Oligonucleotides comprising signalling pairs and hydrophobic nucleotides, stemless beacons, for detection of nucleic acids, methylation status and mutants of nucleic acids

• US 8,133,984 B2
• Filed: 03/16/2007
• Issued: 03/13/2012
• Est. Priority Date: 03/16/2006
• Status: Active Grant

• Current Assignee: Pentabase APS
• Original Assignee: Pentabase APS
• Inventors: Christensen, Ulf Bech
• Primary Examiner(s): Woolwine, Samuel
• Application Number:
  US12/293,201
• Publication Number:
  US 20100068704A1
• Time in Patent Office: 1,824 Days
• Field of Search: None
• US Class Current: 536/23.1
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases

Abstract

The invention relates to novel oligonucleotides comprising a signalling pair and at least two hydrophobic nucleotides. The oligonucleotide analogues are useful for detecting the status of nucleic acid sequences, such as presence, expression, methylation and/or mutation, in particular single point mutations and other sequences where the variation between the correct target and other targets may vary in as little as one nucleotide. The invention also relates to new ways of detecting sequence differences and optimizing conditions by using oligonucleotide analogues and readily available instruments. In particular the invention relates to specifically detecting quantity of a target nucleic acids or detecting one sequence over others that may vary in as little as one nucleotide using oligonucleotides or oligonucleotide analogues comprising a signalling pair and at least two hydrophobic nucleotides, such as a nucleotide analogue comprising an intercalator.

Claims

1. An oligonucleotide analogue comprising a consecutive sequence of n nucleotides and/or nucleotide analogues and at least two hydrophobic nucleotides, wherein said oligonucleotide analogue is covalently linked to a signalling pair,

wherein the hydrophobic nucleotide has the general structure

X—

Y-Q

wherein

X is a nucleotide or nucleotide analogue or a backbone monomer unit capable of being incorporated into the backbone of a nucleic acid or nucleic acid analogue,

Q is a hydrophobic molecule which is not taking part in Watson-Crick hydrogen bonding; and

Y is a linker moiety linking said nucleotide or nucleotide analogue or backbone monomer unit and said hydrophobic molecule; and

wherein n is an integer of at least 4; and

wherein the at least two hydrophobic nucleotides are positioned at the most 9 nucleotides and/or nucleotide analogues from at least one end of the oligonucleotide analogue; and

wherein the hydrophobic nucleotide closest to the 3′

end and the hydrophobic nucleotide closest to the 5′

end of the oligonucleotide analogue are positioned in approximately the same distance from the respective parts of the signalling pair, and

wherein the signalling pair consists of a two part system, wherein one part is positioned at the most 6 nucleotides and/or nucleotide analogues from the 5′

end and the other part is positioned at the most 6 nucleotides or nucleotide analogues from the 3′

end of the oligonucleotide analogue;

wherein the parts of the signalling pair are not identical to the hydrophobic nucleotide,

with the proviso that when one part of the signalling pair is positioned as a dangling end at the 5′

end of the oligonucleotide analogue, then the first nucleotide or nucleotide analogue at the 5′

end is not a hydrophobic nucleotide and when one part of the signalling pair is positioned as a dangling end at the 3′

end of the oligonucleotide analogue, then the first nucleotide or nucleotide analogue at the 3′

end is not a hydrophobic nucleotide.

2. The oligonucleotide analogue according to claim 1 wherein at least one hydrophobic molecule, Q, has a logP value above 0 when a methyl group is added at the position of attachment to the linker, Y.

3. The oligonucleotide analogue according to claim 1, wherein at least one hydrophobic molecule, Q, is selected from the group consisting of polyaromates and heteropolyaromates optionally substituted with one or more selected from the group consisting of hydroxyl, bromo, fluoro, chloro, iodo, mercapto, thio, cyano, alkylthio, heterocycle, aryl, heteroaryl, carboxyl, carboalkoyl, alkyl, alkenyl, alkynyl, nitro, amino, alkoxyl and amido.

4. The oligonucleotide analogue according to claim 1, wherein the signaling pair is capable of a change in a detectable signal depending on the physical distance between the parts of the pair.

5. The oligonucleotide analogue according to claim 4, wherein one part of the signaling pair is a fluorophore and the other part of said signaling pair is a quencher, wherein said fluorophore and said quencher are capable of forming an intramolecular FRET complex.

6. The oligonucleotide analogue according to claim 1, wherein at least two of said hydrophobic nucleotides are positioned in opposite halves of said oligonucleotide analogue.

7. The oligonucleotide analogue according to claim 6, wherein the hydrophobic molecules of the hydrophobic nucleotides are capable of having hydrophobic interactions with each other thereby bringing the two parts of the signalling pair into close proximity within 40 Å

.

8. The oligonucleotide nucleotide analogue according to claim 7, wherein no hydrophobic nucleotide is positioned closer to the 5′

end than a part of the signalling pair and no hydrophobic nucleotide is positioned closer to the 3′

end than the other part of the signalling pair.

9. The oligonucleotide analogue according to claim 1, wherein the oligonucleotide is immobilized to a solid support.

10. A method of detecting and/or quantifying hybridisation between an oligonucleotide analogue according to claim 1 and a target sequence during amplification, said method comprising the steps of

a) providing a mixture of nucleic acids, which potentially comprises a target sequence;

and

b) providing an amplification buffer comprising an amplification enzyme, a set of primers, and an oligonucleotide analogue according to claim 1, wherein said primers and said oligonucleotide analogue are capable of hybridizing with said target sequence or a sequence complementary thereto, said amplification enzyme is capable of extending a primer in a template directed manner and said amplification buffer provides conditions for the amplification enzyme to perform such an extension in the presence of primers and template; and

c) incubating the primers and oligonucleotide analogue with the mixture of nucleic acids under conditions allowing for hybridization of said primers; and

d) extending the 3′

end of said hybridized primers using the target sequence as template using said amplification enzyme; and

e) incubating the nucleic acids, extension products and the oligonucleotide analogue under conditions allowing for hybridisation; and

f) detecting hybridisation and optionally quantifying hybridisation; and

g) optionally repeating step c) to f).

11. The method according to claim 10, wherein nuclease activity removes the most 5′

part of the signaling pair.

12. The method according to claim 10, wherein the oligonucleotide analogue is complementary to said target sequence and able to differentiate between said target and any other sequences.

13. The method according to claim 10, wherein at least two oligonucleotide analogues according to claim 1 are provided, wherein one of said oligonucleotide analogues is complementary to one genotype of the target sequence and the other of said oligonucleotide analogues is complementary to another genotype of said target sequence.

14. The method according to claim 13, wherein the two genotypes differ by a single nucleotide polymorphism at a polymorphic site and wherein one oligonucleotide analogue comprises a “

C”

-nucleobase at the polymorphic site and the other oligonucleotide analogue comprises an “

A”

-nucleobase at the polymorphic site.