# US 10,428,371 B2 · Fluorescent labeled single-stranded nucleic acid and use thereof

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- Number: US 10,428,371 B2
- Document ID: US10428371B2
- Status: Granted
- Kind: B2
- Application number: 15/300,183
- Filed: 2015-03-27
- Published: 2017-05-25
- Granted: 2019-10-01
- Earliest claimed: 2014-03-31
- Assignees: Kabushiki Kaisha Dnaform
- Applicants: Kabushiki Kaisha Dnaform
- Inventors: Takeshi Hanami; Yoshihide Hayashizaki; Takahiro Soma; Yasumasa Kimura
- Web page: https://patentgrep.com/patent/US10428371B2

## CPC

- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C09B 23/04: one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
- C09B 23/06: three >CH- groups, e.g. carbocyanines
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- 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
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)

## Abstract

The present invention is intended to provide a novel fluorescent labeled single-stranded nucleic acid, by which the background of an exciton oligomer can be further reduced and the novel use thereof. The present invention relates to a labeled single-stranded nucleic acid having at least two fluorescent atomic group pairs that exhibit an exciton effect. The labeled single-stranded nucleic acid is characterized in that the emission peak wavelength of one of the fluorescent atomic group pairs (fluorescent atomic group pair A) is shorter than the excitation peak wavelength of the other fluorescent atomic group pair (fluorescent atomic group pair B), and the fluorescent atomic group pairs A and B have a Förster resonance energy transfer (FRET) effect. This fluorescent labeled single-stranded nucleic acid is usable as a primer for amplifying a target nucleic acid or a probe to be hybridized with a target nucleic acid.

## Claims

### Claim 1

A labeled single-stranded nucleic acid comprising at least two fluorescent atomic group pairs that exhibit an exciton effect within each fluorescent atomic group pair, wherein
an emission peak wavelength of a fluorescent atomic group pair J is shorter than an excitation peak wavelength of a fluorescent atomic group pair K,
the fluorescent atomic group pair J and the fluorescent atomic group pair K are both cations,
the fluorescent atomic group pairs J and K have a Förster resonance energy transfer (FRET) effect between each other,
each of a base having the fluorescent atomic group pair J or K that exhibits an exciton effect has a structure represented by the following formula (16), (16b), (17), or (17b):
where in the formulae (16), (16b), (17), and (17b),
Z11 and Z12 are each a fluorescent atomic group that exhibits an exciton effect, and may be identical to or different from each other,
Z11 and Z12 constitute a fluorescent atomic group pair,
B is an atomic group having a natural nucleobase skeleton or an artificial nucleobase skeleton, wherein the natural nucleobase is adenine, guanine, cytosine, thymine or uracil,
E is:
(i) an atomic group having a deoxyribose skeleton, a ribose skeleton, or a structure derived from either one of them, or
(ii) an atomic group having a peptide structure or a peptoid structure,
L1, L2, and L3 are each a linking atom or a linking atomic group, having any number of main chain atoms, each may or may not contain each of C, N, O, S, P, and Si in the main chain, each may or may not contain each of a single bond, a double bond, a triple bond, an amide bond, an ester bond, a disulfide bond, an imino group, an ether bond, a thioether bond, and a thioester bond in the main chain, and may be identical to or different from each other,
D is CR, N, P, P═O, B, or SiR where R is a hydrogen atom, an alkyl group, or any substituent, and
b is a single bond, a double bond, or a triple bond, or alternatively,
in the formulae (16) and (16b), L1- and L2 are each a linker, L3, D, and b may not be present, and L1 and L2 may be bound directly to B, provided that:
in the formulae (16) and (17), E is an atomic group described in the item (i), and at least one O atom in a phosphoric acid linkage may be substituted with an S atom;
in the formulae (16b) and (17b), E is an atomic group described in the item (ii);
in the formulae (17) and (17b), the respective Bs may be identical to or different from each other, and the respective Es may be identical to or different from each other; and
the base having the fluorescent atomic group pair J and the base having the fluorescent atomic group pair K are contained in the labeled single-stranded nucleic acid at a distance at which there are from 1 to 5 bases between the base having the fluorescent atomic group pair J and the base having the fluorescent atomic group pair K.

