Methods For Directed Folding Assembly Or Dimerization Of Proteins By Templated Assembly Reactions

Abstract

The present disclosure provides nucleic acid molecules, compositions, and kits comprising the same, and methods for producing templated assembly products.

What is claimed is: 
     
         1 . A haplomer-ligand complex comprising:
 a haplomer, wherein the haplomer comprises a polynucleotide that is substantially complementary to a target nucleic acid molecule; and   a ligand linked to the 5′ or 3′ terminus of the haplomer, wherein the ligand comprises a ligand partner binding site.   
     
     
         2 . A composition or kit comprising a first haplomer-ligand complex of  claim 1  and a second haplomer-ligand complex of  claim 1 , wherein:
 the ligand of the first haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex; and 
 the ligand of the second haplomer-ligand complex as linked to the 3′ terminus of the polynucleotide of the second haplomer-ligand complex. 
 
     
     
         3 . A bottle haplomer-ligand complex comprising:
 a) a bottle haplomer wherein the bottle haplomer comprises a polynucleotide, wherein the polynucleotide comprises:
 i) a first stem portion comprising from about 10 to about 20 nucleotide bases; 
   ii) an anti-target loop portion comprising from about 16 to about 40 nucleotide bases and having a first end to which the first stein portion is linked, wherein the anti-target loop portion is substantially complementary to a target nucleic acid molecule; and   iii) a second stem portion comprising from about 10 to about 20 nucleotide bases linked to a second end of the anti-target loop portion, wherein the first stem portion is substantially complementary to the second stem portion; and   b) a ligand linked to the terminal end of either the first stem portion or the second stem portion, wherein the ligand comprises a ligand partner binding site;   wherein the T m  of the anti-target loop portion:target nucleic acid molecule is greater than the T m  of the first stem portion:second stem portion.   
     
     
         4 . A composition or kit comprising:
 a bottle haplomer-ligand complex of  claim 3 ; and   a second haplomer-ligand complex comprising:
 a nucleotide portion comprising from about 6 to about 20 nucleotide bases that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex; and 
 a ligand linked to the 5′ or 3′ terminus of the nucleotide portion of the second haplomer-ligand complex, wherein the ligand comprises a ligand partner binding site; 
   wherein the T m  of the second haplomer-ligand complex:first or second stem portion linked to the ligand of the bottle haplomer-ligand complex is less than or equal to the T m  of the first stem portion:second stem portion of the bottle haplomer-ligand complex.   
     
     
         5 . A compound having the formula: 
       
         
           
           
               
               
           
         
       
       where m is from 3 to 6; having the formula: 
       
         
           
           
               
               
           
         
       
       where m is from 3 to 6; having the formula: 
       
         
           
           
               
               
           
         
       
       where n is from 1 to 6; having the formula: 
       
         
           
           
               
               
           
         
       
       where x is from 1 to 6; having the formula: 
       
         
           
           
               
               
           
         
       
       where x is from 1 to 6; or having the formula: 
       
         
           
           
               
               
           
         
       
     
     
         6 . A fusion protein comprising a fragment of a protein of interest fused to a ligand binding domain, wherein the ligand binding domain is an FKBP domain or an FRB domain. 
     
     
         7 . A composition or kit comprising a first fusion protein of  claim 6  and a second fusion protein of  claim 6 , wherein the protein of interest of the first fusion protein and the protein of interest of the second fusion protein can dimerize or fold together. 
     
     
         8 . A method for the directed assembly of a protein comprising:
 contacting a target nucleic acid molecule with a first haplomer-ligand complex of  claim 1 ;   contacting the target nucleic acid with a second haplomer-ligand complex of  claim 1 ;   contacting the first haplomer-ligand complex with a first fusion protein of  claim 6 ; and   contacting the second haplomer-ligand complex with a second fusion protein of  claim 6 ;   wherein the ligand of ate first haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex;   wherein the ligand of the second haplomer-ligand complex is linked to the 3 terminus of the polynucleotide of the second haplomer-ligand complex;   wherein the polynucleotide of the first haplomer-ligand complex is substantially complementary to a target nucleic acid molecule;   wherein the polynucleotide of the second haplomer-ligand complex is substantially complementary to the target nucleic acid molecule at a site in spatial proximity to the polynucleotide of the first haplomer-ligand complex;   wherein the ligand of the first haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and   wherein the ligand of the second e ligand binding domain of the second fusion protein can interact;   thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.   
     
