Pumilio domain-based modular protein architecture for rna binding

Abstract

A programmable modular protein architecture for RNA binding comprises a set of modules, derived from RNA-binding protein Pumilio, that can be concatenated into chains of varying composition and length. When bound into a chain, each module has a preferred affinity for a specific RNA base. The chains can bind arbitrary RNA sequences with high specificity and fidelity by varying the sequence of modules within the chains. Each module contains at least 6 amino acids, with the amino acids in positions 1 and 5 providing the preferred affinity for the specific base, and the amino acid at position 2 serving as a stacking unit between concatenated modules. The modules may have four canonic forms, each having a preferred affinity for a different base and characterized by the base with which it has affinity, the two amino acids that provide the affinity, and the amino acid that serves as a stacking unit.

What is claimed is: 
     
         1 . An RNA binding fusion protein comprising a modular  Pumilio  protein architecture for RNA binding and an endonuclease,
 wherein the modular  Pumilio  protein comprises a set of protein modules concatenated into chains of varying composition and length,   wherein each protein module, when bound into a chain, has a preferred affinity for a specific target RNA base,   wherein the concatenated chains of protein modules provide the ability to bind arbitrary RNA sequences, with high specificity and fidelity, by varying the sequence of the protein modules within the chains, and   wherein each protein module comprises at least 6 amino acids selected such that the amino acids in positions 1 and 5 provide the preferred affinity of the protein module for the specific RNA base and the amino acid at position 2 serves as a stacking unit between concatenated modules.   
     
     
         2 . The RNA binding fusion protein of  claim 1 , wherein the endonuclease is a PIN domain endonuclease. 
     
     
         3 . A universal programmable RNA-binding fusion protein comprising a  Pumilio  modular unit and an endonuclease,
 wherein the  Pumilio  modular unit is concatenated with other modular units,   wherein the  Pumilio  modular unit has a preferred affinity for a specific RNA base with high specificity and fidelity, and   wherein the  Pumilio  modular unit comprises at least 6 amino acids selected such that the amino acids in positions 1 and 5 provide the preferred affinity of the modular unit for the specific RNA base and the amino acid at position 2 is configurable to serve as a stacking unit between concatenated modular units.   
     
     
         4 . The universal programmable RNA-binding fusion protein of  claim 3 , wherein the endonuclease is endonuclease is a PIN domain endonuclease. 
     
     
         5 . The RNA binding fusion protein of  claim 1 , wherein the protein modules have four canonic forms, with each canonic form having a preferred affinity for a different RNA base. 
     
     
         6 . The RNA binding fusion protein of  claim 5 , wherein the four canonic forms are characterized by the RNA base with which they have a preferred affinity, the two amino acids of the six-residue peptide modular unit that provide the preferred affinity for that base, and the amino acid of the six-residue peptide modular unit that serves as a stacking unit between concatenated six-residue peptide modular units. 
     
     
         7 . The RNA binding fusion protein of  claim 6 , the four canonic forms consisting of:
 Form 1, which has a preferred affinity for RNA base Adenine, and has Cysteine at position 1, Tyrosine at position 2, and Glutamine at position 5;   Form 2, which has a preferred affinity for RNA base Uracil, and has Asparagine at position 1, Tyrosine at position 2, and Glutamine at position 5;   Form 3, which has a preferred affinity for RNA base Guanine, and has Serine at position 1, Tyrosine at position 2, and Glutamic Acid at position 5; and   Form 4, which has a preferred affinity for RNA base Cytosine, and has Serine at position 1, Tyrosine at position 2, and Arginine at position 5.   
     
     
         8 . The RNA binding fusion protein of  claim 1 , wherein the stacking unit at position 2 is Tyrosine or Arginine. 
     
     
         9 . The RNA binding fusion protein of  claim 1 , wherein the chain has between six and eighteen six-residue peptide modular units. 
     
