Oligonucleotide library encoding randomised peptides

Abstract

The invention relates to a method of producing an oligonucleotide library comprising a plurality of oligonucleotides, each oligonucleotide in the library having at least one predetermined position, a randomisation codon selected from a defined group of codons, the codons within said defined group coding for different amino acids. Vector, host cells containing such libraries and kits for the production of such libraries are also provided.

1 .- 20 . (canceled) 
     
     
         21 . A kit for producing an oligonucleotide library by a method, said method comprising a plurality of oligonucleotides, each oligonucleotide in the library having at least one predetermined position, a randomisation codon selected from a defined group of codons, the codons within said defined group coding for different amino acids, said method comprising the steps of:
 (a) Providing one or more double-stranded starter oligonucleotides, wherein the starter oligonucleotides have one or more blunt ends;   (b) Providing a plurality of different double stranded randomisation oligonucleotides comprising:
 (i) a coding strand, the coding strand comprising a randomisation codon; and 
 (ii) a substantially complementary non-coding strand, wherein each double stranded randomisation oligonucleotide comprises a nucleotide sequence coding for a restriction endonuclease recognition site capable of being recognised by a restriction endonuclease, the restriction endonuclease capable of cleaving the randomisation oligonucleotide upstream or downstream of the endonuclease recognition site at a predetermined cleavage site to create a blunt ended cut; 
   (c) Ligating each double-stranded starter oligonucleotide to a double-stranded randomisation oligonucleotide to form ligated oligonucleotides;   (d) Amplifying the ligated oligonucleotides;   (e) Digesting the ligated oligonucleotide with the restriction endonuclease to form a plurality of randomised double-stranded oligonucleotides, each of which comprise, at one end, a randomisation codon; and, optionally,   (f) Using the randomised double-stranded oligonucleotides as starter oligonucleotides and repeating method steps (a) to (e) and optional step (f) to produce a plurality of randomised double-stranded oligonucleotides, each comprising an additional randomisation codon;
 the kit comprises a plurality of different randomisation oligonucleotides comprising: 
 (i) a coding strand, the coding strand comprising a randomisation codon; and 
 (ii) a substantially complementary non-coding strand, 
 wherein each double stranded randomisation oligonucleotide comprises a nucleotide sequence coding for a restriction endonuclease recognition site capable of being recognised by a restriction endonuclease, the restriction endonuclease capable of cleaving the randomisation oligonucleotide upstream or downstream of the endonuclease recognition site at a predetermined cleavage site to create a blunt ended cut. 
   
     
     
         22 . A kit according to  claim 21 , wherein the restriction endonuclease recognition site is: 
       
         
           
                 
                 
               
                     
                   (SEQ. ID. No. 1) 
                 
                     
                   5′-GAGTCNNNNN{circumflex over ( )}-3′ 
                 
                     
                     
                 
                     
                   (SEQ. ID. No. 2) 
                 
                     
                   3′-CTCAGNNNNN{circumflex over ( )}-5′ 
                 
             
                
                
                
                
                
               
            
           
         
       
       where—N=any nucleotide
 ̂=the restriction endonuclease cleavage site. 
 
     
     
         23 . A kit according to  claim 21  comprising a restriction enzyme capable of cleaving the randomisation oligonucleotide at the predetermined cleavage site. 
     
     
         24 . A kit according to  claim 23 , wherein the restriction endonuclease is selected from SchI and MlyI. 
     
     
         25 . A kit according to  claim 21  wherein the randomisation codons consist of MAX codons which represent the optimum codon usage of a predetermined organism of interest or a predetermined selection of said MAX condons. 
     
     
         26 . A kit according to  claim 21 , the coding strand of the double-stranded randomisation oligonucleotide comprising a 5′ end and a 3′ end, the 3′ end of the coding strand comprising a blocking group. 
     
     
         27 . A kit according to  claim 26 , wherein the blocking group is selected from an amino group, a phosphate group, a glycerol moiety, a thiol group and a polyethylene glycol moiety. 
     
     
         28 . A kit according to  claim 21 , wherein the non-coding strand comprises a 3′ end and a 5′ end, the 5′ end of the non-coding strand extending one or more nucleotides beyond the 3′ end of the complementary coding strand. 
     
     
         29 . A kit according to  claim 21 , wherein the coding strand comprises the sequence: 
       
         
           
                 
                 
                 
               
                     
                   (I) 
                   5′ XXX{circumflex over ( )}(N) a R(N) b -B 3′ 
                 
                     
                   or 
                     
                 
                     
                     
                 
                     
                   (ii) 
                   5′-(N) b R(N) a {circumflex over ( )}XXX-3′ 
                 
             
                
                
                
                
               
            
           
         
       
       where: XXX is the randomisation codon,
 N is any nucleotide, 
 a is an integer of 0 to 10, preferably 5, 
 b is an integer of 0 to 40, preferably 1 to 20, or preferably 1 to 10, 
 ̂ is the restriction endonuclease cleavage site, 
 R′ is the reverse complement of the restriction endonuclease recognition site sequence, 
 R is the restriction endonuclease recognition site sequence, 
 B may or may not be present and when present may be selected from —OH, —NH 2 , phosphate, a glyceryl moiety, a thiol group and a polyethylene glycol moiety. 
 
     
     
         30 . A kit according to  claim 21  additionally comprising a predefined oligonucleotide comprising:
 (i) a coding strand, the coding strand comprising a predefined codon coding for a predefined amino acid; and 
 (ii) a substantially complementary non-coding strand, 
 
       wherein the predefined oligonucleotide comprises a nucleotide sequence coding for a restriction endonuclease recognition site capable of being recognised by a restriction endonuclease site, the restriction endonuclease capable of cleaving the predefined oligonucleotide upstream or downstream of the endonuclease recognition at a predetermined cleavage site to create a blunt ended cut. 
     
     
         31 . A kit according to  claim 21  additionally comprising a completion oligonucleotide having a predefined sequence. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled)