Genetic encoding of chemical post-translational modification for phage-displayed libraries
The present application provides a method of synthesizing a genetically-encoded chemical modification of a peptide library. A vector in a substrate, such as a phage, is modified to include a peptide linker and a modification to form a genetic “barcode”. The barcode is screened against potential targets which may be used in drug discovery.
1. A method for genetic encoding of chemical modifications in genetically encoded libraries of chemically modified peptides expressed on a genetically encoded display system, comprising the steps of:
(a) producing multiple peptides expressed on a genetically encoded display system with different chemical modifications, wherein said multiple peptides have identical amino acid sequences but different genetic sequences due to the presence of a silent barcode region in genetic sequences that produce identical transcriptional products, wherein the genetically encoded display system that encode different silent barcode regions are produced separately, wherein identical peptides expressed on the genetically encoded display system that encode different silent barcode regions are modified by different chemical modifications,
(b) repeating step (a) by producing a peptide with a different amino acid sequence in a region encoding a peptide variable region compared to the peptide amino acid sequence produced in step (a), wherein the peptide with a different amino acid sequence produced in step (b) has a substantially similar amino acid sequence compared to the amino acid sequence of the peptide produced in step (a),
(c) repeating step (b) multiple times, and
(d) pooling the peptides with different chemical modifications expressed on the genetically encoded display system produced in steps (a), (b) and (c) to create a library in which the peptide amino acid sequences can be determined by genetic sequencing of the region encoding the peptide variable region and the modification can be determined by genetic sequencing of the silent barcode region.
2. The method of claim 1 , wherein different peptides with a different amino acid sequences are generated by random mutagenesis.
3. The method of claim 1 , wherein each of the different chemical modifications of said multiple peptides have identical amino acid sequences comprise introducing a different small molecule at any amino acid residue in the peptide.
4. The method of claim 3 , wherein the chemical modifications of introducing a different small molecule at any amino acid residue in the peptide comprise site-specific chemical conjugation.
5. The method of claim 4 , wherein the small molecule is a carbohydrate, biotin, or sulphonamide.
6. The method of claim 4 , wherein the chemical modification is formation of oxime at the N-terminal serine or alkylation of cysteine.
7. The method of claim 1 , wherein the chemical modifications are chemical reactions that insert one or more linkers, one or more cross-linkers or one or more chemical staples to convert a peptide into a macrocycle with one of more bridges.
8. The method of claim 4 , wherein the small molecule is a diastereomer or enantiomer.
9. The method of claim 1 , wherein the chemical modifications are enzymatic modifications.
10. The method of claim 1 , wherein genetically encoded display system is phage, mRNA, ribosome, bacteria, or yeast.
11. The method of claim 1 wherein the identical transcriptional product is a peptide linker.
12. The method of claim 11 wherein the peptide linker comprises an amino acid sequence as set forth in SEQ ID NO: 10, or is encoded by a nucleotide sequence as set forth in any one of SEQ ID NOs: 11, 12, 13, 14, 15, or 16.
13. The method of claim 11 wherein the genetic barcodes do not reside within hamming distance Hd=1 from each other.
14. The method of claim 13 wherein the genetic barcodes are Hd=2 or Hd=3 away from each other.