Modified oligonucleotides comprising thiol functions and use thereof for detecting nucleic acids

Abstract

The present invention relates to a modified oligonucleotide having two or more thiol functions, which can be immobilized on a gold surface or on a grafted surface, in particular a surface comprising at least one carbon-carbon double bond or carbon-carbon triple bond or haloacetamide functions, preferably maleimide or acrylamide functions. The invention also relates to a method for detecting a nucleic acid in a biological sample comprising a step of detecting hybridization between a modified oligonucleotide and a target nucleic acid amplified from the biological sample. The invention relates more particularly to a method for detecting, genotyping or sequencing a pathogenic organism, preferably a virus.

1 . Modified oligonucleotide corresponding to formula (XIIb):
   N 1 -N 2 - . . . -N n-1 -N n -(I′ b ) y -(M 1 - . . . -M m-1 -M m ) p -(I′ b ) y′   (XIIb)
   or to formula (XIIIb):
   (I c ′)-(I′ b ) y-1 -N 1 -N 2 - . . . -N n-1 -N n -(I′ b ) y′ -(M 1 -M 2 - . . . -M m-1 -M m ) p -(I′ b ) y″   (XIIIb)
 
   in which,   N 1 , . . . , N n  represent, independently of one another, a nucleotide,   M 1 , . . . , M m  represent, independently of one another, a nucleotide,   (I′b) represents a compound of formula:   
       
         
           
           
               
               
           
         
         (Ic′) represents a compound of formula: 
       
       
         
           
           
               
               
           
         
         n is an integer ranging from 4 to 100, 
         m is an integer ranging from 4 to 100, 
         y is an integer ranging from 2 to 12, 
         p represents 0 or 1, 
         y′ is an integer ranging from 0 to 12 if p has the value 1 and y′ is equal to 0 if p has the value 0, 
         y″ is an integer ranging from 0 to 12 if p has the value 1 and if p has the value 0 then y″ has the value 0, 
         the sum of the integers (y+y′) or (y+y′+y″) is not greater than 12, 
         X is selected from the linear or branched C1-C12 alkyl groups, C1-C12 aminoalkyl groups, C1-C12 alkoxy groups, C3-C12 cycloalkyl groups, oxygen-containing or nitrogen-containing C3-C12 cycloheteroalkyl groups, 
         Y is selected from the linear or branched C1-C12 alkyl groups, C1-C12 aminoalkyl groups, C1-C12 alkoxy groups, C3-C12 cycloalkyl groups, oxygen-containing or nitrogen-containing C3-C12 cycloheteroalkyl groups, 
         Z is selected from the C1-C12 alkoxy groups, oxygen-containing or nitrogen-containing C3-C12 cycloheteroalkyl groups, C1-C12 NCO-alkyl groups, C1-C12 CON-alkyl groups, 
         W is selected from the C1-C12 alkane triyl groups, the C6-C18 aryl triyl groups and the C6-C18 aralkane triyl groups, 
         R is H or is selected from the C1-C12 acyl, C1-C12 S-alkyl, C6-C12 S-aryl, S-2-pyridine, oxygen-containing or nitrogen-containing C1-C12 S-heteroalkyl, C3-C12 S-cycloalkyl, oxygen-containing or nitrogen-containing C3-C12 S-cycloheteroalkyl groups, and 
         R1 is selected from the 2-cyanoethyl or R′ 1 R′ 2 R′ 3 SiCH 2 CH 2  groups, in which R′ 1 , R′ 2  and R′ 3  may be identical or different and represent a group selected from the linear or branched alkyls comprising from 1 to 12 carbon atoms and the C6-C12 aryls. 
       
     
     
         2 . Modified oligonucleotide according to  claim 1 , corresponding to the formula: 
       
         
           
           
               
               
           
         
       
       in which n, y, N 1 , . . . , N n-1 , X, Y, Z, W and R have the same definition as in  claim 1 , and Bn represents the base of the n-th nucleotide. 
     
     
         3 . Modified oligonucleotide according to  claim 1 , corresponding to the formula: 
       
         
           
           
               
               
           
         
       
       in which n, y, N 2 , . . . , N n , X, Y, Z, W and R have the same definition as in  claim 1 , and B 1  represents the base of the 1st nucleotide. 
     
     
         4 . Modified oligonucleotide according to  claim 1 , in which the nucleotide sequence (N 1 -N 2 - . . . -N n-1 -N n ) and, optionally, the nucleotide sequence (M 1 -M 2 - . . . -M m-1 -M m ) are specific to a virus, a bacterium or a gene responsible for or involved in a disease. 
     
