# US 8,465,921 B2 · Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders

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- Number: US 8,465,921 B2
- Document ID: US8465921B2
- Status: Granted
- Kind: B2
- Application number: 12/847,148
- Filed: 2010-08-31
- Published: 2011-11-10
- Granted: 2013-06-18
- Earliest claimed: 1995-04-03
- Assignees: Elitech Holding B.V.
- Applicants: Joel Hedgpeth; Irina A. Afonina; Igor V. Kutyavin; Eugeny A. Lukhtanov; Evgeniy S. Belousov; Rich B. Meyer, Jr.
- Inventors: Joel Hedgpeth; Irina A. Afonina; Igor V. Kutyavin; Eugeny A. Lukhtanov; Evgeniy S. Belousov; Rich B. Meyer, Jr.
- Web page: https://patentgrep.com/patent/US8465921B2

## 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
- C07D 207/34: 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 ring carbon atoms
- C07D 403/12: linked by a chain containing hetero atoms as chain links
- C07D 487/04: Ortho-condensed systems
- C07H 19/073: with 2-deoxyribosyl as the saccharide radical
- C07H 19/173: with 2-deoxyribosyl as the saccharide radical
- C07H 19/23: Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12Q 1/6813: Hybridisation assays
- 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)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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

Conjugates between a minor groove binding molecule, such as the trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3), and an oligonucleotide form unusually stable hybrids with complementary target sequences, in which the tethered CDPI3 group resides in the minor groove of the duplex. These conjugates can be used as probes and primers. Due to their unusually high binding affinity, conjugates as short as 8-mers can be used as amplification primers with high specificity and efficiency. MGB conjugation also increases the discriminatory power of short oligonucleotides, providing enhanced detection of nucleotide sequence mismatches by short oligonucleotides. The MGB-conjugated probes and primers described herein facilitate various analytic and diagnostic procedures, such as amplification reactions, PCR, detection of single-nucleotide polymorphisms, gene hunting, differential display, fluorescence energy transfer, hydrolyzable probe assays and others; by allowing the use of shorter oligonucleotides, which have higher specificity and better discriminatory power.

## Claims

### Claim 1

A method for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, the method comprising:
(a) contacting the mixture of polynucleotides with at least one Minor Groove Binder (MGB)-oligonucleotide conjugate,
wherein the MGB-oligonucleotide conjugate comprises a 5′-end, a 3′-end, a Minor Groove Binder (MGB), one or more detectable fluorescent labels, and a quenching agent which quenches the fluorescence emission of one or more of the fluorescent labels; and
(b) measuring hybrid formation, whereby hybrid formation is indicative of the presence of said target sequence.

### Claim 2 (depends on 1)

The method of claim 1, further comprising the step of releasing one or more labels from the MGB-oligonucleotide conjugate subsequent to hybrid formation.

### Claim 3 (depends on 1)

The method of claim 1, wherein the detectable labels are fluorescein, cyanine, rhodamine, or combinations thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein at least one of the detectable labels is a fluorescein.

### Claim 5 (depends on 1)

The method of claim 1, wherein the quenching agent is tetramethylrhodamine.

### Claim 6 (depends on 1)

The method of claim 1, wherein the MGB-oligonucleotide conjugate comprises at least two fluorescent labels.

### Claim 7 (depends on 6)

The method of claim 6, wherein the emission wavelengths of one of the fluorescent labels overlaps the absorption wavelengths of another of the fluorescent labels.

### Claim 8 (depends on 1)

The method of claim 1, wherein the MGB is a molecule having a molecular weight of approximately 150 to approximately 2,000 Daltons that binds into the minor groove of a double-stranded nucleic acid with an association constant of greater than approximately 103 M−1.

### Claim 9 (depends on 1)

The method of claim 1, wherein the MGB is located at the 5′ end of the MGB-oligonucleotide conjugate.

### Claim 10 (depends on 1)

The method of claim 1, wherein the MGB is located at the 3′ end of the MGB-oligonucleotide conjugate.

### Claim 11 (depends on 1)

The method of claim 1, wherein at least one of the detectable fluorescent labels is located at the 5′ end of the MGB-oligonucleotide conjugate.

### Claim 12 (depends on 1)

The method of claim 1, wherein at least one of the detectable fluorescent labels is located at the 3′ end of the MGB-oligonucleotide conjugate.

### Claim 13 (depends on 1)

The method of claim 1, wherein the MGB is selected from the group consisting of a dimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI2), a trimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3), tetramer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI4) and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC5).

### Claim 14 (depends on 1)

The method of claim 1, further comprising the step of altering the spatial relationship between at least one of the detectable fluorescent labels and the quenching agent.

### Claim 15 (depends on 1)

The method of claim 1, wherein the MGB-oligonucleotide conjugate is a primer in an amplification reaction.

### Claim 16 (depends on 15)

The method of claim 15, wherein the amplification reaction is a polymerase chain reaction.