Methods for cyclizing synthetic polymers
View Patent ↗The invention provides methods and compositions for production of a cyclic polymer in a cell free system. In general, the methods of the invention involve ligating first and second recombinant intein domains to a linear synthetic polymer to form a compound containing the structure: D 1 -X (n) -D 2 , where D 1 is a first catalytic domain of an intein; D 2 is a second catalytic domain of an intein; where the second catalytic domain has at its N-terminus a first reactive site for the intein; and X (n) is a polymer of a number n of monomer X, where the polymer N-terminus has a second reactive site for the intein. D 1 -X (n) -D 2 compounds autocatalytically cyclize the X (n) polymer to produce a cyclic polymer. The invention finds use in a variety of drug discovery, clinical and therapeutic applications.
1. A method comprising:
native chemical ligating a first isolated intein domain D 1 and a second isolated intein domain D 2 to-polymer X (n) in a cell-free system to produce a compound comprising the structure:
D 1 -X (n) -D 2
wherein:
polymer X (n) comprises n residues of a monomer X, wherein n is at least 2, and polymer X (n) is not made up of only naturally-occurring amino acid monomers;
said compound comprises a first intein reactive site at the N-terminus of polymer X (n) ;
said compound comprises a second intein reactive site at the N-terminus of D 2 ; and
said compound produces a cyclic product comprising said polymer, wherein said cyclic product is cyclized via a reaction that joins said first intein reactive site and said second intein reactive site.
2. The method of claim 1 , wherein said ligating comprises:
ligating said first intein domain D 1 with said polymer X (n) to produce a compound of the formula:
D 1 -X (n)
wherein D 1 and X (n) are joined by a first peptide bond; and,
ligating said second isolated intein domain D 2 with said polymer X (n) to produce a compound of the formula:
D 1 -X (n) -D 2
wherein D 1 and X (n) are joined by a second peptide bond.
3. The method of claim 1 , wherein said ligating comprises:
ligating a first intein domain D 1 having a C-terminal reactive thioester with said polymer X (n) , said polymer X (n) having an N-terminal sulfhydryl group and a protected C-terminal thioester group, said ligating producing a compound of the formula:
D 1 -X (n) —Z
wherein polymer X (n) comprises n residues of a monomer X, wherein n is at least 2, and Z is said protected C-terminal thioester group,
deprotecting said protected C-terminal thioester group to produce a C-terminal reactive thioester group; and
ligating said C-terminal reactive thioester group with said second intein domain D 2 having an N-terminal sulfhydryl group to produce a compound of the formula:
D 1 -X (n) -D 2
4. The method of claim 1 , wherein said polymer comprises at least one non-naturally occurring monomer.
5. The method of claim 1 , wherein said polymer comprises at least one amino acid monomer.
6. The method of claim 1 , wherein said polymer comprises at least one nucleotide monomer.
7. The method of claim 1 , wherein said first and second intein reactive sites comprise sulfhydryl or hydroxyl moieties.
8. The method of claim 7 , wherein said first and second intein reactive sites comprise cysteine, alanine or serine, or an analog thereof.
9. The method of claim 1 , wherein said polymer is a homopolymer.
10. The method of claim 1 , wherein said polymer is a heteropolymer.
11. The method of claim 1 , further comprising incubating said compound under intein reaction conditions to produce said cyclic product.
12. The method of claim 1 , wherein said polymer is synthetically made.