified positions for the C0 aptamer. The C0 sequence was randomized to its three uracil positions (25, 32, and 51). The Carb-modified doped C0 library was subjected to a SELEX run with or without Streptavidin M270 beads. A control experiment was also performed against Streptavidin M270 beads using the C0 library in EdU chemistry. High-throughput sequencing allowed quantification of the uridine enrichment relative to the starting library. Figure 6. CO affinity curve. A fluorescently labeled CO DNA aptamer in Carb-dU chemistry (black curve) or EdU chemistry (gray curve) was incubated with increasing amounts of Streptavidin beads, and the amount of aptamer remaining bound to the beads after several washes was measured.

[0374] Figure 7. Yields for different PCR conditions (n=l). PCR product concentrations were obtained by gel electrophoresis quantification. The structures of the alkyne-modified triphosphate nucleotides (EdUTP and C8-EdUTP) used in the experiments are shown below the legend. All conditions contain natural nucleotides except for those indicated in the figure.

[0375] EXAMPLES

[0376] The examples below illustrate the present invention.

[0377] EXAMPLE 0 - Synthesis of chemical precursors

[0378] The synthesis of the chemical precursors is presented below.

[0379] Reagents and solvents

[0380] Unless otherwise stated, all reactions were carried out in oven-dried glassware.

[0381] Commercially available chemicals were purchased from ABCR, Acros Organics, Sigma-Aldrich, Alfa Aesar, Combi-Blocks, Carbolution, Fluorochem, and TCI Europe and used as is, unless otherwise specified. The following solvents were dried by distillation over the drying agents indicated in parentheses: THF (sodium), dichloromethane (CaH2). Other anhydrous solvents were purchased from Acros Organics, Sigma-Aldrich, and Alfa Aesar and stored on molecular sieves under an argon atmosphere.

[0382] Purifications

[0383] Flash chromatography was performed on silica gel (Merck Kieselgel 60, particle size 40-63 pm).

[0384] Analyses

[0385] The reactions were monitored by TLC on 0.25 mm silica (60 F254, Merck) using UV light as the visualizing agent. Nuclear magnetic resonance (NMR) spectroscopy: ¹H NMR (400 MHz) and NMR 13C (100 MHz) were measured on a Brucker Avance 400 MHz spectrometer. Chemical shifts are given in parts per million (ppm) downstream of the residual solvent peaks, and coupling constants are given in Hertz (Hz). Signals are designated as singlet (s), broad singlet (br.s), doublet (d), triplet (t), quadruplet (q), quintuplet (quint), and multiplet (m). Signals that could not be interpreted or easily visualized are designated as multiplet (m).

[0386] Electrospray mass spectra were obtained using an ESI-Quadripole autopurify mass spectrometer, Waters (pump: 2545, mass: ZQ2000).

[0387] The LC-MS spectra were recorded on a Waters Acquity UPLC® equipped with a PDA ek detector and an SQ 2 detector, mobile phase A: H2O + 0.1% formic acid, mobile phase B: acetonitrile + 0.1% formic acid.

[0388] High-resolution mass spectrometry (HRMS) was performed on a Waters Xevo® G2-XS QTof mass spectrometer.

[0389] Infrared (IR) spectra were obtained on a Perkin Elmer UATR TWO FTIR spectrophotometer and are indicated in wavelengths (cm⁻¹). 1 ).

[0390] Synthesized compounds

[0391] 2-((4-iodophenyl)amino)acetonitrile 1

[0392] 1

[0393] A suspension of 4-iodoaniline (5 g, 22.8 mmol, 1 equiv.) in MeCN (70 mL), K₂CO₃ (3.8 g, 27.4 mmol, 1.2 equiv.), and Nal (3.4 g, 22.8 mmol, 1 equiv.) was added. Chloroacetonitrile (2.89 mL, 45.7 mmol, 2 equiv.) was then added dropwise, and the mixture was refluxed with stirring for 65 hours under an argon atmosphere. The dark mixture was then cooled to room temperature and filtered through Celite® buffer (rinsing with EtOAc, 30 mL). The filtrate was washed with brine and then extracted with EtOAc, 20 mL (3 times). The combined organic phases were dried over MgSCU before being concentrated under reduced pressure. The residue was purified by column chromatography (SiCl, 9:1 to 7:3 heptane / EtOAc) to obtain the desired product as a beige solid (3.7 g, 14.3 mmol, 64%). The spectroscopic data are consistent with previous reports in the literature (Bernard et al. 2017).

[0394] RMN ‘H (400 MHz, DMSO-d6) ô 7.47 (d, J= 8.8 Hz, 2H), 6.57 (d, J = 8.9 Hz, 2H), 6.45 (t, J = 6.8 Hz, 1H), 4.25 (H, 8). J= 6.2.

[0395] LCMS (ESI) m / z [M+H] + 259 .

[0396] 5-amino-3-(4-iodophenyl)-l,2,3-oxadiazol-3-ium chloride 2

[0397] To a stirred solution of 1 (1.6 g, 6.4 mmol, 1 equiv.) in dry THF (15 mL), 1 / c / V-butyl nitrite (2.2 mL, 18.7 mmol, 3 equiv.) was added dropwise. The mixture was stirred at room temperature for 1 hour. After complete conversion, the solvent was evaporated under reduced pressure. The residue was used without further purification and dissolved directly in a commercial 4 M HCl in dioxane solution (20 mL). The mixture was stirred overnight at room temperature under an argon atmosphere. Then, Et2O was added, and the precipitate was collected by filtration and washed with Et2O (20 mL) to obtain the product as a beige powder (1.5 g, 4.7 mmol, 75%). The spectroscopic data are consistent with previous reports in the literature (Bernard et al. 2017).

[0398] 'H NMR (400 MHz, DMSO-d6) ô 9.88 (s, 2H), 8.64 (s, 1H), 8.15 (d, J = 8.9 Hz, 2H), 7.81 (d, J = 8.9 Hz, 2H).

[0399] LCMS (ESI) m / z [M-C1'] + 288.

[0400] ((3-azidopropyl)carbamoyl) (3-(4-iodophenyl)-l,2,3-oxadiazol-3-ium-5-yl)amide 3

[0401] Warning: This reaction requires the use of tryphosgene and produces phosgene, both of which are lethal if inhaled. The tryphosgene was weighed under a fume hood (double weighing), the hood was kept closed throughout the procedure, and a phosgene detector was attached to the hood. The released phosgene was neutralized in two consecutive NaOH (IM) traps connected to the experimental setup.

[0402] To triphosgene (242.2 mg, 0.82 mmol, 0.33 equiv.) in a 250 mL round-bottom flask, a solution of 3-azidopropane-l-amine (337.6 g, 2.47 mmol, 1 equiv.) dissolved in DCM (24 mL) was added, and the solution was cooled to 0°C. Next, 25 mL of an aqueous solution of NaHCCl (831 mg, 9.89 mmol, 4 equiv.) was added to the flask. The mixture was stirred for 30 minutes at room temperature. Then, a solution of iminosydnone 2 (800 mg, 2.47 mmol, 1 equiv.) in DCM (35 mL) was slowly added. The reaction mixture was stirred for 5 hours at room temperature. After completion, the reaction was cooled with brine (30 mL) and any remaining phosgene was carefully removed by bubbling argon through the mixture (15 min). The DCM was evaporated under reduced pressure. The solid was filtered and washed with water.The crude product was then purified by column chromatography (SiCl, from 100 / 0 to 95 / 5 DCM / MeOH) to obtain the desired product in the form of a yellow solid (355 mg, 0.680 mmol, 35%).

[0403] RMN 'H (400 MHz, MeOD) ô 8.23 ​​(s, 1H), 8.08 (d, J= 8.9 Hz, 2H), 7.73 (d, J= 8.9 Hz, 2H), 3.37 (t, J = 6.8 Hz, 2H), 3.27 (m, 2H) signal below the reference peak, 1.78 (quint, J = 6.8 Hz, 2H).

[0404] NMR 13 C (101 MHz, CDCh) ô 172.2, 160.8, 139.9 (2C), 133.5, 122.9 (2C), 102.2, 99.6, 49.2, 37.9, 29.5.

[0405] IR (cm' 1 ) 32512, 2092 (N3), 1630, 1597, 1585, 1550, 1488, 1430, 1359, 1278, 1221, 1118, 1079, 1059, 1028, 1007, 954, 936, 853, 822, 807, 715, 677, 653, 632, 502, 483.

[0406] HRMS (ESI-TOF) m / z [M+H] + Calculated for C12H12IN7O2 414.0175; Identified 414.0178.

[0407] (3-(4'-(9H-carbazol-9-yl)-[1,1'-biphenyl]-4-yl)-1,2,3-oxadiazol-3-ium-5-yl) ((3-azidopropyl)carbamoyl)amide 4a - Carb

[0408]

[0409] A round-bottom flask under an argon atmosphere was loaded with 3 (56.34 mg, 0.136 mmol, 1 equiv.), Pd(OAc)2 (3.06 mg, 0.0136 mmol, 0.1 equiv.), dppe (5.44 mg, 0.0136 mmol, 0.1 equiv.), K2CO3 (56.56 mg, 0.409 mmol, 3 equiv.) and a MeCN / water mixture (1 mL / 1.5 mL). After stirring for 15 minutes at room temperature, (4-(9H-carbazol-9-yl)phenyl)boronic acid (43.1 mg, 0.150 mmol, 1.1 equiv.) was added and the mixture was heated to 60 °C overnight. The crude product was then purified by column chromatography (SiC>2, from 100 / 0 to 10 / 90 Heptane / EtOAc) to obtain the desired product as a bright yellow solid (35 mg, 0.0663 mmol, 48%).

