# US 2014/0194611 A1 · Stabilized nucleic acid dark quencher-fluorophore probes

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- Number: US 2014/0194611 A1
- Document ID: US20140194611A1
- Status: Published application
- Kind: A1
- Application number: 14/215,272
- Filed: 2014-03-17
- Published: 2014-07-10
- Earliest claimed: 2008-04-01
- Assignees: Biosearch Technologies, Inc.
- Applicants: Biosearch Technologies, Inc.
- Inventors: Ronald M. Cook; Matt Lyttle
- Web page: https://patentgrep.com/patent/US20140194611A1

## CPC

- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C07D 219/04: with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
- C07H 19/06: Pyrimidine radicals
- C07H 19/073: with 2-deoxyribosyl as the saccharide radical
- C09B 31/02: Disazo dyes
- 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
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)
- 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 provides a new class of solids supports for synthesis of modified oligomers of nucleic acids, and nucleic acid probes that have a format expediently synthesized on the new supports. Exemplary solid supports include at least one quencher bound through a linker to the solid support. Various exemplary embodiments include a moiety that stabilizes a duplex, triplex or higher order aggregation (e.g., hybridization) of nucleic acids of which the oligomer of the invention is a component. Other components of the solid support include moieties that stabilize aggregations of nucleic acids, e.g., intercalators, minor groove binding moieties, bases modified with a stabilizing moiety (e.g., alkynyl moieties, and fluoroalkyl moieties), and conformational stabilizing moieties, such as those described in commonly owned U.S. Patent Application Publication No. 2007/0059752.

## Claims

### Claim 1

A nucleic acid oligomer comprising at least one base, having a formula selected from:
wherein
R10 is an alkynyl moiety; and
a quencher of fluorescence energy comprising at least three residues, each independently selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and combinations thereof, wherein at least two of said residues are covalently linked via an exocyclic diazo bond.

### Claim 2 (depends on 1)

The nucleic acid oligomer according to claim 1, wherein said quencher is of a structure according to Formula V:
wherein
R1, R2 and R3 are members independently selected from substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;
X and Y are members independently selected from a reactive functional group and a linkage fragment covalently binding said quencher to the nucleic acid oligomer, with the proviso that at least one of X and Y is said linkage fragment;
m is an integer from 0 to 5, such that when m is greater than 1, the X groups are the same or different; and
v is an integer from 1 to 10.

### Claim 3 (depends on 2)

The nucleic acid oligomer according to claim 2, wherein v is 1.

### Claim 4 (depends on 2)

The nucleic acid oligomer according to claim 2, wherein said linkage fragment is NH or NRt, wherein Rt is selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

### Claim 5 (depends on 1)

The nucleic acid oligomer according to claim 1, wherein said quencher is of a structure according to Formula VI:
wherein
R5, R6 and R7 are members independently selected from —NR′R″, substituted or unsubstituted aryl, nitro, substituted or unsubstituted C1-C6 alkyl, and substituted or unsubstituted C1-C6 alkoxy,
wherein R′ and R″ are independently selected from H and substituted or unsubstituted C1-C6 alkyl;
X and Y are members independently selected from a reactive functional group and a linkage fragment covalently binding said quencher to the nucleic acid oligomer, with the proviso that at least one of X and Y is said linkage fragment;
n is an integer from 0 to 1;
a is an integer from 0 to 4, such that when a is greater than 1, the R5 groups are the same or different;
b is an integer from 0 to 4, such that when (v×b) is greater than 1, the R6 groups are the same or different;
c is an integer from 0 to 5, such that when c is greater than 1, the R7 groups are the same or different; and
v is an integer from 1 to 10, such that when v is greater than 1, the value of b on each of the v phenyl rings is the same or different.

### Claim 6 (depends on 5)

The nucleic acid oligomer according to claim 5, wherein v is 1.

### Claim 7 (depends on 6)

The nucleic acid oligomer according to claim 6, wherein said quencher has a structure selected from:
wherein X5 is a linkage fragment covalently binding said quencher to the nucleic acid oligomer.

