US 2003/0143612 A1 · Collections of binding proteins and tags and uses thereof for nested sorting and high throughput screening

Abstract

Provided herein are addressable collections of anti-tag capture agents, such as antibodies, that are used as tools for sorting proteins containing polypeptide tags for which the capture agents are specific. Also provided are methods of nested sorting using the collections. The methods include the steps of creating tagged collections of molecules by introducing a set of nucleic acid molecules that encode unique preselected polypeptides to create a library of tagged molecules; either before or after introducing the tags, dividing the library into N divisions; translating each division and reacting each with one of N capture agent collections, identifying the capture agents bound to the polypeptide tags linked to molecules of interest, and thereby identifying the one of the divided collections that contains the molecules of interest. The method can further include adding a new set of tags and repeating the sorting process with the same or a different collection capture agents and thereby identifying a protein or molecule of interest.

Claims31

Dependent claims

A method for screening a nucleic acid library, comprising:

a) creating a tagged library by a method comprising:
incorporating each one of a set of oligonucleotides that each comprises a region Em into a nucleic acid molecule in a library of nucleic acid molecules to create a tagged library, wherein:
the oligonucleotide comprises the formula:
5′-Em-3′;
each E encodes a sequence of amino acids to which a capture agent specifically binds;
each such sequence of amino acids is unique in the set;
and
m is, independently, an integer of 2 or higher;
b) translating the library or a sublibrary thereof;
c) contacting proteins from the translated library or sublibrary with a collection of capture agents to produce complexes between the tagged proteins and capture agents, wherein:
each of the capture agents specifically binds to a polypeptide encoding an Em;
and
each of the capture agents is identifiable;
d) screening the complexed capture agents to identify those that have bound to a translated protein of interest, thereby identifying the Em that is linked to the protein of interest.

11 more elements

A method for nested sorted, comprising:

a) creating tagged collections of nucleic acid molecules by incorporating each one of the set of oligonucleotides at one end of each nucleic acid molecule to create a master collection comprising N members, wherein the oligonucleotides have the formula:
5′-Dn-Em-3′
wherein:
each D is a unique sequence among the set of oligonucleotides and contains at least about 10 nucleotides;
each E encodes a sequence of amino acids that comprises epitope;
each epitope is unique in the set;
each epitope is a sequence to which a capture agent binds;
each of n and m is, independently, an integer of 2 or higher;
and
the oligonucleotides are single-stranded, double-stranded, and/or partially double-stranded;
b) amplifying each of n samples with a primer that comprises Dn to produce n sets of amplified nucleic acid reactions, wherein each reaction comprises amplified sequences that comprise a single Dn and all of the Em's;
c) translating each sample to produce n translated samples;
d) contacting proteins from each translated reaction with one of n collections of capture agents to produce complexes thereof, wherein each of the capture agents in the collection specifically reacts with a sequence of amino acids encoded by an Em;
and each of the antibodies can be identified;
e) screening the complexes to identify those that have bound to a protein of interest, thereby identifying the Em and Dn that is linked to nucleic acid molecules that encode the protein of interest.

12 more elements

Dependencies are inferred from the claim text.

Collections of binding proteins and tags and uses thereof for nested sorting and high throughput screening

Life of this patent

Collections of binding proteins and tags and uses thereof for nested sorting and high throughput screening

Life of this patent

Abstract

Claims31