Oligonucleotides comprising signalling pairs and hydrophobic nucleotides, stemless beacons, for detection of nucleic acids, methylation status and mutants of nucleic acids
First Claim
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-QwhereinX 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, andwherein 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.
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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.
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Citations
14 Claims
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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, andwherein 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
- Y-Q
Specification