# US 2019/0376151 A1 · Modified oligonucleotides comprising thiol functions and use thereof for detecting nucleic acids

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- Number: US 2019/0376151 A1
- Document ID: US20190376151A1
- Status: Published application
- Kind: A1
- Application number: 16/271,581
- Filed: 2019-02-08
- Published: 2019-12-12
- Earliest claimed: 2012-04-04
- Assignees: Etablissement Francais Du Sang; Centre National De La Recherche Scientfique (Cnrs); Universite De Montpellier; Universite Claude Bernard Lyon 1
- Applicants: Etablissement FrançAis Du Sang; Centre National De La Recherche Scientfique (Cnrs); Universite De Montpellier; Universite Claude Bernard Lyon 1
- Inventors: Chantal Fournier-Wirth; Myriam Lereau; Jean-François Cantaloube; Jean-Jacques Vasseur; François Morvan; Albert Meyer; Julie Mayen; Carole Chaix; Carole Farre
- Web page: https://patentgrep.com/patent/US20190376151A1

## CPC

- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12Q 1/6832: Enhancement of hybridisation reaction
- C12Q 1/707: {non-A, non-B Hepatitis, excluding hepatitis D}
- C12Q 2600/158: Expression markers
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 1

Modified oligonucleotide corresponding to the formula:
in which,
n is an integer ranging from 4 to 100,
y is an integer ranging from 2 to 12,
N1, . . . , Nn represent, independently of one another, a nucleotide,
X is selected from 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 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 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 C1-C12 alkane triyl groups, C6-C18 aryl triyl groups and C6-C18 aralkane triyl groups,
R is H or is selected from 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
Bn represents the base of the n-th nucleotide.

### Claim 2 (depends on 1)

Modified oligonucleotide according to claim 1, in which the nucleotide sequence (N1-N2- . . . -Nn-1-Nn) is specific to a virus, a bacterium or a gene responsible for or involved in a disease.

### Claim 3 (depends on 2)

Modified oligonucleotide according to claim 2, in which the nucleotide sequence (N1-N2- . . . -Nn-1-Nn) is 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).

### Claim 4 (depends on 1)

Modified oligonucleotide according to claim 1, in which the nucleotide sequence (N1-N2- . . . -Nn-1-Nn) has a structure of the alpha anomer, beta anomer, linear, or “snail” type.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 5)

Grafted substrate according to claim 5, 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.

### Claim 7 (depends on 5)

Grafted substrate according to claim 5, in which said substrate is non-planar, and is preferably a microparticle or a nanoparticle.

### Claim 8 (depends on 5)

Use of a grafted substrate according to claim 5 for detecting at least one target nucleic acid in a biological sample.

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1, 9)

Detection method according to claim 9, 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.

### Claim 11 (depends on 10)

Method according to claim 10, for determining a 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 virus bearing information relating to the genotype and/or subtype, and
detection of the hybridization of said amplicon with said at least one detection probe is carried out with a probe specific to a viral genotype and/or subtype.

### Claim 12 (depends on 11)

Method according to claim 11, 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.

### Claim 13 (depends on 9)

Method according to claim 9, wherein the method is applied for diagnostics, genotyping or sequencing of viral strains, preferably of hepatitis viruses, or of arboviruses, preferably the Flaviviridae, the Togaviridae or the Bunhyaviridae.

### Claim 14 (depends on 13)

Method according to claim 13, wherein the method is applied for diagnostics, genotyping or sequencing of HCV, HBV, dengue viruses or West Nile virus.

### Claim 15 (depends on 1, 5)

Kit for detecting at least one target nucleic acid in a biological sample comprising:
at least one modified oligonucleotide according to claim 1 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 5.

### Claim 16

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.

### Claim 17 (depends on 16)

Use of an oligonucleotide according to claim 16 for detecting at least one target nucleic acid in a biological sample.