### Claim 8 (depends on 5)

The nucleic acid oligomer according to claim 5, wherein said linkage fragment is NH or NRt, wherein Rt is selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

### Claim 9 (depends on 1)

The nucleic acid oligomer according to claim 1, wherein said quencher is of a structure according to Formula (II)
wherein
R1, R2 and R3 are members independently selected from substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;
wherein a member selected from R1, R2 and R3 includes a structure according to Formula IV:
wherein R4 is a member selected from alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;
X, Y and Y′ are members independently selected from a reactive functional group and a linkage fragment covalently binding said quencher to the nucleic acid oligomer, with the proviso that at least one of X, Y and Y′ is said linkage fragment;
f is an integer from 0 to 4, such that when (f×s) is greater than 1, the Y′ groups are the same or different;
m is an integer from 0 to 5, such that when m is greater than 1, the X groups are the same or different;
n is an integer from 0 to 6, such that when (n×t) is greater than 1, the Y groups are the same or different;
s is an integer from 0 to 6, such that when s is greater than 1 the R3 groups are the same or different; and
t is an integer from 1 to 6, such that when t is greater than 1 the R2 groups are the same or different, and when t is 1 and s is 0, a member selected from R1, R2 and combinations thereof is a member selected from substituted or unsubstituted polycyclic aryl and substituted or unsubstituted polycyclic heteroaryl groups.

### Claim 10 (depends on 9)

The nucleic acid oligomer according to claim 9, wherein said quencher is of a structure according to Formula (III):

### Claim 11 (depends on 9)

The nucleic acid oligomer according to claim 9, wherein said quencher has the structure:
wherein
X5 and X6 are members independently selected from H, a reactive functional group and a linkage fragment covalently binding said quencher to the nucleic acid oligomer, with the proviso that at least one of X5 and X6 is said linkage fragment.

### Claim 12 (depends on 9)

The nucleic acid oligomer according to claim 9, wherein said linkage fragment is NH or NRt, wherein Rt is selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

### Claim 13 (depends on 1)

The nucleic acid oligomer according to claim 1, wherein said nucleic acid oligomer is a probe, preferably wherein said probe is selected from molecular beacons, scorpion probes, sunrise probes, conformationally assisted probes, light up probes, invader detection probes, and taqman probes.

### Claim 14 (depends on 1)

The nucleic acid oligomer according to claim 1, wherein said quencher is attached at the 3′ end, the 5′ end, or at an internal position of said nucleic acid oligomer.

### Claim 15 (depends on 1)

The nucleic acid oligomer according to claim 1, wherein said alkynyl moiety is selected from the group consisting of: ethynyl, 1-propynyl, 1-butynyl, 1-pentynyl, 1,3-pentadiynyl, phenylethynyl, phenylethynyl, pyridine-ethynyl, pyrimidine-ethynyl, triazine-ethynyl, thiophene-ethynyl, thiazole-ethynyl, imidazole-ethynyl and substituted or unsubstituted C1-C10 alkynyl groups.

### Claim 16 (depends on 15)

The nucleic acid oligomer according to claim 15, wherein the substituents for the C1-C10 alkynyl groups are selected from the group consisting of: 2-, 3-, and 4-pyridinyl; triazine; 2-, 4- and 5-pyrimidinyl; 2-, 4- and 5-thiazolyl; 1-methyl-2-imidazolyl; 2- and 4-imidazolyl; 2-, 4- and 5-oxazolyl; 3-pyridinyl; 4-pyridinyl; 2-pyridinyl; 2- and 3-thienyl; 2- and 3-furanyl; 2- and 3-pyrrolyl; propenyl (—CH═CH—CH3); vinyl; and —C≡C—Z′ where Z′ is hydrogen (H) or C1-C10 alkyl, C1-C10 alkyl with 1 to 6 halogen atoms; or C1-C10 heteroalkyl having 1 to 3 heteroatoms selected from the group consisting of O, N and S.

### Claim 17 (depends on 15)

The nucleic acid oligomer according to claim 15, wherein said alkynyl moiety is 1-propynyl.

### Claim 18 (depends on 1)

The nucleic acid oligomer according to claim 1, further comprising a fluorophore.