In vivo oligonucleotide generator, and methods of testing the binding affinity of triplex forming oligonucleotides derived therefrom
AssigneeThe Regents of the University of California
InventorsSarah B. Noonberg, C. Anthony Hunt
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Abstract
The present invention encompasses improved methods and materials for the delivering of antisense, triplex, and/or ribozyme oligonucleotides intracellularly, and RNA polymerase III-based constructs termed "oligonucleotide generators" to accomplish the delivery of oligonucleotides. Also encompassed by the present invention are methods for screening oligonucleotide sequences that are candidates for triplex formation.
Claims28
A construct for generating a specific oligonucleotide within a cell, which construct comprises a nucleotide sequence from which the transcript is the specific oligonucleotide, said nucleotide sequence being flanked in the 5' direction by a stabilizing region and in the 3' direction by a termination sequence, and a promoter, which initiates transcription by RNA polymerase III, and which promoter is in the 5' direction from the stabilizing region.
An oligonucleotide generator, comprising from 5' to 3':
- (a) an U6-type RNA polymerase III promoter;
- (b) a specific nucleotide sequence from which a specific oligonucleotide can be transcribed;
- and
- (c) a termination sequence;
- wherein the components of the oligonucleotide generator are operably linked;
- and wherein the oligonucleotide generator is capable of being transcribed by RNA polymerase III to produce a transcript comprising the specific oligonucleotide.
A generator vector, comprising from 5' to 3':
- (a) a U6-type promoter;
- (b) a stabilizing region from which a hairpin-forming sequence can be transcribed;
- and
- (c) a termination sequence;
- wherein the components of the generator vector are operably linked.
A method of measuring triplex formation, which method comprises:
- (a) attaching a single-stranded nucleic acid to a solid support;
- (b) contacting the solid support with a fluid comprising a labeled double-stranded probe;
- (c) separating the unbound probe from the solid support;
- and
- (d) quantifying the amount of labeled double-stranded probe bound to the solid support.
A method of measuring triplex blotting, which method comprises:
- (a) attaching a double-stranded nucleic acid to a solid support;
- (b) contacting the solid support with a fluid comprising a labeled single-stranded probe;
- (c) separating the unbound probe from the solid support;
- and
- (d) quantifying the amount of labeled single-stranded probe bound to the solid support.
Dependencies are inferred from the claim text.