In vivo oligonucleotide generator, and methods of testing the binding affinity of triplex forming oligonucleotides derived therefrom
First Claim
1. 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.
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Reexamination
Accused Products
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.
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Citations
28 Claims
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1. 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.
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2. An oligonucleotide generator, comprising from 5'"'"' to 3'"'"':
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(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. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A generator vector, comprising from 5'"'"' to 3'"'"':
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(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. - View Dependent Claims (22, 23, 24, 25, 26)
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27. A method of measuring triplex formation, which method comprises:
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(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.
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28. A method of measuring triplex blotting, which method comprises:
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(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.
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Specification