[0410] RMN 'H (400 MHz, CDCh) ô 8.29 (s, 1H), 8.17 (d, J= 7.7 Hz, 2H), 7.95 (s, 4H), 7.87 (d, J= 8.6 Hz, 2H), 7.74 (d, J= 8.6 Hz, 2H), 7.46 (ddd, J= 11.2, 9.4, 4.5 Hz, 4H), 7.33 (ddd, J= 8.0, 6.9, 1.3 Hz, 2H), 5.89 (br. s, 1H), 3.41 (dt, J= 10.9, 6.6 Hz, 4H), 1.90 - 1.83 (m, 2H).

[0411] RMN 13 C (101 MHz, CDCh) ô 172.9, 145.1, 140.7 (2C), 138.6, 137.5, 134.1, 133.2, 129.0 (2C), 128.9 (2C), 127.7 (2C), 126.2 (2C), 123.8 (2C), 122.1 (2C), 120.6 (2C), 120.5 (2C), 109.8 (2C), 102.0, 49.3, 38.0, 29.6.

[0412] IR (cm’ 1 ) 3062, 2089 (N3), 1633, 1597, 1527, 1447, 1362, 1229, 1173, 958, 860, 825, 748, 724, 513.

[0413] HRMS (ESI-TOF) m / z [M+H] + calculé pour C30H24N8O2 529.2100; Identifié 529.2199.

[0414] ( 3-( 4-( lH-indol-5-yl)phenyl)-l , 2, 3-oxadiazol-3-ium-5-yl) ( 3-azidopropyl)carbamoyl)amide

[0415] 4b - Ind

[0416] A round-bottom flask under an argon atmosphere was loaded with 3 (78 mg, 0.189 mmol, 1 equiv.), Pd(OAc)2 (4.24 mg, 0.0189 mmol, 0.1 equiv.), dppe (7.52 mg, 0.0189 mmol, 0.1 equiv.), K2CO3 (78.3 mg, 0.567 mmol, 3 equiv.) and a MeCN / water mixture (1 mL / 1.5 mL). After stirring for 15 minutes at room temperature, (lH-indol-5-yl)boronic acid (50.51 mg, 0.240 mmol, 1.1 equiv.) was added and the mixture was heated to 60°C overnight. The crude product was then purified by column chromatography (SiCl, from 100 / 0 to 0 / 100 Heptane / EtOAc). A second purification by prep-HPLC was required to obtain the desired product as a bright yellow solid (25.1 mg, 0.0624 mmol, 33%), under the conditions described below.

[0417] 'H NMR (400 MHz, CDCh) ô 8.32 (s, 1H), 8.18 (s, 1H), 7.99 (d, J= 7.4 Hz, 1H), 7.92 (s, 1H), 7.88 (d, J= 8.7 Hz, 2H), 7.83 (d, J= 8.3 Hz, 2H), 7.52 (d, J= 8.0 Hz, 1H), 7.47 (d, J= 9.3 Hz, 1H), 7.31 (s, 1H), 6.66 (s, 1H), 3.40 (dt, J= 11.4, 5.7 Hz, 4H), 1.85 (dt, J= 13.7, 7.0 Hz, 2H). IR (cm' 1 ) 3243, 2115 (N3), 1626, 1585, 1551, 1472, 1433, 1370, 1286, 1227, 1121, 964, 936, 896, 840, 795, 762, 728, 684, 621, 563, 535.

[0418] LCMS (ESI) m / z [M+H] + 403.

[0419] HRMS (ESI-TOF) m / z [M+H] + Calculated for C2oHi8N802403.1631; Identified 403.1638.

[0420] HPLC prep conditions:

[0421] - Column: HDO (HyPURITY C18 (250 mm x 10 mm))

[0422] - Flow rate: 1 mL / min

[0423] - Gradient: according to Table 6

[0424] [Table 6] 35 95 5

[0425] EXAMPLE 1

[0426] Materials and methods

[0427] To validate our method, we have more specifically:

[0428] (1) generated a DNA library in which thymidine is replaced by 5-ethynyl-2'-deoxyuridine (EdU),

[0429] (2) conjugated the bank by a copper(I)-catalyzed azide-alkyne (CuAAC) bioorthogonal cycloaddition reaction with a chemical group R containing an iminosydnone ring,

[0430] (3) selected modified sequences that meet a selection criterion,

[0431] (4) cleaved the chemical group R by a bioorthogonal SPICC (Strain-Promoted Iminosydnone-Cycloalkyne Cycloaddition) reaction to produce DNA containing 5-triazole-C3-urea-uri dines (TdU),

[0432] (5) used these sequences (sense) as a template for the synthesis of a complementary DNA strand (antisense) containing EdU,

[0433] (6) amplified by PCR these sequences and their complementary strand into EdU-containing DNA,

[0434] (7) retrieved the simple strand sequences meaning and

[0435] (8) repeated steps (2) to (7) to enable Darwinian-type molecular evolution enrichment of chemically modified sequences.

[0436] This method is presented in Figure IA.

[0437] Equipment :

[0438] The chemical reactions were controlled using an analytical HPLC system: e2695 separation module and 2998 photodiode array detector (Waters) and an Atlantis reverse-phase HPLC column: dC18, 3 pm, 2.1 mm x 150 mm (Waters). The HPLC data were acquired and processed using Empower 3 software.

[0439] Polyacrylamide gel electrophoresis and dot blotting were performed using the Chemidoc MP imaging system (Bio-Rad). Agarose gels were visualized using the Vilber Lourmat FUSION-Fx7 (Thermo Fisher Scientific). All gels were analyzed with ImageJ software version 1.53k.

[0440] Oligonucleotides were quantified using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). PCR experiments were performed using the Veriti® 96-well thermocycler (model 9902, 0.2 ml) (Applied Biosystems), and qPCR experiments were performed using the Eppendorf Mastercycler® ep gradient S. Sequencing experiments were performed using the iSeqlOO instrument (Illumina).

[0441] Reagents:

[0442] Unless otherwise noted, chemical reagents were purchased from Sigma-Aldrich and molecular biology reagents from Thermo Fisher Scientific. dNTPs, OneTaq Hot Start DNA polymerase, and the nucleoside digestion mix were purchased from New England Biolabs. Single-stranded DNA and streptavidin-agarose beads were purchased from Sigma-Aldrich. Micro Bio-Spin®6 columns were purchased from Bio-Rad. Oligonucleotides (primers and candidate templates) were purchased from Eurogentec. The primer sequences used here, with the exception of those used for HTS, are summarized in Table 7 below:

[0443] [Table 7]

[0444] The bank's starting matrix was purchased from EllaBiotech. The sequence of the purchased model is given in Table 8 below.

[0445] [Table 8]

[0446] N = 20% U, 26.7% A, G and C.

[0447] EdU triphosphate was purchased from Baseclick. Dynabeads™ M-270 Streptavidin and Dynabeads™ M-270 Amine were purchased from Thermo Fisher Scientific. Nucleospin Gel and PCR Clean-up were purchased from Macherey-Nagel.

[0448] Sample preparation for high-throughput sequencing (HTS): HTS sample preparation begins with amplification of the template using an appropriate PCR. This PCR is performed with two primer pairs: adapters (R2P3X and R1N0P5X) and indexes. The adapters are the same for each sample, but the index pair must be different for each sample. The adapter pair used is given in Table 9 below: [Table 9]

[0449] Amplification is performed in a 20 µl PCR using adapters and indexes, natural dNTPs and OneTaq Hot Start polymerase with its standard buffer shown in Table 10 below: [Table 10]

[0450] Unless otherwise indicated, the PCR program is that presented in the following Table 11: [Table 11] The final steps in sample preparation are as follows: each crude PCR product is quantified on a 3% agarose gel and then pooled to obtain an equimolar mixture. This mixture is purified on a 3% agarose gel before being extracted from the agarose gel using the NucleoSpin Gel and PCR Clean-up Kit (ref 740609.250). The purified pool is then quantified on a 3% agarose gel, visualized under UV light, and sequenced using the iSeqlOO instrument.

[0451] Modification of oligonucleotides by CuAAC. The following general procedure was used to perform CuAAC on oligonucleotides. All solvents used in the reaction were degassed with argon before use. First, a catalyst solution was prepared by mixing CuSCU (3 pL of a 100 mM solution in water), THPTA (15 pL of a 100 mM solution in water), DMSO (75 pL), and water (57 pL). The resulting CuSO₄ THPTA (2 mM / 10 mM) DMSO / water (50:50 v / v) solution was incubated for 15 minutes at 37 °C to form the CuI-THPTA complex.

[0452] 200 pmoles of oligonucleotide (bearing one or more alkyne groups) (13.3 pL of a 105 pM alkyne solution), azide (7 pL of a 10 mM DMSO solution), water (3.7 pL), and HEPES (4 pL of a 1 mM water solution) were mixed. 10 pL of the previously prepared CuI-THPTA solution were added to the mixture. Finally, 2 pL of freshly prepared sodium ascorbate (Na. Asc) solution (100 mM in water) were added to obtain 40 pL of reaction mixture (alkyne / azide / CuSO₄ HEPES / THPTA / Na. Asc: 35 pM / 1.75 mM / 100 pM / 500 pM / 2.5 mM) which was incubated at 37°C, 300 revolutions per minute (rpm) for 1 hour.

