Compositions and methods for the identification and selection of nucleic acids and polypeptides

Abstract

This invention relates generally to systems and methods for identifying and selecting, desired proteins or nucleic acid molecules by linking mRNA, with known or unknown sequences, to its translated protein to form a cognate pair. The cognate pair is selected based upon desired properties of the protein or the nucleic acid. This method also includes the evolution of a desired protein or nucleic acid molecule by amplifying the nucleic acid portion of the selected cognate pair, introducing variation into the nucleic acid, translating the nucleic acid, attaching the nucleic acid to its protein to form a second cognate pair, and re-selecting this cognate pair based upon desired properties. Modified mRNAs operable to crosslink to tRNAs are also provided. Methods of producing a psoralen monoadduct or a crosslink are also provided.

What is claimed is:  
     
         1 . A modified mRNA molecule operable to crosslink to a tRNA molecule, wherein the modified mRNA molecule comprises a crosslinker located on or near a stop codon.  
     
     
         2 . The modified mRNA molecule of  claim 1 , wherein the crosslinker is an agent that can be activated to form one or more covalent bonds with the tRNA.  
     
     
         3 . The modified mRNA molecule of  claim 1 , wherein the crosslinker is an agent that is activated to form one or more covalent bonds with the tRNA using light.  
     
     
         4 . The modified mRNA molecule of  claim 1 , wherein the crosslinker is a modified base that is incorporated directly into the mRNA.  
     
     
         5 . The modified mRNA molecule of  claim 1 , wherein the crosslinker is selected from the group consisting of one or more of the following 2-thiocytosine, 2-thiouridine, 4-thiouridine, 5-iodocytosine, 5-iodouridine, 5-bromouridine and 2-chloroadenosine, aryl azides, and modifications or analogues thereof.  
     
     
         6 . The modified mRNA molecule of  claim 1 , wherein the crosslinker is psoralen.  
     
     
         7 . A modified mRNA molecule operable to crosslink to a tRNA molecule, wherein the modified mRNA molecule comprises a crosslinker located on or near a pseudo stop codon.  
     
     
         8 . The modified mRNA molecule of  claim 7 , wherein the crosslinker is an agent that can be activated to form one or more covalent bonds with the tRNA.  
     
     
         9 . The modified mRNA molecule of  claim 7 , wherein the crosslinker is an agent that is activated to form one or more covalent bonds with the tRNA using light.  
     
     
         10 . The modified mRNA molecule of  claim 7 , wherein the crosslinker is a modified base that is incorporated directly into the mRNA.  
     
     
         11 . The modified mRNA molecule of  claim 7 , wherein the crosslinker is selected from the group consisting of one or more of the following 2-thiocytosine, 2-thiouridine, 4-thiouridine, 5-iodocytosine, 5-iodouridine, 5-bromouridine and 2-chloroadenosine, aryl azides, and modifications or analogues thereof.  
     
     
         12 . The modified mRNA molecule of  claim 7 , wherein the crosslinker is psoralen.  
     
     
         13 . A kit to generate cognate pairs comprising at least one psoralen monoadduct attached to a nonadducted stable aminoacyl tRNA analog or at least one psoralen monoadduct attached to an oligonucleotide.  
     
     
         14 . A method for evolving a desired protein sequence comprising: 
 providing at least two candidate mRNA molecules, wherein at least one of said mRNA molecules contains at least one codon selected from the group consisting of a stop codon and a pseudo stop codon;    translating at least two of said candidate mRNA molecules to generate at least one translated protein;    linking at least one of said candidate mRNA molecules to its corresponding translated protein via a tRNA molecule to form at least one cognate pair, wherein at least one of said candidate mRNA molecules is connected to said tRNA molecule by a crosslinker;    identifying one or more of said cognate pairs based upon the properties of said translated protein or said mRNA molecule;    identifying a molecule selected from the group consisting of one or more of the following: an mRNA molecule of said selected cognate pair, a nucleic acid molecule complementary to said mRNA molecule and a nucleic acid molecule homologous to said mRNA molecule, thereby identifying said desired protein or said desired nucleic acid molecule;    providing a plurality of cognate pairs,    binding at least of said plurality of cognate pairs with one or more binding agents;    selecting said desired or protein nucleic acid molecule based upon a reaction or lack of a reaction to said one or more binding agents, thereby selecting a first desired cognate pair;    recovering said first desired cognate pair to generate a recovered cognate pair;    amplifying a first nucleic acid component of said recovered cognate pair;    producing a second nucleic acid component, wherein said second nucleic acid component comprises said first nucleic acid component with one or more variations;    producing a second protein by translating said second nucleic acid component;    linking said second protein with said second nucleic acid component to generate a second desired cognate pair; and    obtaining the desired protein sequence by re-selecting said second desired cognate pair based upon at least one desired property.    
     
     
         15 . The method of  claim 14 , wherein said desired property is selected from the group consisting of one or more of the following: binding properties, enzymatic reactions and chemical modifications.  
     
     
         16 . The method of  claim 14 , wherein said desired property is an ability to resist binding, enzymatic reaction or chemical modification.  
     
     
         17 . The method of  claim 14 , wherein the step of selecting said first desired cognate pair comprises: 
 providing a first ligand with a desired binding characteristic;    contacting one or more of said first cognate pairs with said first ligand to generate unbound complexes and bound complexes;    recovering either the bound complexes or the unbound complexes;    amplifying at least one nucleic acid component of the recovered complexes;    introducing variation to a sequence of said nucleic acid component of said recovered complexes;    translating one or more second proteins from said nucleic acid components,    linking at least one of said second proteins with at least one of said second nucleic acid components to generate one or more second cognate pairs; and    obtaining the desired protein sequence by contacting said at least one of said second cognate pairs with at least one second ligand to select one or more of said second cognate pairs, wherein said second ligand is the same or different than said first ligand.    
     
     
         18 . A method of forming a psoralen monoadduct on a nucleic acid, comprising: 
 providing a first nucleic acid and a second nucleic acid, 
 wherein said first nucleic acid and said second nucleic acid are substantially complementary to each other,  
 wherein said first nucleic acid comprises one or more uridine monoadduct targets, and  
 wherein said second nucleic acid comprises at least one pseudoridine hybridizing said first nucleic acid and said second nucleic acid in the presence of psoralen to form a hybrid;  
   irradiating said hybrid with ultraviolet light, thereby forming said psoralen monoadduct on said first nucleic acid.    
     
     
         19 . The method of  claim 18 , wherein said one or more uridine monoadduct targets comprises a uridine located adjacent to an adenosine  
     
     
         20 . The method of  claim 18 , wherein said one or more uridine monoadduct targets comprises a uridine located adjacent to and 3′ from an adenosine  
     
     
         21 . A method of producing a psoralen monoadduct or a crosslink, comprising: 
 providing a first nucleic acid and a second nucleic acid;    wherein said first nucleic acid and said second nucleic acid are substantially complementary to each other;    wherein said first nucleic acid comprises one or more uridine monadduct targets or crosslink targets and one or more uridine monoadduct non-targets or crosslink non-targets;    wherein said uridine monoadduct non-targets or crosslink non-targets are operable to be replaced with one or more pseudouridines;    replacing one or more of said uridine monoadduct non-targets or crosslink non-targets with pseudouridine;    hybridizing said first nucleic acid and said second nucleic acid in the presence of psoralen to form a hybrid;    irradiating said hybrid, thereby forming said psoralen monoadduct or said crosslink on said first nucleic acid on said targets, while protecting said nontargets.