     
         9 . A method for the directed assembly of a protein comprising:
 contacting a target nucleic acid molecule with a first haplomer-ligand complex of  claim 1 ;   contacting the target nucleic acid with haplomer-ligand complex of  claim 1 ;   contacting the first haplomer-ligand complex with a first fusion protein of  claim 6 ; and   contacting the second haplomer-ligand complex with a second fusion protein of  claim 6 ;   wherein the ligand of the first haplomer-ligand complex is finked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex;   wherein the ligand of the second haplomer-ligand complex is linked to the 3′ terminus of the polynucleotide of the second haplomer-ligand complex;   wherein the polynucleotide of the first haplomer-ligand complex is substantially complementary to a target nucleic acid molecule;   wherein the polynucleotide of the second haplomer-ligand complex is substantially complementary to the target nucleic acid molecule at a site in spatial proximity to the polynucleotide of the first haplomer-ligand complex;   wherein the ligand of the first haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and   wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;   thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.   
     
     
         10 . A method for the directed assembly of a protein comprising:
 contacting a target nucleic acid molecule with a complex formed by the interaction of a first haplomer-ligand complex of  claim 1  with a first fusion protein of  claim 6 , wherein the ligand of the first haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex, and wherein the ligand of the first haplomer-ligand complex interacts with the ligand binding domain of the first fusion protein; and   contacting the target nucleic acid molecule with a complex formed by the interaction of a second haplomer-ligand complex of  claim 1  with a second fusion protein of  claim 6 , wherein the ligand of the second haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the second haplomer-ligand complex, and wherein the ligand of the second haplomer-ligand complex interacts with the ligand binding domain of the second fusion protein;   thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.   
     
     
         11 . A method for the directed assembly of a protein comprising:
 contacting a target nucleic acid molecule with a complex formed by the interaction of a first haplomer-ligand complex of  claim 1  with a first fusion protein of  claim 6 , wherein the ligand of the first haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the first haplomer-ligand complex, and wherein the ligand of the first haplomer-ligand complex interacts with the ligand binding domain of the first fusion protein; and   contacting the target nucleic acid molecule with a complex formed by the interaction of a second haplomer-ligand complex of  claim 1  with a second fusion protein of  claim 6 , wherein the ligand of the second haplomer-ligand complex is linked to the 5′ terminus of the polynucleotide of the second haplomer-ligand complex, and wherein the ligand of the second haplomer-ligand complex interacts with the ligand binding domain of the second fusion protein;   thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.   
     
     
         12 . A method for he directed assembly of a protein comprising:
 contacting a target nucleic acid molecule with a bottle haplomer-ligand complex of  claim 3 ;   contacting the target nucleic acid with a second haplomer-ligand complex of  claim 1 , wherein the second haplomer-ligand complex comprises a nucleotide portion that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex;   contacting the bottle haplomer-ligand complex with a first fusion protein of  claim 6 , wherein the ligand of the bottle haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and   contacting the second haplomer-ligand complex with a second fusion protein of  claim 6 , wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;   thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.   
     
     
         13 . A method for the directed assembly of a protein comprising:
 contacting a target nucleic acid molecule with a bottle haplomer-ligand complex of  claim 3 ;   contacting the target nucleic acid with a second haplomer-ligand complex of  claim 1 , wherein the second haplomer-ligand complex comprises a nucleotide portion that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex;   contacting the bottle haplomer-ligand complex with a first fusion protein of  claim 6 , wherein the ligand of the bottle haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and   contacting the second haplomer-ligand complex with a second fusion protein of  claim 6 , wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;   thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.   
     
     
         14 . A method for the directed assembly of a protein comprising:
 contacting a target nucleic acid molecule with a bottle haplomer-ligand complex of  claim 3 ;   contacting the target nucleic acid molecule with a second haplomer-ligand complex of  claim 1 , wherein the second haplomer-ligand complex comprises a nucleotide portion that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex;   contacting the bottle haplomer-ligand complex with a first fusion protein of  claim 6 , wherein the ligand of the bottle haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and   contacting the second haplomer-ligand complex with a second fusion protein of  claim 6 , wherein the ligand of the second haplomer-ligand and complex and the ligand binding domain of the second fusion protein can interact;   thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.   
     
     
         15 . A method for the directed assembly of a protein comprising:
 contacting a target nucleic acid molecule with a bottle haplomer-ligand complex of  claim 3 ;   contacting the target nucleic acid molecule with a second haplomer-ligand complex of  claim 1 , wherein the second haplomer-ligand complex comprises a nucleotide portion that is substantially complementary to the stem portion of the bottle haplomer-ligand complex that is linked to the ligand of the bottle haplomer-ligand complex;   contacting the bottle haplomer-ligand complex with a first fusion protein of  claim 6 , wherein the ligand of the bottle haplomer-ligand complex and the ligand binding domain of the first fusion protein can interact; and   contacting the second haplomer-ligand complex with a second fusion protein of  claim 6 , wherein the ligand of the second haplomer-ligand complex and the ligand binding domain of the second fusion protein can interact;   thereby resulting in the folding or dimerization of the fragment of the protein of interest of the first fusion protein with the fragment of the protein of interest of the second fusion protein.