[0453] The resulting modified bank was purified by ethanol precipitation (centrifugation at 4°C, 20,000 g for 15 min after being placed at -80°C for approximately 1 hour).

[0454] SPICC on oligonucleotides. The following general procedure was used to perform SPICC on iminosydnone-containing oligonucleotides. The modified oligonucleotides were incubated with cycloalkyne to obtain the final reaction mixture (iminosydnone / cycloalkyne: 20 pM / 1 mM). This mixture was incubated for 2 hours at 50 °C at 900 rpm, yielding triazole-urea oligonucleotides. DBCO-amine, DBCO-Peg (also known as m-dPEGn-DBCO), or BCN was used as the cycloalkyne.

[0455] Synthesis of a random DNA library. A random DNA library was synthesized and purified as follows: 10 nmoles of commercial single-stranded synthetic DNA were purified by denaturing polyacrylamide gel electrophoresis containing 6% acrylamide and 7 M urea in sodium-borate IX buffer. 2 nmoles of the library were first extended for 1 hour in a double-stranded DNA template using the HeavyP5X primer and EdU triphosphate instead of thymidine, resulting in a heavier second strand. The double-stranded DNA template was purified by 6% denaturing PAGE in 7 M urea SB IX buffer, recovering the 101-nucleotide fluorescent strand containing the alkyne groups. This library was quantified and used for subsequent steps.

[0456] Optimization of PCR conditions by HTS. PCR conditions were studied on (a) the EdU random DNA library, (b) the Carb random DNA library, (c) the Ind random DNA library, (d) the TdU random DNA library and (e) the Benzyl random DNA library in order to determine those which offer the best fidelity.

[0457] First, 20 pmoles of the random EdU library were modified with CuAAC (see previous paragraph) using Carb, Ind, and Benzyl (benzyl azide) azides to obtain libraries (b), (c), and (e), respectively. After DNA recovery by ethanol precipitation, half of the sample was subjected to the SPICC reaction. HEPES and DBCO-amine were added to 10 pL of DNA to obtain a 15 pL reaction mixture (EdU / HEPES / DBCO-amine: 5 pM / 10 mM / 1 mM).

[0458] The reaction was incubated for 2 hours at 50 °C with stirring (300 rpm). Once again, the DNA was recovered by ethanol precipitation, yielding libraries (d) from two different matrices.

[0459] To analyze the fidelity of the amplification under different conditions, PCR was performed with natural dNTPs and OneTaq in its GC buffer. The adapters and indexes required for Illumina multiplex sequencing were also added to the mixture (see Sample Preparation for High-Throughput Sequencing (HTS)).

[0460] The PCR program parameters were as follows: one cycle consisting of 95 °C for 2 min (initial denaturation), 95 °C for 15 s (denaturation), 57 °C for 30 s (hybridization) and 68 °C for 30 min (elongation), followed by 25 cycles consisting of 95 °C for 15 s (denaturation), 57 °C for 30 s (hybridization) and 68 °C for 1 min (elongation).

[0461] After PCR, 5 µl of each PCR product were pooled to obtain an equivolume mixture instead of the equimolar mixture described. Purification and sequencing preparation were performed in the same manner (see Sample Preparation for High-Throughput Sequencing (HTS)). High-throughput sequencing provided access to the concentration of each base in each sample. Thus, we were able to calculate the percentage of U in each sample and compare it to that of the control, giving us the results in Table 16.

[0462] Selection of DNA aptamers against Streptavidin. Selection was performed against Streptavidin M270 Dynabeads (ref. 65306) which can be isolated from the supernatant using a magnetic separation support (ref. S1506S). The Streptavidin M270 Dynabeads were first equilibrated by 2 washes of 200 pL of selection buffer (TS / IGEPAL selection buffer: 10 mM HEPES pH 7-7.6, 150 mM NaCl, 5 mM KCl, 1.5 mM CaCl2 and 1 mM MgCl2, IGEPAL 0.05%).

[0463] Three SELEX experiments were performed simultaneously: EdU-Selex with the EdU-DNA starting library, Carb-Selex with the Carb-DNA library, and Ind-Selex with the Ind-DNA library. First, the starting library was modified with CuAAC (see the section "Modification of oligonucleotides by CuAAC").

[0464] Before use, 200 pmoles of each library (approximately 10 14Separate sequences) in 200 µl of screening buffer were heated to 85 °C for 5 minutes, cooled on ice for 2 minutes, and left at 37 °C until use to disrupt intermolecular interactions between the sequences. For the RI of each SELEX, the library was incubated for 1 hour at 37 °C with 900 rpm shaking in 20 µl of Streptavidin magnetic beads (10 mg / mL). The supernatant was then collected by isolating the targets using a magnet.

[0465] Unbound aptamers were removed by 2 additional 30-second washes with 200 pL of selection buffer.

[0466] For EdU-Selex, the bound DNA was eluted from the beads by heating the reaction to 85°C for 5 minutes and precipitated with ethanol.

[0467] The elution of bound DNA in Carb-Selex and Ind-Selex consisted of incubating the beads with 100 µL of an equimolar solution of BCN / DBCO-Peg in water and DMSO (1 mM final solution in cycloalkyne, water / DMSO 92.5:7.5 vol / vol). This reaction was incubated for 2 hours at 50 °C with stirring (900 rpm). The resulting triazole-urea (TdU) DNA was recovered by ethanol precipitation and dissolved in 50 µL of water.

[0468] Before performing PCR on all eluted sequences, the appropriate number of PCR cycles for each SELEX was determined using qPCR with 25 pL, engaging only 1 pL of DNA out of 50. qPCR allows for the determination of the cycle threshold (Ct). The number of cycles chosen for large-scale PCR was Ct + 5 cycles. The candidate sequences were then each amplified by 1 ml-PCR (10 reactions of 100 pL) using HeavyP5X and P3X primers and EdU instead of natural thymidine. The PCR program parameters were those obtained during the previous optimization, presented in Tables 12 and 13 below: [Table 12]

[0469] [Table 13]

[0470] After concentration, the PCR product was purified on 6% denaturing PAGE in 7M urea SB IX buffer and the gel was revealed by Chemidoc (BioRad) in Cy5.5 mode allowing recovery of the heaviest fluorescent strand corresponding to candidate sequences and containing alkyne functions.

[0471] After concentration and precipitation, the EdU-DNA candidates of each SELEX were quantified using a NanoDrop. The EdU-SELEX candidates were ready to be directly committed to the next selection cycle, and the Carb-SELEX and Ind-SELEX candidates were modified with their corresponding azide before the start of the next selection cycle.

[0472] Selection pressure was progressively increased over the cycles by increasing the number and duration of washes from two 30-s washes in RI to three 30-s washes and two 5-min washes under 900 rpm stirring in R6. 0.1 pg of ssDNA (ref. D7656) per pL was added as a competitor during selections R5 and R6. The amount of DNA pool decreased from 200 pmoles in RI to approximately 20 pmoles in R6. However, the amount of target was kept constant during the 6 rounds (20 pL of 10 mg / mL magnetic beads). The conditions used for each cycle are shown in Table 14 below: [Table 14]

[0473] High-throughput sequencing of selected DNA candidates. Library preparation for DNA candidate sequencing was performed following the high-throughput sequencing (HTS) sample preparation procedure. However, amplification was carried out in a 20 µL PCR using adapters and indexing sequences, natural dNTPs, and OneTaq Hot Start polymerase with its GC buffer. The PCR program was the one determined during development (1 h elongation in the first cycle).

[0474] High-throughput sequencing allowed access to the concentration of each base in each sample, as well as the enrichment of SELEX over time. The results are shown schematically in Figure 2.

[0475] R07 Screening for evaluating the role and specificity of chemical modifications against streptavidin. The enriched sequences obtained at the end of the 6th round of each SELEX were grouped according to their abundance to form pool R06. This pool was divided to be either left in the EdU chemistry or modified by CuAAC with the Carb and Ind modifications (27 pmoles for each chemistry). To evaluate the specificity of the candidates against streptavidin, a 7th screening round was performed using Streptavidin M270 Dynabeads, Streptavidin-Agarose (ref. SI 638), and Amino M270 Dynabeads (ref. 14307D). Nine SELEX experiments were performed simultaneously, each chemistry with each target.

[0476] A few pmoles from each modified pool were retained for direct sequencing for EdU-SELEX and cycloalkyne treatment prior to sequencing for Carb-SELEX and Ind-SELEX, thus providing 3 control experiments.

[0477] Target balancing was performed as previously described for ROI at R06.

[0478] Before use, the 9 pmoles of each library in 200 µL of screening buffer were heated to 85 °C for 5 minutes, cooled on ice for 2 minutes, and left at 37 °C until use to disrupt intermolecular interactions between sequences. 2 µL of ssDNA were added to the mixture as a competitor. The library was incubated for 1 hour at 37 °C with 900 rpm shaking in either 20 µL of Streptavidine M270 Dynabeads, 100 µL of Streptavidine-Agarose gel, or 20 µL of Amino M270 Dynabeads (the same quantity of beads as Streptavidine M270 Dynabeads). The supernatant was then collected by isolating the targets using a magnet.

[0479] Unbound aptamers were removed by three additional 30-second washes with 200 µL of selection buffer and two 5-minute washes with shaking at 900 rpm in 200 µL of selection buffer. qPCR experiments were performed to determine the appropriate number of PCR cycles required for HTS-PCR. The conditions used for each experiment are shown in Table 15 below:

[0480] [Table 15]

[0481] For EdU-Selex, the bound DNA was eluted from the beads by heating the reaction to 85°C for 5 minutes and precipitated by ethanol.

