Library Compositions and Methods for Acyclic Identification of Aptamers
AssigneeAptamatrix, Inc.
InventorsPhilip N. Borer, Mark P. McPike
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Abstract
The present invention provides improved aptamer libraries useful for discovery of aptamers that have high binding affinity for a single or a plurality of targets. The libraries contain higher copies of each member candidate such that they are more likely to be available to the application of acyclic identification methods that obviate the most time-consuming and costly step in traditional SELEX method, the multiple cycles of evolutionary selection.
Claims27
A method for identifying an aptamer that binds to a target comprising the steps of:
- (a) providing a starting library of a starting pool of aptamer candidates;
- (b) reducing the sequence complexity of said starting library of aptamer candidates by a factor of at least about 100 to an intermediary pool of surviving aptamer candidates;
- (c) amplifying said intermediary library of surviving aptamer candidates to an over-represented library of said surviving aptamer candidates such that, for every 100 pmol of said surviving aptamer candidates, each surviving aptamer candidate has, on average, at least about three copies;
- (d) contacting said over-represented library with a target under a buffer condition that allows binding between members of said over-represented library and said target;
- (e) isolating at least one aptamer candidate that is bound to said target;
- and
- (f) determining the sequence of said bound aptamer candidate.
7-8. (canceled)
21-22. (canceled)
A method for identifying an aptamer that binds to a target comprising the steps of:
- (a) providing a plurality of starting libraries each comprising a starting pool of aptamer candidates, each of said aptamer candidates of substantially the same length while having a primary structure and at least one common pre-selected secondary structure, said primary structure comprising at least a variable nucleotide sequence where nucleotides at m number of positions are being varied, said common pre-selected secondary structure varying among said plurality of starting libraries;
- (b) reducing the sequence complexity in each of said plurality of starting libraries by a factor of at least about 100 to a plurality of intermediary libraries of surviving aptamer candidates;
- (c) amplifying each of said plurality of intermediary libraries to a plurality of over-represented libraries of said surviving aptamer candidates such that, for every 100 pmol of said surviving aptamer candidates, each surviving aptamer candidate has, on average, at least about three copies;
- (d) mixing said plurality of over-represented libraries into a comprehensive library with surviving aptamer candidates of varying secondary structures;
- (e) contacting said comprehensive library with a target under a buffer condition that allows binding between members of said comprehensive library and said target;
- (f) isolating at least one aptamer candidate that is bound to said target;
- and
- (g) determining the sequence of said bound aptamer candidate.
An aptamer library biased towards multiple pre-selected targets, comprising an over-represented library of aptamer candidates produced by amplifying a sparsely represented library such that each aptamer candidate has, on average, at least about three copies for every 100 pmol of said aptamer candidates, said sparsely represented library having been reduced of its sequence complexity through at least one round of refinement against at least about ten pre-selected targets.
Dependencies are inferred from the claim text.