Platform for discovery and analysis of therapeutic agents

Abstract

A method of characterizing candidate agents including steps of (a) providing a library of candidate agents attached to nucleic acid tags; (b) contacting the library with a solid support to attach the candidate agents to the solid support, whereby an array of candidate agents is formed; (c) contacting the array with a screening agent, wherein one or more candidate agents in the array react with the screening agent; (d) detecting the array to determine that at least one candidate agent in the array reacts with the screening agent; (e) sequencing the nucleic acid tag to determine the tag sequences attached to candidate agents in the array; and (f) identifying the at least one candidate agent in the array that reacts with the screening agent based on the tag sequence that is attached to the at least one candidate agent.

What is claimed is: 
     
         1 . A method for producing an array of proteins, comprising
 (a) providing a library of mRNA molecules, wherein individual mRNA molecules in the library comprise a target sequence and a tag sequence,   (b) deriving a first sub-library from the library, the first sub-library comprising nucleic acids having the tag sequences or complements thereof, wherein the nucleic acids are attached to individual features on a solid support,   (c) deriving a second sub-library from the library, the second sub-library comprising nucleic acids having the target sequences and the tag sequences or complements thereof;   (d) contacting the second sub-library with the first sub-library, thereby attaching nucleic acids of the second sub-library to the solid support via hybridization of the tag sequences and the complements thereof; and   (e) translating the target sequences on the solid support to produce an array of proteins attached to the individual features.   
     
     
         2 . A method for producing an array of proteins, comprising
 (a) providing a library of cDNA molecules that are attached to a solid support;   (b) amplifying the cDNA molecules on the solid support to form clusters, wherein each cluster comprises multiple copies of a particular cDNA molecule from the library;   (c) transcribing the multiple copies at the clusters to produce multiple mRNA molecules attached to each of the clusters; and   (d) translating the mRNA molecules at the clusters to produce multiple proteins attached to each of the clusters.   
     
     
         3 . The method of  claim 1 , further comprising a step of sequencing the tag sequences or complements thereof on the solid support, thereby determining locations of the tag sequences or complements thereof at the individual features on the solid support; and/or wherein the nucleic acids of the first sub-library further comprise the target sequences. 
     
     
         4 . The method of  claim 3 , further comprising a step of sequencing the target sequences on the solid support. 
     
     
         5 . The method of  claim 2 , further comprising a step of sequencing at least a portion of each of the mRNA molecules on the solid support. 
     
     
         6 . A method of screening proteins comprising
 (i) producing an array of proteins according to  claim 1 ;   (ii) contacting the array of proteins with a screening agent, wherein one or more proteins in the array react with the screening agent; and   (iii) detecting the array of proteins during or after the contacting with the screening agent, thereby determining that at least one protein in the array reacts with the screening agent.   
     
     
         7 . A method of screening proteins comprising
 (i) producing an array of proteins according to  claim 2 ;   (ii) contacting the array of proteins with a screening agent, wherein one or more proteins in the array react with the screening agent; and   (iii) detecting the array of proteins during or after the contacting with the screening agent, thereby determining that at least one protein in the array reacts with the screening agent.   
     
     
         8 . The method of  claim 6 , further comprising a step of sequencing the tag sequences or complements thereof on the solid support, thereby determining locations of the tag sequences or complements thereof at the individual features on the solid support. 
     
     
         9 . The method of  claim 8 , further comprising a step of identifying the at least one protein in the array that reacts with the screening agent based on the tag sequence that is attached to the at least one protein. 
     
     
         10 . The method of  claim 7 , further comprising a step of sequencing at least a portion of each of the mRNA molecules on the solid support. 
     
     
         11 . The method of  claim 10 , further comprising a step of identifying the at least one protein in the array that reacts with the screening agent based on the sequence for at least a portion of the mRNA molecule that is attached to the at least one protein. 
     
     
         12 . The method of  claim 6  or  7 , wherein:
 (a) the screening agent reacts with the one or more proteins by binding to the one or more proteins or by blocking binding between the one or more proteins and an analyte having affinity for the one or more proteins; or 
 (b) the screening agent reacts with the one or more proteins by chemically modifying the one or more proteins or 
 (c) the screening agent reacts with the one or more proteins by producing an analyte product. 
 
     
     
         13 . The method of  claim 12 , wherein the screening agent reacts with the one or more proteins by binding to the one or more proteins or by blocking binding between the one or more proteins and an analyte having affinity for the one or more proteins and wherein the detecting of the array comprises detecting the screening agent that is bound to the one or more proteins. 
     
     
         14 . The method of  claim 12 , wherein the screening agent reacts with the one or more proteins by chemically modifying the one or more proteins, and wherein the detecting of the array comprises detecting the one or more modified proteins. 
     
     
         15 . The method of  claim 12 , wherein the screening agent reacts with the one or more proteins by producing an analyte product, and wherein the detecting of the array comprises detecting the analyte product. 
     
     
         16 . The method of  claim 1  or  claim 2 , wherein the library of mRNA molecules comprises a plurality of variants of the same gene. 
     
     
         17 . The method of  claim 16  wherein the variants are produced by random mutagenesis. 
     