[0482] The elution of bound DNA in Carb-Selex and Ind-Selex consisted of incubating the beads with 100 µL of an equimolar solution of BCN / DBCO-Peg in water and DMSO (1 mM final solution in cycloalkyne, water / DMSO 92.5:7.5 vol / vol). This reaction was incubated for 2 hours at 50 °C with stirring (900 rpm). The resulting triazole-urea DNA (TDU-DNA) was recovered by ethanol precipitation.

[0483] High-throughput sequencing allowed access to the enrichment based on the samples. The results are shown schematically in Figure 3.

[0484] Candidate Synthesis. The 11 candidates were synthesized and purified as follows: For each sequence, 2 nmol of commercial single-stranded synthetic DNA were purified by denaturing polyacrylamide gel electrophoresis containing 6% acrylamide and 7M urea in sodium-borate IX buffer. The resulting sequences were first extended for one hour into a double-stranded DNA template using the HeavyP5X primer and EdU triphosphate instead of thymidine, resulting in a heavier second strand. The double-stranded DNA template was purified by 6% denaturing PAGE in 7M urea SB IX buffer, recovering the 101-nucleotide fluorescent strand containing the alkyne groups. The sequences were then quantified and used for subsequent steps.

[0485] Screening of aptamer candidates. The candidates were modified with CuAAC (see the section on CuAAC modification of oligonucleotides). Four rounds of screening were performed simultaneously: one with the 11 candidates in EdU chemistry, one with the 11 candidates in Carb chemistry, one with the 11 candidates in Ind chemistry, and one with the 11 candidates modified in their original chemistry. For each pool, each sequence was at a final concentration of 2 pM. The screening conditions were strictly similar to those of R07 (buffer, amount of competitor, target Streptavidin-M270 beads, incubation conditions, number of washes, and elution), as was the recovery of bound DNA. Control experiments were performed for each of the four experiments, consisting of a starting pool appropriately modified for Carb and Ind chemistry, and a pool of mixed chemistries (CuAAC and SPICC). High-throughput sequencing has made it possible to access enrichment based on samples.The results are shown schematically in the diagram.

[0486] Figure 4.

[0487] Synthesis of the C0-doped bank. The synthesis of the C0-doped bank was carried out following exactly the same procedure as that used for the synthesis of the 11 candidates (see the section Synthesis of candidates).

[0488] SELEX experiment with the doped C0 library. 10 pmoles of the doped C0 library modified with EdU or Carb were subjected to a selection cycle under the same conditions as for R07 against Streptavidine-M270 beads. Two controls were used for these experiments: one directly consisting of the initial doped C0 library and the other consisting of the doped C0 library modified with Carb chemistry and directly treated with cycloalkyne (without selection).

[0489] High-throughput sequencing allowed access to the base percentages of the sequences that were enriched in each experiment. The results are shown schematically in Figure 5.

[0490] Affinity measurement. The affinity measurement for C0 was performed by quantifying the aptamer remaining bound to the target after selecting a constant amount of aptamer on different concentrations of streptavidin.

[0491] The target was first equilibrated by washing 50 µL of selection buffer. Before use, 10 pmoles of carb-modified CO₂ in 50 µL of selection buffer were heated to 85 °C for 5 minutes, cooled on ice for 2 minutes, and left at 37 °C until use to disrupt intermolecular interactions between sequences. 0.5 µL of ssDNA was added to the mixture as a competitor. The library was incubated for 1 hour at 37 °C with 900 rpm shaking using 0, 1, 2, 4, 8, 10, 15, or 20 µL of Streptavidin M270 Dynabeads. The supernatant was then collected by isolating the targets using a magnet.

[0492] Unbound aptamers were removed by two additional 30-second washes with 50 µl of selection buffer.

[0493] Elution of the bound DNA consisted of incubating the beads with 50 µL of an equimolar solution of BCN / DBCO-peg in water and DMSO (1 mM final solution in cycloalkyne, water / DMSO 92.5:7.5 vol / vol). This reaction was incubated for 2 hours at 50 °C with shaking (900 rpm). The resulting triazole-urea DNA (TDU-DNA) was recovered by ethanol precipitation. The same experiment was performed using the unmodified control sequence C0, i.e., EdU-C0.

[0494] The eludons were analyzed by dot blot: the collected solutions were transferred onto a positively charged nylon transfer membrane (Hybond® from Amersham, GE Healthcare) by aspiration. The membrane was then visualized using Chemidoc in Cy5.5 mode to quantify the DNA in each experiment. The eluted DNA was quantified using ImageJ software. The percentage of bound DNA was set at 100% in the two experiments without Streptavidin. The percentage of bound DNA for each experiment was calculated relative to these two controls. The values ​​were plotted on a graph showing the affinity (given as the percentage of bound DNA relative to the control) for each experiment using a different Streptavidin concentration (Figure 6).

[0495] Results

[0496] We have demonstrated the validity of the method described in Figure IA and its generic nature, using two chemical groups: Azido-C3-amino-iminosydnone-benzyl-carbazole (compound 4a) and Azido-C3-amino-iminosydnone-benzyl-indole (compound 4b). These two molecules are shown in Figure IC. These chemical groups were able to be covalently conjugated by a copper(I)-catalyzed azide-alkyne (CuAAC) bioorthogonal cycloaddition reaction on 5-ethynyl-2'-deoxyuridines (EdU) contained in DNA sequences to generate 5-triazole-C3-amino-iminosydnone-benzyl-Carbazole-2'-deoxyuridines (Carb-dU) and 5-C3-amino-iminosydnone-benzyl-indole-2'-deoxyuridines (Ind-dU), respectively.Then, these groups could be cleaved by a bioorthogonal SPICC (Strain-Promoted Iminosydnone-Cycloalkyne Cycloaddition) reaction using dibenzocyclooctyne-amine (DBCO-amine) to produce DNA containing 5-triazole-C3-urea-uri dines (TdU) (see Figure IB).

[0497] First, we verified that a DNA library containing EdU (with a random 35-nucleotide region) could be conjugated with both compounds (4a, 4b) using the CuAAC bioorthogonal reaction. To this end, the library was digested into single nucleosides before and after the conjugation reaction, and the conjugation yield was calculated by HPLC analysis. Under our conditions, we estimated that approximately 70% of the EdU reacts with compounds 4a and 4b, respectively (data not shown).

[0498] We then used the same method to verify that the conjugated reagents could be cleaved by a bioorthogonal SPICC reaction to provide almost 100% DNA containing TdU in the end (data not shown).

[0499] The possibility of polymerase chain reaction (PCR) amplification of a DNA library containing modified nucleotides of the EdU, Carb-dU, Ind-dU, TdU, and 5-triazole-benzyl-2'-deoxyuridine (5-triazole-benzyl-dU, used as a positive control because this small modification can be amplified by PCR and has already been used for the selection of chemically modified aptamers, see Pfeiffer et al. 2018) was tested using OneTaq from New England Biolabs, a mixture of Taq and Deep Vent DNA polymerases, with its GC buffer. For all PCRs, we performed a 30-minute initial replication phase to increase the chances of synthesizing a complementary DNA strand (cDNA) from the chemically modified DNA template. This first phase is the rate-limiting reaction, because, once produced, the cDNA can be easily amplified by PCR.

[0500] The results are presented in Table 16 below and clearly show that:

[0501] - As expected, 5-triazole-benzyl-2'-deoxyuridine is well tolerated and the PCR products retain under most PCR conditions the same percentage of uridine as the starting EdU library.

[0502] In the absence of SPICC cleavage, PCR of libraries containing Carb-dU-modified uridine resulted in a reduction of at least 20% of the uridines, suggesting that polymerases introduce mutations to change the carbazole-conjugated uridine to another base, or that sequences containing fewer carbazole-conjugated uridines are more likely to be copied into cDNA. The same phenomenon is observed with indole-modified uridine (Ind-dU).

[0503] - On the other hand, when the two modifications were cleaved using the SPICC reaction, the TdU-containing DNA was well amplified, with the percentage of uridines not being significantly reduced (less than 7%).

[0504] This demonstrates that TdU-modified DNA (produced by cleavage via the SPICC reaction) can be efficiently copied into cDNA by the OneTaq polymerase in its GC buffer. These results also demonstrate the advantage of cleavage, since the same sequences before cleavage (modified with carbazole or indole) are much more difficult to replicate.

[0505] [Table 16]

[0506] Following the proof of concept above, we performed molecular evolutions to select ligands capable of binding to streptavidin-coated M270 magnetic beads (Thermo company). Each selection was made using a library of 10 15DNA sequences exhibiting various chemical modifications were selected. Two selections were performed using our method, one with Carb-dU-containing DNA and the other with Ind-dU-containing TdNA (referred to as Carb-SELEX and Ind-SELEX, respectively). A conventional selection using unconjugated EdU-containing DNA (EdU-SELEX) was also performed. Unlike conventional EdU-SELEX, where streptavidin-bound sequences are recovered by heating at 95°C, we used the SPICC reaction to elute the aptamers in the other two selections. We reasoned that cleaving their chemical modification would alter their structure and lead to a loss of affinity for the target, while also providing TdU-containing DNA that could be amplified by PCR. High-throughput sequencing was used to track library evolution over six molecular evolution cycles (Figure 2).

