Transcription-free SELEX

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.

What is claimed is:  
     
         1 . A method of identifying nucleic acid ligands that photocrosslink to a target from a candidate mixture of ribonucleic acids, wherein each member of said candidate mixture comprises one or more photoreactive groups, the method comprising: 
 a) contacting said candidate mixture of nucleic acids with target, wherein nucleic acids having an increased affinity to target relative to the candidate mixture form nucleic acid-protein complexes with the target;    b) irradiating said complexes, wherein said nucleic acid and target photocrosslink;    c) partitioning the photocrosslinked nucleic acid-target complexes from the candidate mixture; and    d) identifying a nucleic acid ligand that photocrosslinked to the target    wherein each member of said candidate mixture is assembled from fragments of RNA comprising randomized sequence.    
     
     
         2 . The method of  claim 1  wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.  
     
     
         3 . The method of  claim 1  further comprising after step c): 
 i) repeating steps a), b) and c); and  
 ii) amplifying the nucleic acid ligands that photocrosslinked to the target.  
 
     
     
         4 . The method of  claim 3  wherein said photoreactive group is 5-bromouracil.  
     
     
         5 . The method of  claim 1  wherein said RNA fragments comprise synthetic RNA molecules.  
     
     
         6 . The method of  claim 5  wherein said synthetic RNA molecules comprise at least one non-naturally occurring ribonucleotide.  
     
     
         7 . The method of  claim 6  wherein said non-naturally-occurring ribonucleotide is a 2′-OMe ribonucleotide.  
     
     
         8 . The method of  claim 1  wherein said candidate mixture of nucleic acids is assembled from fragments of RNA by annealing said RNA fragments to a complementary DNA template, and then ligating said annealed RNA fragments.  
     
     
         9 . A method for the preparation of nucleic acid ligands that photocrosslink to in 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, each RNA fragment comprising one or more photoreactive groups, 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, wherein RNA nucleic acids ligands having an increased affinity to target relative to the candidate mixture form nucleic acid-protein complexes with the target;    e) irradiating said complexes, wherein said RNA nucleic acid ligand and said target photocrosslink;    f) partitioning the photocrosslinked nucleic acid-target complexes from the candidate mixture;    g) reverse transcribing those RNA nucleic acid ligands that photocrosslinked to the target to form DNA templates;    h) 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;    i) optionally repeating steps (b)-(h) for a desired number of repetitions.    
     
     
         10 . The method of  claim 9  wherein a first, second and a third library comprising synthetic randomized RNA fragments are used in step (b), wherein said first library further comprises a fixed RNA sequence complementary to the 5′ fixed regions of said DNA template, and wherein said second library further comprises a fixed RNA sequence complementary to the 3′ fixed sequence region of said DNA template.  
     
     
         11 . The method of  claim 10  wherein said first library comprises X ribonucleotides of fixed sequence and Y ribonucleotides of randomized sequence; wherein said second library comprises A ribonucleotides of fixed sequence and B ribonucleotides of randomized sequence; wherein said third library comprises Z ribonucleotides of randomized sequence; and wherein X+Y>Z, A+B>Z, Y>X, B>A, Z>Y, and Z>B.  
     
     
         12 . A method for the preparation of RNA nucleic acid ligands that photocrosslink 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 RNA library comprising synthetic randomized RNA fragments, each RNA fragment comprising one or more photoreactive groups, 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, wherein RNA nucleic acids ligands having an increased affinity to target relative to the candidate mixture form nucleic acid-protein complexes with the target;    e) irradiating said complexes, wherein said RNA nucleic acid ligand and said target photocrosslink;    f) partitioning the photocrosslinked nucleic acid-target complexes from the candidate mixture;    g) contacting those RNA nucleic acid ligands that photocrosslinked to the target with one or more DNA libraries, each said DNA 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;    h) ligating said DNA fragments together to form new DNA templates;    i) 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;    j) optionally repeating steps (b)-(i) for the desired number of repetitions.