     
         5 . Modified oligonucleotide according to  claim 4 , in which the nucleotide sequence (N 1 -N 2 - . . . -N n-1 -N n ) and, optionally, the nucleotide sequence (M 1 -M 2 - . . . -M m-1 -M m ) are selected from:
 the sequences SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 35 or SEQ ID NO: 36 specific to the hepatitis C virus (HCV),   the sequences SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 40, specific to the flaviviruses,   the sequence SEQ ID NO: 18 or SEQ ID NO: 41, specific to the dengue viruses, or   the sequence SEQ ID NO: 19, specific to the West Nile viruses (WNV).   
     
     
         6 . Modified oligonucleotide according to  claim 1 , in which the nucleotide sequence (N 1 -N 2 - . . . -N n-1 -N n ) and, optionally, the nucleotide sequence (M 1 -M 2 - . . . -M m-1 -M m ) have a structure of the alpha anomer, beta anomer, linear, or “snail” type. 
     
     
         7 . Substrate grafted with at least one modified oligonucleotide according to  claim 1 , said substrate comprising at least one receiving zone coated with a substance that tolerates the grafting of said modified oligonucleotide. 
     
     
         8 . Grafted substrate according to  claim 7 , in which:
 said receiving zone is coated with a gold or platinum film, and said substrate is of metal, preferably of copper or titanium, or   said receiving zone comprises on its surface at least one carbon-carbon double bond or carbon-carbon triple bond or haloacetamide functions, preferably maleimide or acrylamide functions, and said substrate is of plastic, preferably of polystyrene.   
     
     
         9 . Grafted substrate according to  claim 7 , in which said substrate is non-planar, and is preferably a microparticle or a nanoparticle. 
     
     
         10 . Method for detecting at least one target nucleic acid in a biological sample, comprising a step of:
 detecting said target nucleic acid with at least one detection probe formed by a modified oligonucleotide according to  claim 1 .   
     
     
         11 . Detection method according to  claim 10 , comprising the steps of:
 obtaining at least one source nucleic acid from said biological sample,   producing an amplicon by the amplification of said target nucleic acid from the source nucleic acid, and   detecting the hybridization of said amplicon with at least one detection probe formed by a modified oligonucleotide according to  claim 1 .   
     
     
         12 . Method according to  claim 11 , for determining the genotype and/or subtype of a virus present in a biological sample, in which
 the amplicon is generated by the amplification of a target nucleotide sequence, corresponding to a genomic region of the virus bearing information relating to the viral genotype and/or subtype, and   detection is carried out with a probe specific to a viral genotype and/or subtype.   
     
     
         13 . Method according to  claim 12 , in which the step of production of the amplicon is carried out by amplifying a target nucleotide sequence corresponding to a genomic region of the virus bearing information relating to the viral genotype and/or subtype, with a mixture of nucleotide primers, preferably selected from the primer pairs:
 SEQ ID NO: 8 and SEQ ID NO: 9, when the amplicon is generated starting from any genotype of HCV;   SEQ ID NO: 10 and SEQ ID NO: 9, when the amplicon is generated starting from an HCV of genotype 1a/1b;   SEQ ID NO: 29 and SEQ ID NO: 9, when the amplicon is generated starting from an HCV of genotype 2;   SEQ ID NO: 8 and SEQ ID NO: 11, when the amplicon is generated starting from an HCV of genotype 3a;   SEQ ID NO: 8 and SEQ ID NO: 30, when the amplicon is generated starting from an HCV of genotype 4a/4b;   SEQ ID NO: 20 and SEQ ID NO: 21, or SEQ ID NO: 22 and SEQ ID NO: 21 when the amplicon is generated from a flavivirus.   
     
     
         14 . Kit for detecting at least one target nucleic acid in a biological sample comprising:
 at least one modified oligonucleotide according to one of  claims 1  to  6  and at least one substrate comprising at least one receiving zone coated with a substance that tolerates the grafting of said modified oligonucleotide, said receiving zone preferably being coated with gold, with platinum or comprising on its surface at least one carbon-carbon double bond or carbon-carbon triple bond or haloacetamide functions, preferably maleimide or acrylamide functions, or   at least one grafted substrate according to  claim 7 .   
     
     
         15 . Oligonucleotide having a nucleotide sequence selected from the sequences SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40 and SEQ ID NO: 41. 
     
     
         16 . Use of a modified oligonucleotide according to  claim 1  for detecting at least one target nucleic acid in a biological sample. 
     
     
         17 . Use according to  claim 16 , for diagnostics, genotyping or sequencing of viral strains, preferably of hepatitis viruses, or of arboviruses, preferably the Flaviviridae, the Togaviridae or the Bunhyaviridae. 
     
     
         18 . Use according to  claim 17 , for diagnostics, genotyping or sequencing of HCV, HBV, dengue viruses or West Nile virus. 
     
     
         19 . Use of an oligonucleotide according to  claim 15  for detecting at least one target nucleic acid in a biological sample. 
     
     
         20 . Use of a grafted substrate according to  claim 7  for detecting at least one target nucleic acid in a biological sample.