[0507] For each selection, the frequency of several sequences increased considerably over the cycles. We chose to focus on sequences with a frequency greater than 0.005% (50 copies per million) in the library after one cycle of SELEX. These sequences were grouped into families based on their similarity. We observed that one family (named C0) was particularly enriched after four cycles of Carb-SELEX, representing up to 98% of the library in the final round (Figure 2). Several families were also enriched in EdU-SELEX and Ind-SELEX, where the most abundant family represented 73% and 6% of the library, respectively, in the final cycle. With the exception of the El family, which appears to be enriched in both In-SELEX and especially EdU-SELEX, different families were selected for each SELEX, demonstrating that each chemistry enabled the identification of different sequences.

[0508] It is worth noting that prior to the enrichment of certain families, we observed that the frequency of uridines in the libraries did not decrease in the first cycles, regardless of the chemistry used (Figure 2). This frequency even increased slightly in the first cycles of Carb-SELEX.

[0509] These results confirm that our method is capable of amplifying sequences independently of how they have been chemically modified for selection.

[0510] To validate these enrichments, we conducted a series of experiments. First, we mixed the libraries from the last cycle of each SELEX so that the most abundant families in each SELEX had approximately the same frequency in the mixture. This mixture was divided into three parts. The first part remained unconjugated with DNA sequences containing EdU, while the other two parts were conjugated by the CuAAC bioorthogonal reaction to contain either DNA sequences containing Carb-dU or DNA sequences containing Ind-dU. Each part was incubated with streptavidin-coated M270 magnetic beads, and the sequences retained on the beads after several washes were recovered either by heating at 95°C for the EdU-containing DNA or by SPICC reaction for the other two chemistries. The recovered sequences were then amplified and analyzed by NGS.A very strong enrichment of certain DNA families was observed depending on their chemistry. It is striking to note that the families highly enriched when the mixture contains DNA sequences with Carb-dU (CO, C12, and Cl8) correspond to those previously enriched in Carb-SELEX. Similarly, the families highly enriched when the mixture contains DNA sequences with Ind-dU (16, Il1, and 113) correspond to those previously enriched in Ind-SELEX, and the families highly enriched when the mixture contains DNA sequences with EdU (El to El5) correspond to those previously enriched in EdU-SELEX (Figure 3). A similar experiment was performed against streptavidin immobilized on beads of a different nature (agarose-streptavidin beads) or against the same type of beads but without streptavidin (M270 magnetic beads with amine groups).The same experiments were also performed without beads to see if certain sequences are preferentially amplified by PCR. NGS analysis showed that after selection without beads or with M270 magnetic beads containing amine groups, the frequency of each family in the library remained approximately the same regardless of the chemistry used, and no sequence showed a significant increase in frequency compared to the others (data not shown). These results suggest that no sequence has a significantly higher affinity for M270 beads with amine groups or a higher PCR amplification yield.

[0511] Similar results were also obtained using a library consisting of an equimolar mixture of 11 candidate aptamers instead of a mixture of libraries from SELEX (Figure 4).

[0512] All these results confirm that each SELEX has made it possible to identify aptamers capable of binding to streptavidin and that their binding depends on the chemical modifications used to select them.

[0513] To further confirm the chemistry dependence on aptamer binding, we synthesized a library of 64 CO aptamer variants with mutations at positions corresponding to uridines: 25, 32, and 51 (Figure 5). When the EdU DNA variant library was conjugated with reagent 4a via the CuAAC bioorthogonal reaction and then transformed into a TdU DNA library via the SPICC bioorthogonal reaction, the percentage of uridines remained constant regardless of position. This again demonstrates the unbiased amplification of TdU DNA sequences following a conjugation and cleavage process. This library of 64 variants was then split into two parts. The first part remained in DNA chemistry with EdU while the other part was conjugated with reagent 4a by the bioorthogonal CuAAC reaction (allowing us to obtain variants with Carb-dU).Each library with a different chemistry was then incubated with streptavidin-coated M270 magnetic beads, and the sequences retained on the beads after several washes were recovered either by heating at 85°C for EdU-coated DNA or by the SPICC reaction for Carb-dU-coated DNA. The recovered sequences were then amplified and analyzed by NGS (Figure 5).

[0514] When variants are in DNA with EdU, the frequency of each variant remains virtually unchanged in the library, suggesting that none of these variants has a higher affinity for the beads compared to the others. In contrast, when variants are in DNA with Carb-dU, variants containing uridines were significantly more amplified, demonstrating that the Carb-dU modification is important for binding to streptavidin beads. Only variants with a single substitution of a uridine by a guanine or adenine at position 51 were enriched, but only half as much as the unmutated aptamer. This indicates that carbazole could be removed from this position, but this would still reduce the aptamer's affinity. This also indicates that the aptamer's affinity is highly dependent on the presence of Carb-dU at positions 25 and 32.We have also observed that the modification at position 32 appears to be crucial for the interaction, as the frequency of all variants with a uridine substitution at this position has been considerably reduced, by at least -90% (Figure 5).

[0515] Finally, we confirmed that the C0 aptamer had a significantly higher affinity for streptavidin-coated M270 magnetic beads when in Carb-dU chemistry compared to EdU chemistry (Figure 6). This demonstrates that we were able to use our method to identify a DNA aptamer containing 5-triazole-C3-amino-iminosydnone-benzyl-Carbazole-2'-deoxyuridines (Carb-dU), while we showed that DNA sequences containing this chemical modification are poorly replicated by DNA polymerases (see Table 16 above).

[0516] Conclusion

[0517] The results presented above show that a DNA polymerase is able to efficiently and unbiasedly use as a template DNA strand a DNA comprising nucleotides (here, uridines, but it is possible to use similar modifications for other bases) modified (TdU) with a limited chemical group (5-triazole-C3-urea-), resulting from the SPICC reaction cleavage of much bulkier chemical groups (5-triazole-C3-amino-iminosydnone-benzyl-Carbazole- of Carb-dU and 5-C3-amino-iminosydnone-benzyl-indole- of Ind-dU) comprising a linker cleavage by SPICC. These modified nucleotides (TdU), as well as the corresponding nucleosides and nucleobases, are therefore an object of the present invention, as are methods for reverse transcription of RNA (since a similar result can be expected with reverse transcriptases) and for amplification of DNA (or RNA) comprising these modified nucleotides.

[0518] On this basis, it is now possible to select aptamers, or any other type of functional nucleic acid, comprising nucleotides chemically modified by any group of interest, provided that this is attached to the modified nucleotide via a group cleavable by SPICC.

[0519] EXAMPLE 2

[0520] Materials and methods

[0521] The materials and methods used are the same as in Example 1, except that in addition to a natural dUTP and the EdUTP (alkyne directly at the C5 position of the base) described in Example 1, another modified dUTP, called C8-EdUTP, was used. In this modified dUTP, the final alkyne is separated from the C5 position of the base by an 8-carbon alkyl spacer. This was ordered from BaseClick.

[0522] - Several different DNA polymerases were tested in addition to One Taq from New England Biolabs (NEB; ref M0481S):

[0523] Vent, ordered from New England Biolabs (ref M0254S),

[0524] Deep Vent: ordered from New England Biolabs (ref M0258S), PWO: ordered from Sigma-Aldrich (ref 11644955001), and Q5: ordered from New England Biolabs (ref M0491S).

[0525] Results

[0526] The PCR efficiency observed with the three types of nucleotide triphosphates (natural dUTP, EdUTP and C8-EdUTP) and the different DNA polymerases is shown in Figure 7.

[0527] The results obtained show that the different DNA polymerases tested are able to incorporate EdUTP with an efficiency generally greater than the incorporation of natural UTP, and are able to incorporate C8-EdUTP with an efficiency that varies depending on the DNA polymerase and amplification buffer used, but sufficient to allow amplification by PCR with this modified nucleotide.

[0528] Based on these results and those presented in Example 1 in a SELEX-type selection using coupling and decoupling based on bioorthogonal chemistry reactions with EdUTP, we can expect that a SELEX-type selection using coupling and decoupling based on bioorthogonal chemistry reactions with C8-EdUTP (or another similarly modified type of dNTP) is also possible.

[0529] Bibliographical references

[0530] EP2074211,

[0531] EP3201353,

[0532] US9228179B2,

[0533] US9988623B2,

[0534] US10125162B2,

[0535] US10221207B2,

[0536] US10239908B2,

[0537] US10316321B2,

[0538] US2021 / 0171951A1,

[0539] WO20 15 / 084846,

[0540] WO20 18 / 152470,

[0541] Bernard S., Audisio D., Riomet M., Bregant S., Sallustrau A., Plougastel L., Decuypere E., Gabillet S., Kumar R. A., Elyian J., Nguyet Trinh M., Koniev O., Wagner A., Kolodych S., Taran F., Angew. Chem. Int. Ed. 2017, 56, 15612-15616,

[0542] Pfeiffer F, Toile F, Rosenthal M, Brandie GM, Ewers J, Mayer G. Identification and characterization of nucleobase-modified aptamers by click-SELEX. Nat Protoc 2018;13: 1153-80.

