US 11,718,849 B2
Phosphopeptide-encoding oligonucleotide libraries and methods for detecting phosphorylation-dependent molecular interactions
Jesse Rinehart, Guilford, CT (US); Karl Barber, New Haven, CT (US); Farren Isaacs, Stamford, CT (US); and Jeffrey R Sampson, San Jose, CA (US)
Assigned to AGILENT TECHNOLOGIES, INC., Santa Clara, CA (US)
Filed by Yale University, New Haven, CT (US); and Agilent Technologies, Inc., Santa Clara, CA (US)
Filed on Feb. 18, 2019, as Appl. No. 16/278,610.
Claims priority of provisional application 62/639,279, filed on Mar. 6, 2018.
Claims priority of provisional application 62/632,144, filed on Feb. 19, 2018.
Prior Publication US 2019/0256843 A1, Aug. 22, 2019
Int. Cl. C12N 15/10 (2006.01)
CPC C12N 15/1082 (2013.01) [C12N 15/1093 (2013.01)] 7 Claims
 
1. A method of preparing a library of phosphopeptide-encoding oligonucleotides, the method comprising:
selecting a plurality of phosphopeptide sequences from full-length native protein sequences, comprising:
identifying, in a full-length native protein sequence, a plurality of phosphorylation-susceptible amino acid sequences, wherein each phosphorylation-susceptible amino acid sequence comprises a single phosphorylation-susceptible residue;
elongating each phosphorylation-susceptible amino acid sequence to include up to 15 residues from the full-length native protein sequence on each side of the single phosphorylation-susceptible residue, wherein each phosphopeptide sequence, of the plurality of phosphopeptide sequences, comprises 16 to 31 residues;
in silico converting each phosphopeptide sequence, of the selected plurality of phosphopeptide sequences, into a phosphopeptide-encoding nucleic acid sequence, wherein the single phosphorylation-susceptible residue is encoded as a codon that does not encode a phosphorylation-residue in nature, and wherein the single phosphorylation-susceptible residue is encoded by a codon of thymine, adenine, and/or guanine; and
synthesizing a library of phosphopeptide-encoding oligonucleotides having the phosphopeptide-encoding nucleic acid sequences, wherein each phosphopeptide-encoding oligonucleotide, of the library of phosphopeptide-encoding oligonucleotides has one of the phosphopeptide-encoding nucleic acid sequences.