# US 8,465,920 B2 · Method for hybridizing nucleic acids

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- Number: US 8,465,920 B2
- Document ID: US8465920B2
- Status: Granted
- Kind: B2
- Application number: 12/735,261
- Filed: 2008-09-12
- Published: 2010-12-09
- Granted: 2013-06-18
- Earliest claimed: 2007-12-27
- Assignees: Polyplus Transfection; Centre National De La Recherche Scientifique; Universite De Strasbourg
- Applicants: Nathalie Lenne; Patrick Erbacher
- Inventors: Nathalie Lenne; Patrick Erbacher
- Web page: https://patentgrep.com/patent/US8465920B2

## CPC

- C12Q 1/6832: Enhancement of hybridisation reaction
- 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 invention relates to a method for manipulating, isolating, detecting or amplifying a target nucleic acid in a sample by hybridization with an oligonucleotide-oligocation conjugate, comprising allowing said nucleic acid to react with an oligonucleotide-oligocation conjugate comprising at least A1 and Bj linked together directly or via a linker, wherein. A, is an i-mer oligonucleotides, with i=3 to 50, where Ai is an oligomer with naturally or non naturally occurring nucleobases and/or pentafuranosyl groups and/or native phosphodiester bonds, optionally comprising a marker group. Bj is a j-mer organic oligocation moiety, with j=1 to 50, where B is —HPO3—R1—(NH—R2)n—NH—R3—O—, where R1, R2 and R3 are lower alkylene, identical or different, NH—R2 moieties being identical or different when n is >1; HPO3—R1—CH(X)—R3—O—, where Ri and R3, identical or different, are lower alkylene and X is putrescine, spermidine or spermine residue.

## Claims

### Claim 1

A method for detecting or amplifying a target nucleic acid in a sample of genomic DNA or of total RNA, comprising at least an oligonucleotide-oligocation conjugate Ai-Bj and comprising the steps of:
extending an oligonucleotide-oligocation conjugate Ai-Bj of structure I to III with said target nucleic acid in a sample of genomic DNA as a template;
detecting said target nucleic acid in said sample of genomic DNA with an oligonucleotide-oligocation conjugate Ai-Bj of structure I to X; or
reverse transcribing said target nucleic acid of a sample of total RNA with an oligonucleotide-oligocation conjugate Ai-Bj of structure I to III;
wherein in said conjugate Ai-Bj
Ai is an i-mer oligonucleotide with i=3 to 50, where Ai is an oligomer with naturally or non naturally occurring nucleobases and/or pentafuranosyl groups and/or native phosphodiester bonds,
Bj, moiety is attached to Ai moiety or to linker to Ai via a phosphodiester link,
Bj, being a j-mer organic oligocation moiety, with j=1 to 50, where B is:
—HPO3—R1—(NH—R2)n—NH—R3—O—, where R1, R2 and R3 identical or different, are a C1-C6 alkylene radical, the NH—R2 moieties being identical or different when n is >1;
—HPO3—R1—CH(X)—R3—O— where R1 and R3, identical or different, are a C1-C6 alkylene radical and X is putrescine, spermidine or a spermine residue,
said structures Ito X being as follows:
HO—3′Ai5′-Bj-R4 structure I
HO—3′Ai5′-R5—Bj-R4 structure II
HO—3′Ai15′—Bj-Ai2-R4 structure III
R4—Bj-3′Ai5′—R6 structure IV
R4—Bj-R53′Ai5′-R6 structure V
R7-3′Ai5′-Bj—R4 structure VI
R7-3′Ai5′-R5—Bj—R4 structure VII
R7-3′Ai15′-Bj-3′Ai25′-R4 structure VIII
R7-3′Ai15′-Bj-5′Ai23′-R8 structure IX
R4—Bj1-3′Ai5′-Bj2-R6 structure X
wherein
Ai1 and Ai2, identical or different are i-mer oligonucleotides with i=3 to 50, where Ai1 and Ai2 are oligomers with naturally or non naturally occurring nucleobases and/or pentafuranosyl groups and/or native phosphodiester bonds;
R4 and R6, identical or different, are H or a linker, a quencher, a marker, a chromophore group, a fluorophore group, a chemical moiety, a biotin, a hydrophobic chain, a cholesterol derivative, an antigen, a protein, a peptide, a sugar group and a phosphate group;
R5, different from H, Ai and Bj, is a chemically stable or cleavable linker between Ai and Bj;
R7 and R8, identical or different, are different from H and are selected in the group comprising a linker, a quencher, a marker, a chromophore group fluorophore group, a chemical moiety, a biotin, a hydrophobic chain, a cholesterol derivative, an antigen, a protein, a peptide, a phosphate group and a sugar group.

### Claim 2 (depends on 1)

The method of claim 1, wherein said oligonucleotide-oligocation conjugate of structures I-III are used as primers for DNA or RNA polymerase.

### Claim 3 (depends on 1)

The method of claim 1, allowing nucleic acid amplification using at least one oligonucleotide-olgocation conjugate of structure I, II or III.

### Claim 4 (depends on 1)

The method of claim 1, wherein an oligonucleotide-oligocation conjugate Ai-B3 of structure IV to X is used as a probe to detect said target nucleic acid.

### Claim 5 (depends on 4)

The method of claim 4, wherein said oligonucleotide-oligocation is used as a probe in PCR or Real Time-PCR.

### Claim 6 (depends on 5)

The method of claim 5, wherein said oligonucleotide-oligocation probe is a dual labeled probe.

### Claim 7 (depends on 1)

The method of claim 1, wherein at least one of structures IV to X is used as a clamp for inhibiting the detection or amplification of a target nucleic acid.

### Claim 8 (depends on 1)

The method of claim 1, wherein at least one oligonucleotide-oligocation conjugate of structure I to X is used for distinguishing between a wild-type and a mutant target nucleic acid.

### Claim 9 (depends on 1)

The method of claim 1, wherein at least one oligonucleotide-oligocation conjugate of structure I to X is used in a multiplex assay.

### Claim 10 (depends on 1)

The method of claim 1, further comprising purifying, capturing and modifying said target nucleic acid, using at least one oligonucleotide-oligocation of structure I to X.

### Claim 11 (depends on 1)

The method of claim 1, wherein Ai is selected from the group comprising deoxyribonucleotides, ribonucleotides and non naturally occurring nucleobases.

### Claim 12 (depends on 11)

The method of claim 11, wherein said non naturally occurring nucleobases are locked (LNA) nucleotides, PNA, phosophorothioate modifications or substitutions or 2′-fluoro or 2′-O-alkyl groups modifications or substitutions.