Claims

DEMANDS 1. Modified nucleoside consisting of a modified nucleobase bonded to the anomeric carbon atom at position 1' of a pentose residue, wherein the modified nucleobase is a nucleobase in which a hydrogen atom is replaced by Ro or a nitrogen atom is replaced by CRo, where Ro is a substitution group of formula -A0-X1-A1-Y1-R1 or -A0-X1-A1-Y2, in which: - Ao represents a single bond or a Ci-10-alkanediyl chain possibly preceded and / or interrupted and / or followed and / or replaced by one or more motifs chosen from the group consisting of -C=C-, -C(R2)=C(R3)-, -O-, -S-, - Xi represents a group chosen from connection with Ao and the dotted line indicates the point of connection with Ai; - Ai represents a Ci-20-alkanediyl chain possibly preceded and / or interrupted and / or followed and / or replaced by one or more motifs chosen from the group consisting of -C=C-, - C(R8)=C(R9)-, -O-, -S-, -NR10-, -C(O)-, -C(S)-, and -C(NRn)-; - Yi represents a group , in which X2 represents -NH- CO-, -O-CO-, -SO2-, or -P(O)(Ci-6-alkyl)-, the -NH-CO- and -O-CO- groups being linked to Ai by the NH or O motif respectively and to the rest of the Yi group by the CO motif, R31 represents a hydrogen (H) or a halogen, the dotted line indicates the point of bonding with Ai and the wavy line indicates the point of bonding with Ri; - Ri represents a group of formula A2-R30, in which A2 is a single bond or a Ci-20-alkanediyl chain possibly preceded and / or interrupted and / or followed and / or replaced by one or more motifs selected from the group consisting of an arylene, a C3-12- divalent carbocycle, -OC-, -C(Ri2)=C(Ri3)-, -O-, -S-, -NR14-, -C(O)-, -C(S)-, and -C(NRis)-, and R30 is a Ci-20-alkanediyl aryl, Cs-n-carbocyclyl, heteroaryl, or heterocycle group, said group possibly being substituted by one or more groups selected from ORIÔ, SR17, SOR18, SO2R19, OSO2R20, SO3R21, NR22R23, CO2R24, CONR25R26, NH-(C=NH)-NH2, N + R27R28R29G , NH-(C=NH2 + G2')-NH2, CO2 G3 + , SO3 G4 +, a metal complex derivative, an amino acid derivative, a cyanine derivative, a rhodamine derivative, a BODIPY derivative, a fluorophore group, and a photobridging group, or R30 is a group selected from ORIÔ, SR17, SOR18, SO2R19, OSO2R20, SO3R21, NR22R23, CO2R24, CONR25R26, NH-(C=NH)- NH2, N + R27R28R29G , NH-(C=NH2 + G2)-NH2, CO2G3 + , SO3G4 + , a metal complex derivative, an amino acid derivative, a cyanine derivative, a rhodamine derivative, a BODIPY derivative, a fluorophore group, and a photobridging group; - R2 to R29 each represent, independently of each other, H or Ci-6-alkale; - Gf and G2 represent, independently of each other, a negatively charged counter-ion; - G3 and G4 + represent, independently of each other, a positively charged counterion; and \,x2^ / NH - Y2 represents ' in which the dotted line indicates the point of connection with Ai.

2. Modified nucleoside according to claim 1 wherein Ai represents a C1-10- alkanediyl chain optionally preceded and / or interrupted and / or followed and / or replaced by one or more motifs selected from the group consisting of -OC-, -C(R8)=C(R9)-, -O-, -S-, -NR10-, -C(O)-, -C(S)-, and -C(NRn)-.

3. Modified nucleoside according to claim 1 in which Ai represents -(CH2) m o- with mO representing an integer from 1 to 20, in particular from 1 to 10; or a chain - (O)mi(CH2CH2O)m2(CH2CH2)m3- with ml = 0 or 1, m3 = 0 or 1 and m2 representing an integer from 1 to 10 with the condition that m2+m3 does not exceed 10, very preferably Ai represents -(CH2) m o- with mO representing an integer from 1 to 20, in particular from 1 to 10, such as 3.

4. A modified nucleoside according to any one of claims 1 to 3, wherein Ao represents a single bond or a Ci-io-alkanediyl chain optionally preceded and / or interrupted by one or more motifs selected from the group consisting of -C=C- and -O-, preferably -C=C-, 5. A modified nucleoside according to any one of claims 1 to 4, wherein Ao represents a single bond; a Ci-io-alkanediyl chain optionally preceded and / or interrupted by one or more -C=C- motifs; or a -(O) chain n i(CH2CH2O) n 2(CH2CH2) n 3- with ni = 0 or 1, n3 = 0 or 1 and n2 representing an integer from 1 to 5 with the condition that n2+n3 does not exceed 5.

6. Modified nucleoside according to any one of claims 1 to 5 in which Ao is selected from the group consisting of -OC-, and -C=C-(CH2)4-C=C-, 7. A modified nucleoside according to any one of claims 1 to 6, wherein Xi represents a group selected from , in which the wavy line indicates the point of connection with Ao and the dotted line indicates the point of connection with Ai.

8. Modified nucleoside according to any one of claims 1 to 7, wherein Y2 represents 9. Nucleoside modified according to any one of claims 1 to 8, wherein the substitution group is of formula -A0-X1-A1-Y1-R1 and R30 is a heteroaryl.

10. Modified nucleoside according to any one of claims 1 to 9, wherein the modified nucleobase is derived: (a) of a pyrimidine, advantageously of a uracil or a cytosine; or (b) of a purine, advantageously of an adenine or a guanine.

11. Modified nucleoside according to claim 10, wherein: (a) in the case of a nucleobase derived from a pyrimidine, the nucleobase is modified by replacing the hydrogen atom attached to the carbon in position 5 of the pyrimidine ring with Ro; or (b) in the case of a nucleobase derived from a purine, the nucleobase is modified by replacing the nitrogen atom located at position 7 of the purine ring with CRo.

12. Modified nucleoside according to any one of claims 1 to 11 wherein the pentose residue is selected from deoxyribose residues, ribose residues and ribose derivatives.

13. Modified nucleotide consisting of a nucleoside modified according to any one of claims 1 to 12 wherein the OH group in position 5' of the pentose residue is replaced by an -O-(Z3i) group q i-(Z32)q2-Z33-H or a salt thereof for which: - q1 and q2 independently represent 0 or 1, and - Z31, Z32 and Z33 represent, independently of each other, a phosphate group - P(O)(OH)-O- or a derivative of a phosphate group advantageously chosen from -P(O)(SH)- O-, -P(O)(CH3)-O-, -P(S)(OH)-O-, -P(S)(SH)-O-, and -P(S)(CH3)-O-, 14. Modified nucleic acid comprising at least one nucleotide modified according to claim 13.

15. Modified nucleic acid according to claim 14, which is a functional nucleic acid, advantageously selected from nucleic acids capable of binding specifically to a given ligand such as aptamers and catalytic nucleic acids such as ribozymes and deoxyribozymes.

16. Modified nucleic acid according to claim 14 or claim 15, which is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA).

17. A method for reverse transcribing into DNA an RNA modified according to claim 16, wherein Ro is a substitution group of formula -A0-X1-A1-Y2, where A0, X1, A1, and Y2 are as defined in any one of claims 1 to 8, comprising contacting the RNA with a reverse transcriptase in the presence of a primer and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under appropriate conditions.

18. A method for amplifying a modified RNA or DNA according to claim 16, wherein Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined in any one of claims 1 to 8, comprising the following steps: a) where the nucleic acid to be amplified is a modified RNA in which Ro is a substitution group of formula -A0-X1-A1-Y2, where Ao, Xi, Ai and Y2 are as defined in any one of claims 1 to 8, the reverse transcription of the RNA into DNA by contacting it with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under appropriate conditions;b) contacting the DNA obtained in step a) or the DNA modified according to claim 16, wherein Ro is a substitution group of formula -A0-X1-A1-Y2, where A0, Xi, Ai, and Y2 are as defined in any one of claims 1 to 8, with a DNA polymerase in the presence of a sense primer capable of hybridizing to the 5' end of one of the two DNA strands, an antisense primer capable of hybridizing to the 5' end of the other DNA strand, the sense primer comprising a sequence for generating a promoter for an RNA polymerase when the nucleic acid to be amplified is RNA modified according to claim 16, wherein Ro is a substitution group of formula -A0-X1-A1-Y2, where A0, Xi, Ai, and Y2 are as defined in any one of claims 1 to 8, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;and c) DNA amplification by polymerase chain reaction comprising the following substeps:; (i) a DNA denaturation step enabling the separation of the two DNA strands, during which the mixture obtained in step b) is advantageously heated to a temperature of 90°C to 100°C; (ii) a step of pairing the sense and antisense primers to the separated DNA strands, during which the mixture obtained in substep (i) is advantageously heated to a temperature of 45°C to 72°C, advantageously from 45°C to 65°C; (iii) an elongation step of a complementary strand of each DNA strand comprising a primer by DNA polymerase, during which the mixture obtained in substep ii) is advantageously heated to a temperature of 65°C to 80°C; d) Optionally, N iterations of step c), N being an integer greater than 1, in which for each iteration n (l <n<N-l), la sous-étape (i) de l’itération n+1 est mise en œuvre sur le mélange obtenu à la sous-étape (iii) de l’itération n au lieu du mélange obtenu à l’étape b), et e) lorsque l’acide nucléique à amplifier est un ARN, la transcription des ADN amplifiés à l’étape c) et éventuellement à l’étape d) en ARN avec une ARN polymérase en présence d’un mélange de ribonucléotides comprenant trois groupements phosphate ou dérivé de phosphate dans des conditions appropriées.