     
         10 . The RNA binding fusion protein of  claim 1 , wherein the residues of the six-residue peptide modular unit are defined in one of the thirty-two amino acid sequences in  FIG. 3  at amino acid positions 1, 2, and 5. 
     
     
         11 . The RNA binding fusion protein of  claim 1 , wherein at least one of the modular units has a preferred affinity for RNA base Adenine, modular unit amino acid 1 is Cysteine, modular unit amino acid 2 is Tyrosine, and modular unit amino acid 5 is Glutamine. 
     
     
         12 . The RNA binding fusion protein of  claim 1 , wherein at least one of the modular units has a preferred affinity for RNA base Uracil, modular unit amino acid 1 is Asparagine, modular unit amino acid 2 is Tyrosine, and modular unit amino acid 5 is Glutamine. 
     
     
         10 . The RNA binding fusion protein of  claim 1 , wherein at least one of the modular units has a preferred affinity for RNA base Guanine, modular unit amino acid 1 is Serine, modular unit amino acid 2 is Tyrosine, and modular unit amino acid 5 is Glutamic Acid. 
     
     
         13 . The RNA binding fusion protein of  claim 1 , wherein at least one of the modular units has a preferred affinity for RNA base Cytosine, modular unit amino acid 1 is Serine, modular unit amino acid 2 is Tyrosine, and modular unit amino acid 5 is Arginine. 
     
     
         14 . A method for targeted silencing of RNA transcripts in living cells, the method comprising the steps of:
 (a) producing an RNA binding fusion protein comprising a modular  Pumilio  protein architecture for RNA binding and an endonuclease   wherein the modular  Pumilio  protein comprises concatenated six-residue peptide modular units in a chain of six or more six-residue peptide modular units of suitable composition and length for binding a target RNA molecule;   wherein each protein module, when bound into a chain, has a preferred affinity for a specific target RNA base,   wherein the concatenated chains of protein modules provide the ability to bind arbitrary RNA sequences, with high specificity and fidelity, by varying the sequence of the protein modules within the chains,   wherein each protein module comprises at least 6 amino acids selected such that the amino acids in positions 1 and 5 provide the preferred affinity of the protein module for the specific RNA base and the amino acid at position 2 serves as a stacking unit between concatenated modules, and   (b) binding the RNA-binding fusion protein to the target RNA molecule, thereby bringing the endonuclease into contact with the RNA molecule and silencing transcription of the RNA molecule.   
     
     
         15 . The method of  claim 14 , wherein the six-residue peptide modular units have four canonic forms, with each canonic form having a preferred affinity for a different RNA base. 
     
     
         16 . The method of  claim 15 , wherein the four canonic forms are characterized by the RNA base with which they have a preferred affinity, the two amino acids of the six-residue peptide modular unit that provide the preferred affinity for that base, and the amino acid of the six-residue peptide modular unit that serves as a stacking unit between concatenated six-residue peptide modular units. 
     
     
         17 . The method of  claim 16 , wherein the four canonic forms are:
 Form 1, which has a preferred affinity for RNA base Adenine, and has Cysteine at position 1, Tyrosine at position 2, and Glutamine at position 5;   Form 2, which has a preferred affinity for RNA base Uracil, and has Asparagine at position 1, Tyrosine at position 2, and Glutamine at position 5;   Form 3, which has a preferred affinity for RNA base Guanine, and has Serine at position 1, Tyrosine at position 2, and Glutamic Acid at position 5; and   Form 4, which has a preferred affinity for RNA base Cytosine, and has Serine at position 1, Tyrosine at position 2, and Arginine at position 5; and   wherein the stacking unit at position 2 is Tyrosine or Arginine.   
     
     
         18 . The method of  claim 14 , wherein the residues of the six-residue peptide modular unit are defined in one of the thirty-two amino acid sequences in  FIG. 3  at amino acid positions 1, 2, and 5. 
     
     
         19 . An expression vector encoding the RNA binding fusion protein of  claim 1 . 
     
     
         20 . An expression vector encoding the universal programmable RNA-binding fusion protein of  claim 3 .