US 11,834,756 B2
Methods and compositions for protein and peptide sequencing
Annalisa Marie Pawlosky, Mountain View, CA (US); Michael Gibbons, Livermore, CA (US); Sara Ahadi, San Mateo, CA (US); Shirley Jing Shao, Sunnyvale, CA (US); Anna Le, San Jose, CA (US); Ali Bashir, San Francisco, CA (US); Marc Berndl, Mountain View, CA (US); Michelle Therese Hoerner Dimon, Redwood City, CA (US); and Lauren Schiff, Encino, CA (US)
Assigned to Google LLC, Mountain View, CA (US)
Filed by Google LLC, Mountain View, CA (US)
Filed on Sep. 11, 2020, as Appl. No. 17/019,121.
Claims priority of provisional application 62/900,440, filed on Sep. 13, 2019.
Prior Publication US 2021/0079557 A1, Mar. 18, 2021
Int. Cl. C12Q 1/68 (2018.01); C40B 30/04 (2006.01); C40B 70/00 (2006.01); C12Q 1/6806 (2018.01); C40B 40/04 (2006.01); C12Q 1/6869 (2018.01)
CPC C40B 30/04 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6869 (2013.01); C40B 40/04 (2013.01); C40B 70/00 (2013.01); C12Q 2525/205 (2013.01)] 21 Claims
 
1. A method of sequencing a peptide, comprising:
(a) incubating the peptide with a library of DNA aptamers exhibiting binding specificity toward at least one N-terminal amino acid under conditions where one or more aptamers bind specifically to at least one N-terminal amino acid of the peptide, wherein each aptamer in the library comprises a peptide binding ssDNA region and a unique barcode sequence indicative of the first binding round and the associated peptide binding ssDNA region;
(b) ligating the DNA aptamer bound to the N-terminal of the peptide onto a proximal DNA barcode construct;
(c) removing the peptide binding sequence from the DNA aptamer, thereby leaving the barcode of the DNA aptamer covalently attached to the DNA barcode construct:
(d) removing the N-terminal amino acid from the peptide to produce an N-terminal amino acid-shortened peptide;
(e) incubating the N-terminal amino acid-shortened peptide with the library of aptamers exhibiting binding specificity toward at least one N-terminal amino acid under conditions where one or more aptamers bind specifically to at least one N-terminal amino acid of the N-terminal amino acid-shortened peptide, wherein each aptamer in the library comprises a peptide binding ssDNA region and a unique barcode sequence indicative of the second binding round and the associated peptide binding ssDNA region;
(f) ligating the DNA aptamer bound to the N-terminal of the peptide onto the proximal DNA barcode construct;
(g) removing the peptide binding sequence from the DNA aptamer, thereby leaving the barcode of the DNA aptamer covalently attached to the DNA barcode construct;
(h) removing the N-terminal amino acid from the N-terminal amino acid-shortened peptide;
(i) repeating steps (a)-(d) a plurality of times to construct a chain of positional barcodes that correspond to sequential N-terminal amino acids in the peptide; and
(j) sequencing the chain of positional barcodes to thereby obtain the sequence of the peptide.