19. A method according to claim 18, wherein: - one of the deoxyribonucleotides comprising three phosphate groups or phosphate derivatives of the mixture added in step b) comprises a nucleobase in which a hydrogen atom is replaced by a Ro or -Ao-Xn group or in which a nitrogen atom is replaced by a CRo or C-Ao-Xn group where Ro and Ao are as defined in claim 1 and where Xn represents - C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR7=CH2, RÔ and R7 being as defined in claim 1; and / or - one of the ribonucleotides comprising three phosphate groups or phosphate derivatives of the mixture used for transcription in step e) comprises a nucleobase in which a hydrogen atom is replaced by a Ro or -Ao-Xn group or in which a nitrogen atom is replaced by a CRo or C-Ao-Xn group where Ro and Ao are as defined in claim 1 and where Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR7=CH2, RÔ and R7 being as defined in claim 1.

20. A method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a library of natural or modified DNA according to claim 16, wherein Ro is replaced by a group of the formula -Ao-Xn, where Ao is as defined in claim 1 and Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR7=CH2, RÔ and R7 being as defined in claim 1, the sequence of which comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that: - the sequence of the region at the 5' end is known and identical for all DNA, - the sequence of the region at the 3' end is known and identical for all DNA, and - the sequence of the central region is random; b) systematically when the library comprises natural DNA and optionally when the library comprises modified DNA as defined in step a), amplification by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate or phosphate-derived groups, one of which comprises a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group, where Ao is as defined in claim 1 and where Xn represents -C=CH, -N3, -C(O)RO, -O-NH2, -NH-NH2, -SH or -CR?=CH2, RÔ and R7 being as defined in claim 1, c) then, when Xn is -C=CH, -N3, -SH or -C(R?)=CH2,the coupling reaction of the DNA obtained in step a) or b) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R?)-AI-YI-RI, OR SH-A1-Y1-R1 respectively; when Xn is -CO-RÔ, the coupling reaction of the DNA obtained in step a) or b) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1; and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the DNA obtained in step a) or b) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified DNA bearing a substitution group of formula -A0-X1-A1-Y1-R1, Ai, Xi, Yi and Ri being as defined in claim 1; d) the selection of modified DNA bearing a substitution group of formula -A0-X1-A1-Y1-Ri having the function of interest; e) the cleavage of the Yi group to give modified DNA bearing a substitution group of formula -A0-X1-A1-Y2, Y2 being as defined in claim 1; f) optionally, N iterations of steps a) to e),where N is an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN modifiés obtenu à l’étape e) de l’itération n ; et g) le séquençage des ADN obtenus à la fin de l’étape de) ou, lorsqu’elle est présente, à la fin de l’étape f)., 21. A method for selecting modified functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises three distinct regions: a 5' end region, a central region, and a 3' end region, characterized in this that : - the sequence of the region at the 5' end is known and identical for all DNA, - the sequence of the region at the 3' end is known and identical for all DNA, and - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives of which one ribonucleotide comprises a nucleobase of which a hydrogen atom is replaced by an -Ao-Xn group or of which a nitrogen atom is replaced by a C-Ao-Xn group where Ao is as defined in claim 1 and where Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR7=CH2, RÔ and R7 being as defined in claim 1, under appropriate conditions; d) when Xn is -C=CH, -N3, -SH or -C(R7)=CH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R7)-AI-YI-Ri, or SH-A1-Y1-R1 respectively; when Xn is -CO-RÔ, the coupling reaction of the RNAs obtained in step c) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1;and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1, Ai, Xi, Yi and Ri being as defined in claim 1; e) the selection of modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri having the function of interest; f) the cleavage of the Y1 group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2, Y2 being as defined in claim 1; (g) reverse transcription of modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 into DNA by contact with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivative under appropriate conditions;h) Optionally, N iterations of steps a) to g), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape; a) of iteration n+1 corresponds to the mixture of nucleic acids obtained in substep (iii) of step g) of iteration n; and g) the sequencing of the DNAs obtained at the end of step g) or, where present, at the end of step h).

22. A method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that: - the sequence of the region at the 5' end is known and identical for all DNA, - the sequence of the region at the 3' end is known and identical for all DNA, and - the sequence of the central region is random;b) amplification of the DNAs from the library provided in step a) by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives, of which one deoxyribonucleotide comprises a nucleobase in which a hydrogen atom is replaced by a Ro group or in which a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -Ao-Xi-Ai-Yi-Ri, wherein Ao, Xi, Ai, Yi, and Ri are as defined in claim 1 or claim 9, to give modified DNAs bearing a substitution group of formula - A0-X1-A1-Y1-R1, c) the selection of modified DNAs carrying a substitution group of formula -A0-X1-A1-Y1- Ri having the function of interest;d) cleavage of the Yi group to give modified DNA bearing a substitution group of formula -A0-X1-A1-Y2, Y2 being as defined in claim 1; e) optionally, N iterations of steps a) to d), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à la sous-étape (iii) de l’étape d) de l’itération n ; et f) le séquençage des ADN obtenus à la fin de l’étape d) ou, lorsqu’elle est présente, à la fin de l’étape e).; 23. A method for selecting modified functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that: - the sequence of the region at the 5' end is known and identical for all DNA, - the sequence of the region at the 3' end is known and identical for all DNA, and - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives, of which one ribonucleotide comprises a nucleobase in which a hydrogen atom is replaced by a Ro group or in which a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -A0-X1-A1-Y1-R1, in which Ao, Xi, Ai, Yi and Ri are as defined in claim 1 or claim 9 under suitable conditions to give RNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri; d) the selection of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y1-Ri having the function of interest; e) cleavage of the Yi group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2, Y2 being as defined in claim 1;f) reverse transcription of modified RNAs bearing a substitution group of the formula -A0-X1-A1-Y2 into DNA by contact with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under suitable conditions; g) optionally, N iterations of steps a) to f), N being an integer greater than or equal to 1, wherein for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à l’étape f) de l’itération n ; et h) le séquençage des ADN obtenus à la fin de l’étape f) ou, lorsqu’elle est présente, à la fin de l’étape g).; 24. A method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a library of natural or modified DNA according to claim 16, wherein Ro is replaced by a group of the formula -Ao-Xn, where Ao is as defined in claim 1 and Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR=CH2, RÔ and R7 being as defined in claim 1, the sequence of which comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that: - the sequence of the region at the 5' end is known and identical for all DNA, - the sequence of the region at the 3' end is known and identical for all DNA, and - the sequence of the central region is random; b) systematically when the library comprises natural DNA and optionally when the library comprises modified DNA as defined in step a), amplification by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate or phosphate-derived groups, one of which comprises a nucleobase in which a hydrogen atom is replaced by an -Ao-Xn group or in which a nitrogen atom is replaced by a C-Ao-Xn group, where Ao is as defined in claim 1 and where Xn represents -C=CH, -N3, -C(O)RO, -O-NH2, -NH-NH2, -SH or -CR?=CH2, RÔ and R7 being as defined in claim 1, c) then, when Xn is -C=CH, -N3,-SH or -C(R7)=CH2, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R7)-AI-YI-RI, OR SH-A1-Y1-R1 respectively; when Xn is -CO-RÔ, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1; and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the DNAs obtained in step a) or b) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y1-R1, Xi, Ai, Yi and Ri being as defined in claim 1; e) the cleavage of the Yi group to give modified DNAs bearing a substitution group of formula -A0-X1-A1-Y2, Y2 being as defined in claim 1; d) the selection of modified DNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; f) Optionally, N iterations of steps a) to e), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN modifiés obtenu à l’étape e) de l’itération n ; et g) le séquençage des ADN obtenus à la fin de l’étape de) ou, lorsqu’elle est présente, à la fin de l’étape f).

25. A method for selecting functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that: - the sequence of the region at the 5' end is known and identical for all DNA, - the sequence of the region at the 3' end is known and identical for all DNA, and - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives of which one ribonucleotide comprises a nucleobase of which a hydrogen atom is replaced by an -Ao-Xn group or of which a nitrogen atom is replaced by a C-Ao-Xn group where Ao is as defined in claim 1 and where Xn represents -C=CH, -N3, -C(O)RÔ, -O-NH2, -NH-NH2, -SH or -CR7=CH2, RÔ and R7 being as defined in claim 1, under appropriate conditions; d) when Xn is -C=CH, -N3, -SH or -C(R7)=CH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula N3-A1-Y1-R1, CH=C-Ai-Yi-Ri, CH2=C(R7)-AI-YI-Ri, or SH-A1-Y1-R1 respectively; when Xn is -CO-RÔ, the coupling reaction of the RNAs obtained in step c) with a molecule of formula NH2-O-A1-Y1-R1 or NH2-NH-A1-Y1-R1;and when Xn is -O-NH2 or -NH-NH2, the coupling reaction of the RNAs obtained in step c) with a molecule of formula RÔ-CO-AI-YI-RI, to give modified RNAs bearing a substitution group of formula -Ao-Xi-Ai-Yi-Ri, Xi, Ai, Yi and Ri being as defined in; claim 1; e) cleavage of the Yi group to give modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2, Y2 being as defined in claim 1; f) selection of modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; g) reverse transcription of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 into DNA by contacting them with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under suitable conditions; h) optionally, N iterations of steps a) to g), N being an integer greater than or equal to 1, wherein for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’acides nucléiques obtenu à la sous-étape (iii) de l’étape g) de l’itération n ;and g) the sequencing of the DNA obtained at the end of step g) or, where present, at the end of step h).; 26. A method for selecting modified functional DNA having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that: - the sequence of the region at the 5' end is known and identical for all DNA, - the sequence of the region at the 3' end is known and identical for all DNA, and - the sequence of the central region is random; b) amplification of the DNAs from the library provided in step a) by polymerase chain reaction of the DNAs from the library provided in step a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives, of which one deoxyribonucleotide comprises a nucleobase in which a hydrogen atom is replaced by a Ro group or in which a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -A0-X1-A1-Y1-R1, wherein A0, Xi, Ai, Yi, and Ri are as defined in claim 1 or claim 9, to give modified DNAs bearing a substitution group of formula - A0-X1-A1-Y1-R1, c) cleavage of the Yi group to give modified DNA bearing a substitution group of formula -A0-X1-A1-Y2, Y2 being as defined in claim 1; d) selection of modified DNA bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; e) optionally, N iterations of steps a) to d), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à la sous-étape (iii) de l’étape d) de l’itération n ; et f) le séquençage des ADN obtenus à la fin de l’étape d) ou, lorsqu’elle est présente, à la fin de l’étape e).

