Back to results
US 6,387,620 B1Granted
Transcription-free selex
AssigneeGilead Sciences, Inc.
InventorsJonathan Drew Smith, Larry Gold
Life of this patent
Estimating the term…
Abstract
Methods are provided for the production of nucleic acid ligands against target molecules using a procedure known as Transcription-free Systematic Evolution of Ligands by EXponential enrichment (Transcription-free SELEX). The Transcription-free SELEX method assembles nucleic acid ligands from fragments of synthetic nucleic acids by annealing those fragments to a complementary template, and then ligating the fragments together.
Claims9
A method for identifying nucleic acid ligands of a target compound, said method comprising:
- a) preparing a candidate mixture of ribonucleic acids;
- b) contacting the candidate mixture of nucleic acids with said target, wherein nucleic acids having an increased affinity to said target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;
- c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;
- d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said target;
- wherein said candidate mixture comprises ribonucleic acid molecules, each of said ribonucleic acid molecules being assembled from fragments of RNA comprising randomized sequence.
2 more elements
A method for the preparation of nucleic acid ligands to a target, the method comprising:
- (a) providing a DNA template library comprising fixed 3′ and 5′ sequence regions, and random internal sequences;
- (b) contacting said DNA template library with one or more RNA libraries, each said library comprising synthetic randomized RNA fragments, wherein said RNA fragments anneal to said DNA template, and wherein each said RNA fragment is shorter than said DNA template;
- (c) ligating said RNA fragments together to form a candidate mixture of RNA nucleic acid ligands;
- (d) purifying said candidate mixture of RNA nucleic acid ligands from said DNA template library and contacting said candidate mixture of RNA nucleic acid ligands with a target;
- (e) partitioning RNA nucleic acid ligands in said candidate mixture that interact with the target in a desired manner from those that do not;
- (f) reverse transcribing those RNA nucleic acid ligands that interact with the target in a desired manner to form DNA templates;
- (g) amplifying those DNA templates using the Polymerase Chain Reaction with primers that hybridize to said fixed 5′ and 3′ sequence regions to form a new DNA template library;
- (h) optionally repeating steps (b)-(g) for a desired number of repetitions.
5 more elements
A method for the preparation of RNA nucleic acid ligands to a target, the method comprising:
- (a) providing a DNA template library comprising fixed 3′ and 5′ sequence regions, and random internal sequences;
- (b) contacting said DNA template library with one or more RNA libraries, each said library comprising synthetic randomized RNA fragments, wherein said RNA fragments anneal to said DNA template, and wherein each said RNA fragment is shorter than said DNA template;
- (c) ligating said RNA fragments together to form a candidate mixture of RNA nucleic acid ligands;
- (d) purifying said candidate mixture of RNA nucleic acid ligands from said DNA template library and contacting said candidate mixture of RNA nucleic acid ligands with a target;
- (e) partitioning RNA nucleic acid ligands in said candidate mixture that interact with the target in the desired manner from those that do not;
- (f) contacting those RNA nucleic acid ligands that interact with the target in the desired manner with one or more DNA libraries, each said library comprising synthetic randomized DNA fragments, wherein said DNA fragments anneal to said RNA nucleic acid ligands, and wherein each said DNA fragment is shorter than said RNA nucleic acid ligands;
- (g) ligating said DNA fragments together to form new DNA templates;
- (h) amplifying those new DNA templates using the Polymerase Chain Reaction with primers that hybridize to said fixed 5′ and 3′ sequence regions to form a new DNA template library;
- (i) optionally repeating steps (b)-(h) for the desired number of repetitions.
6 more elements
Dependencies are inferred from the claim text.