Locked nucleic acid hybrids and methods of use
AssigneeInvitrogen Corporation
InventorsBoris Belotserkovskii, Gurucharan Reddy, David A. Zarling
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Abstract
The invention relates to methods for inhibiting, cloning, modifying or labelling an endogenous DNA sequence using compositions comprising recombinases in combination with exogenous polynucleotides containing “anchoring” or “locking” sequences. The anchoring sequences serve to stabilize structures formed by the exogenous polynucleotides and the endogenous DNA. The stabilized structure thus can either serve to regulate gene transcription or replication, or can allow the endogenous sequences to be labelled or pulled out, i.e. cloned, or modified.
Claims25
A composition comprising a double D-loop, said double-D loop comprising:
- a target nucleic acid;
- a first single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said first single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- a second single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said second single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- and
- a locking complex formed by the central regions of said first and said second single stranded polynucleotides,
- wherein said locking complex is a triplex or a quadruplex.
A composition comprising a double D-loop, said double D loop comprising:
- a target nucleic acid;
- a first single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said first single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- a second single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said second single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- and
- a locking complex formed by the central regions of said first and said second single stranded polynucleotides,
- wherein said locking complex comprises a quadruplex.
A composition comprising a double D-loop, said double D loop comprising:
- a target nucleic acid;
- a first single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said first single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- a second single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said second single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- and
- a locking complex formed by the central regions of said first and said second single stranded polynucleotides,
- wherein said locking complex comprises Z-form duplex.
A composition comprising a double D-loop, said double D loop comprising:
- a target nucleic acid;
- a first single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said first single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- a second single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said second single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- and
- a locking complex formed by the central regions of said first and said second single stranded polynucleotides,
- wherein said locking complex comprises a triplex.
A composition comprising a double D-loop comprising a target nucleic acid and a first and a second single stranded targeting polynucleotide, wherein said first and said second targeting polynucleotides are substantially complementary to each other and further wherein said first and said second targeting polynucleotides each comprise:
- a first and second homology clamp wherein said first and said second homology clamps substantially correspond to or are substantially complementary to a preselected target nucleic acid sequence;
- and
- a locking nucleic acid sequence positioned between said first and said second homology clamps wherein said locking nucleic acid sequence is capable of stabilizing a locking complex in a double D-loop structure, wherein said locking complex is a triplex or a quadruplex.
A composition comprising a double D-loop, said double-D loop comprising:
- a target nucleic acid;
- a first single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said first single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- a second single stranded targeting polynucleotide comprising:
- a central region and two flanking regions which flank the central region of said second single stranded targeting polynucleotide and which form homology clamps with said target nucleic acid;
- and
- a locking complex formed by the central regions of said first and said second single stranded polynucleotides,
- wherein said locking complex comprises at least one non-Watson-Crick base pair.
A kit comprising a first and a second single stranded targeting polynucleotide, wherein said first and said second targeting polynucleotides are substantially complementary to each other and further wherein said first and said second targeting polynucleotides each comprise:
- a first and a second homology clamp that wherein said first and said second homology clamps substantially correspond to or are substantially complementary to a preselected target nucleic acid sequence;
- and
- a locking nucleic acid sequence positioned between said first and said second homology clamps wherein said locking nucleic acid sequence is capable of stabilizing a locking complex in a double D-loop structure, wherein said locking complex is a triplex or a quadruplex.
Dependencies are inferred from the claim text.