27. A method for selecting functional RNAs having a function of interest, comprising the following steps: a) providing a DNA library whose sequence comprises 3 distinct regions: a 5' end region, a central region and a 3' end region, characterized in that: - the sequence of the region at the 5' end is known and identical for all DNA, - the sequence of the region at the 3' end is known and identical for all DNA, and - the sequence of the central region is random; b) amplification by polymerase chain reaction of the DNAs obtained in substep (a) in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, the sense primer comprising a sequence enabling the generation of a promoter for an RNA polymerase, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives;c) the transcription of the DNA obtained in step b) into RNA with an RNA polymerase in the presence of a mixture of ribonucleotides comprising three phosphate groups or phosphate derivatives, of which one ribonucleotide comprises a nucleobase in which a hydrogen atom is replaced by a Ro group or in which a nitrogen atom is replaced by a CRo group, where Ro is a substitution group of formula -A0-X1-A1-Y1-R1, in which Ao, Xi, Ai, Yi and Ri are as defined in claim 1 or claim 9 under suitable conditions to give RNA bearing a substitution group of formula -A0-X1-A1-Y1-Ri; e) the cleavage of the Yi group to give modified RNA bearing a substitution group of formula -A0-X1-A1-Y2, Y2 being as defined in claim 1; d) the selection of modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 having the function of interest; f) the reverse transcription of the modified RNAs bearing a substitution group of formula -A0-X1-A1-Y2 into DNA by contacting them with a reverse transcriptase in the presence of a primer and deoxyribonucleotides comprising three phosphate groups or phosphate derivatives under suitable conditions; g) optionally, N iterations of steps a) to f), N being an integer greater than or equal to 1, in which for each iteration n (l <n<N-l), la bibliothèque fournie à l’étape a) de l’itération n+1 correspond au mélange d’ADN obtenu à l’étape f) de l’itération n ; et h) le séquençage des ADN obtenus à la fin de l’étape f) ou, lorsqu’elle est présente, à la fin de l’étape g).

28. A method according to any one of claims 20 to 27, wherein: (1) where the process includes a reverse transcription step, the reverse transcriptase is selected from avian myeloblastosis virus reverse transcriptase, murine leukemia virus reverse transcriptase or derivatives thereof; (2) where the process includes a reverse transcription step, the buffer used for the reverse transcription includes: - 10 to 100 mM Tris-HCl, pH 8.3, in particular; - 10 to 150 mM KCl; - 0 to 10 mM MgCh, - 0 to 0.05 M DTT; and - O to lOmM MnCh; (3) when the process includes a reverse transcription step, this is carried out under the following conditions: 30 minutes to 1 hour 30 minutes at a temperature of + / - 2 degrees relative to the optimal temperature of the reverse transcriptase; (4) where the process includes a polymerase chain reaction amplification step, the DNA polymerase comprises a thermostable DNA polymerase from the B family of archaea, advantageously a thermostable DNA polymerase from the GB-D strain of Pyrococcus comprising an amino acid sequence with at least 90% identity with SEQ ID NO:1, alone or in combination with a Taq polymerase comprising an amino acid sequence with at least 90% identity with SEQ ID NO:17); (5) where the process includes a polymerase chain reaction step, the buffer used for the polymerase chain reaction comprises: - a Tris-SCU buffer at a concentration of 5 to 200 mM, advantageously 50 to 150 mM, more advantageously 60 to 100 mM, 70 to 90 mM, in particular about 80 mM; - of (NH4)2SU4 at a concentration of 1 to 100 mM, advantageously of 5 to 60 mM, of 10 to 40 mM, of 15 to 30 mM, in particular about 20 mM; - Mg ions 2+ advantageously in the form of MgSCU at a concentration of 1 to 10 mM, advantageously from 1 to 5 mM, from 1.5 to 3 mM, in particular about 2 mM; - glycerol at a concentration of 0 to 20% (v / v), advantageously 1 to 10% (v / v), 2.5 to 7.5% (v / v), in particular about 5% (v / v), - DMSO (dimethyl sulfoxide) at a concentration of 0 to 20% (v / v), advantageously 1 to 10% (v / v), 2.5 to 7.5% (v / v), in particular about 5% (v / v), - of octylphenoxy poly(ethyleneoxy)ethanol at a concentration of 0 to 1% (v / v), advantageously of 0.01 to 0.1% (v / v), of 0.04 to 0.08% (v / v), in particular of about 0.06% (v / v), - polysorbate 20 at a concentration of 0 to 1% (v / v), advantageously 0.01 to 0.1% (v / v), 0.025 to 0.075% (v / v), in particular about 0.05% (v / v), - a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives as defined above at a concentration of 0.1 to 3 mM, advantageously 0.1 to 1 mM, 0.1 to 0.5 mM, 0.1 to 0.3 mM, in particular about 0.2 mM, and - 20 to 30 units / mL, advantageously 23 to 27 units / mL, in particular about 25 units / mL of DNA polymerase; (6) where the process includes a polymerase chain reaction step, this step is carried out under the following conditions: - at step c): (i) a DNA denaturation step enabling the separation of the two DNA strands, wherein the mixture obtained in step b) is heated to a temperature of 90°C to 100°C for 10 to 30 seconds, preferably to a temperature of 93°C to 97°C for 10 to 30 seconds, in particular to a temperature of about 95°C for about 15 seconds; (ii) a primer-to-separated DNA strands pairing step, during which the mixture obtained in substep (i) is advantageously heated to a temperature of 45°C to 72°C for 15 to 45 seconds, preferably to a temperature of 55°C to 60°C for 10 to 30 seconds, in particular to a temperature of about 57°C for about 15 seconds; (iii) an elongation step of a complementary strand of each DNA strand comprising a primer by DNA polymerase, during which the mixture obtained in substep ii) is advantageously heated to a temperature of 65°C to 80°C for 15 to 90 minutes, preferably at a temperature of 65°C to 72°C for 20 to 60 minutes, especially at a temperature of about 68°C for about 30 or 45 or 60 minutes; - at step d) when it is present: (i) a DNA denaturation step allowing the two DNA strands to be separated, wherein the mixture obtained in step b) is heated to a temperature of 90°C to 100°C for 10 to 30 seconds, preferably to a temperature of 93°C to 97°C for 10 to 30 seconds, in particular to a temperature of about 95°C for about 15 seconds; (ii) a primer-to-separated DNA strands pairing step, during which the mixture obtained in substep (i) is advantageously heated to a temperature of 45°C to 72°C for 15 to 45 seconds, preferably to a temperature of 55°C to 60°C for 10 to 30 seconds, in particular to a temperature of about 57°C for about 15 seconds; (iii) an elongation step of a complementary strand of each DNA strand comprising a primer by DNA polymerase, during which the mixture obtained in substep ii) is advantageously heated to a temperature of 65°C to 80°C for 45 to 75 seconds, preferably to a temperature of 65°C to 72°C for 55 to 65 seconds, in particular to a temperature of about 68°C for about 60 seconds; (7) when the process includes a transcription step, the RNA polymerase is selected from the T7 RNA polymerase comprising the amino acid sequence SEQ ID NO:20 or a sequence with at least 90% identity with SEQ ID NO:20 and the sp6 RNA polymerase comprising the amino acid sequence SEQ ID NO:21 or a sequence with at least 90% identity with SEQ ID NO:21; (8) where the process includes a transcription step, the buffer used for transcription includes: - 10 to 100m HEPES, - 3 to 20 mM MgC12, - 0 to 50mM NaCl, - 0 to 10m Spermidine, - 1 to 100m DTT, and - 0 to 10% triton XI 00; (9) when the process includes a transcription step, this is carried out under the following conditions: 1h to 12h, advantageously 1h to 10h, 1h to 5h, or 1h to 3h, more advantageously 1h30 to 2h30, or 1h45 to 2h5, in particular about 2h, at a temperature of 35 to 39 °C, advantageously 36 to 38 °C, in particular about 37 °C, optionally followed by the addition of DNase A and incubation between 10 and 30 minutes, advantageously between 15 and 25 minutes, in particular about 20 minutes, at a temperature of 35 to 39 °C, advantageously 36 to 38 °C, in particular about 37 °C; (10) The sequencing step is performed: - directly onto natural or modified DNA bearing a substitution group of the formula - A0-X1-A1-Y2 obtained in the previous step, or - when the DNA obtained in the previous step is modified and carries a substitution group of the formula -A0-X1-A1-Y2, after amplification of this DNA by polymerase chain reaction in the presence of a DNA polymerase, a sense primer hybridizing to the 5' end of the sense strand, an antisense primer hybridizing to the 5' end of the antisense strand, and a mixture of deoxyribonucleotides comprising three phosphate groups or phosphate derivatives whose nucleobase is natural, this may facilitate the sequencing step; or (11) any combination of (1) to (10) above.

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