     
         18 . The method of  claim 1 , wherein:
 (a) the first sub-library is derived by a method that includes contacting mRNA molecules of the library with the solid support to attach the mRNA molecules to the solid support; or   (b) the solid support comprises nucleic acid primers and the mRNA molecules attach to the solid support via hybridization to the nucleic acid primers; or   (c) the first sub-library is derived by a method that includes reverse transcribing the individual mRNA molecules or a portion of the individual mRNA molecules comprising the tag sequence; or   (d) the library of mRNA molecules is provided in a fluidic sample, and wherein the first sub-library and the second sub-library are derived from separate fractions of the fluidic sample; or   (e) the solid support is located within a flow cell; or   (f) the proteins are covalently attached to the mRNA molecules; or   (g) the method further comprises a step of selectively removing the mRNA molecules or proteins that are attached to one or more features of the array; or   (h) the selective removal comprises laser mediated cleavage of a bond that attaches the mRNA molecules or proteins to the features.   
     
     
         19 . The method of  claim 18 , wherein the first sub-library is derived by a method that includes contacting mRNA molecules of the library with the solid support to attach the mRNA molecules to the solid support, and wherein the mRNA molecules are amplified on the solid support to produce the complements of the tag sequences. 
     
     
         20 . The method of  claim 18 , wherein the solid support comprises nucleic acid primers and the mRNA molecules attach to the solid support via hybridization to the nucleic acid primers, wherein the mRNA molecules comprise a universal primer binding sequence, the nucleic acid primers comprise a universal primer sequence, and the mRNA molecules attach to the solid support via hybridization of the universal primer binding sequence to the universal primer sequence. 
     
     
         21 . The method of  claim 18 , wherein the first sub-library is derived by a method that includes reverse transcribing the individual mRNA molecules or a portion of the individual mRNA molecules comprising the tag sequence, wherein the reverse transcribed mRNA molecules, or portions thereof, are amplified on the solid support to produce the complements of the tag sequences. 
     
     
         22 . The method of  claim 1 , wherein:
 (i) the first sub-library comprises nucleic acids having complements of the tag sequences, wherein the second sub-library comprises RNA molecules having the target sequences and the tag sequences, and wherein (d) comprises contacting the second sub-library with the first sub-library, thereby attaching mRNA molecules of the second sub-library to the solid support via hybridization of the tag sequences and the complements thereof; or   (ii) the first sub-library comprises nucleic acids having the tag sequences, wherein the second sub-library comprises cDNA molecules having the target sequences and complements of the tag sequences, wherein (d) comprises contacting the second sub-library with the first sub-library, thereby attaching cDNA molecules of the second sub-library to the solid support via hybridization of the tag sequences and the complements thereof, and wherein (e) comprises reverse transcribing the cDNA molecules to produce mRNA molecules on the solid support and translating the mRNA molecules on the solid support to produce an array of proteins attached to the individual features; or   (iii) the target sequences are translated with ribosomes on the solid support and the ribosomes are treated with puromycin to produce an array of proteins attached to the individual features.   
     
     
         23 . An array comprising:
 (i) mRNA molecules,   (ii) a solid support comprising nucleic acids having complements of sequences of the mRNA molecules, wherein the nucleic acids are attached to individual features on a solid support,   wherein the sequences of the individual mRNA molecules are hybridized or attached to respective complementary nucleic acid sequences at the individual features on the solid support, and   wherein proteins derived by translation of the mRNA molecules are attached to respective mRNA molecules;   wherein either:   (A) said mRNA molecules are comprised in a library of mRNA molecules, wherein individual mRNA molecules in the library comprise a target sequence and a tag sequence, the sequences of the mRNA molecules to which the nucleic acids of the solid support are complementary are the tag sequences, and wherein the sequences of the individual mRNA molecules which are hybridized to respective complementary nucleic acid sequences are the tag sequences and the respective complementary nucleic acid sequences are complementary tag sequences; or   (B) said nucleic acids having complements of sequences of the mRNA molecules are members of a library of different cDNA molecules attached to the solid support, wherein each different cDNA molecule is attached to an individual feature on the solid support, and wherein each feature includes multiple copies of a particular cDNA molecule; and said mRNA molecules are attached to the cDNA molecules, wherein each of the cDNA molecules is complementary to the respective attached mRNA molecule.   
     
     
         24 . The array of  claim 23 , wherein:
 (i) the proteins are attached to the respective mRNA molecules via ribosomes; or   (ii) the proteins comprise a luminescent label; or   (iii) the solid support comprises at least 1×10 6  of the features; or   (iv) the average pitch of the features on the solid support is less than 10 microns; or   (v) the features comprise an average area of less than 100 square microns; or   (vi) the array has the features recited as [A] and the library of mRNA molecules comprise a plurality of variants of the same gene; or   (vii) the array has the features recited as [B] and the library of cDNA molecules comprise a plurality of variants of the same gene; or   (viii) the proteins are selected from the group consisting of antibodies, enzymes, receptors, kinases, phosphatases, polymerases, proteases, esterases, histone modifying enzymes and nuclear hormone receptors; or   (ix) the solid support is located within a flow cell.