### Claim 2 (depends on 1)

The labeled single-stranded nucleic acid according to claim 1, wherein
the base having the fluorescent atomic group pair J and the base having the fluorescent atomic group pair K are contained in the labeled single-stranded nucleic acid at a distance at which the fluorescent atomic group pairs J and K have an FRET effect between each other.

### Claim 3 (depends on 1)

The labeled single-stranded nucleic acid according to claim 1, wherein
the structure represented by the formula (16) is a structure represented by the following formula (16-1) or (16-2),
the structure represented by the formula (16b) is a structure represented by the following formula (16b-1) or (16b-2),
the structure represented by the formula (17) is a structure represented by the following formula (17-1), and
the structure represented by the formula (17b) is a structure represented by the following formula (17b-1):
where in the formulae (16-1), (16-2), (16b-1), (16b-2), (17-1), and (17b-1),
1, m and n are any positive integers, may be identical to or different from each other, each may or may not contain each of C, N, O, S, P, and Si in a main chain thereof, and each may or may not contain each of a single bond, a double bond, a triple bond, an amide bond, an ester bond, a disulfide bond, an imino group, an ether bond, a thioether bond, and a thioester bond in the main chain,
B,E,Z11,Z12,and b are identical to those in the formulae (16), (16b), (17), and (17b), and
in the formulae (16-1), (16-2), and (17-1), at least one O atom in a phosphoric acid linkage may be substituted with an S atom.

### Claim 4 (depends on 1)

The labeled single-stranded nucleic acid according to claim 1, wherein
the base having the fluorescent atomic group pair that exhibits an exciton effect has a structure represented by the formula (16).

### Claim 5 (depends on 1)

The labeled single-stranded nucleic acid according to claim 1, wherein
Z11 and Z12 are each independently an atomic group represented by any one of the following formulae (7) to (10):
where in the formulae (7) to (9),
X1 and X2 are S or O
n is 0 or a positive integer,
R1 to R10 and R13 to R21 are each independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group,
one of R11 and R12 is a linking group to be bound to L1- or L2 in the formulae (16), (17), (16b), and (17b), and the other is a hydrogen atom or a lower alkyl group,
when a plurality of R15s are present in the formula (7), (8), or (9), they may be identical to or different from each other,
when a plurality of R16s are present in the formula (7), (8), or (9), they may be identical to or different from each other, and
X1, X2, and R1 to R21 in Z11 and X1, X2, and R1 to R21 in Z12 may be identical to or different from each other, respectively, and where in the formula (10),
E is S or O,
R2 to R12 are each independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group,
R1 is a linking group to be bound to L1 or L2 in the formulae (16), (17), (16b), and (17b),
when a plurality of R3s is present in the formula (10), they may be identical to or different from each other, and
when a plurality of R4s is present in the formula (10), they may be identical to or different from each other.

### Claim 6 (depends on 5)

The labeled single-stranded nucleic acid according to claim 5, wherein
Z11 and Z12 are each independently an atomic group represented by the formula (7) or (8), and
Z11 and Z12 represented by the formula (7) or (8) are each a group represented by the following formula (19) or (20):
where in the formulae (19) and (20),
X1, R1to R10, R13 and R14, R11, and R12 are identical to those in the formulae (7) to (9).

### Claim 7 (depends on 1)

The labeled single-stranded nucleic acid according to claim 1, used as a primer for amplifying a target nucleic acid or a probe to be hybridized with a target nucleic acid.

### Claim 8 (depends on 1)

A method for detecting a target nucleic acid, comprising measuring fluorescence under the conditions where the labeled single-stranded nucleic acid according to claim 1 as a probe is capable of hybridizing with a target nucleic acid, to determine the presence or absence of the hybridization of the target nucleic acid to the probe.

### Claim 9 (depends on 1)

A method for amplifying a target nucleic acid, comprising amplifying a target nucleic acid using the labeled single-stranded nucleic acid according to claim 1 as a primer.