ProtSeq: towards high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes

Jessica Hong
Michael Connor Gibbons
Ali Bashir
Diana Wu
Shirley Shao
Zachary Cutts
Mariya Chavarha
Ye Chen
Lauren Schiff
Mikelle Foster
Victoria Church
Llyke Ching
Sara Ahadi
Anna Hieu-Thao Le
Alexander Tran
Michelle Therese Dimon
Phillip Jess
iScience, 25 (2022), pp. 32

Abstract

We demonstrate early progress toward constructing a high-throughput, single-molecule protein sequencing technology utilizing barcoded DNA aptamers (binders) to recognize terminal amino acids of peptides (targets) tethered on a next-generation sequencing chip. DNA binders deposit unique, amino acid identifying barcodes on the chip. The end goal is that over multiple binding cycles, a sequential chain of DNA barcodes will identify the amino acid sequence of a peptide. Toward this, we demonstrate successful target identification with two sets of target-binder pairs: DNA-DNA and Peptide-Protein. For DNA-DNA binding, we show assembly and sequencing of DNA barcodes over 6 consecutive binding cycles. Intriguingly, our computational simulation predicts that a small set of semi-selective DNA binders offers significant coverage of the human proteome. Toward this end, we introduce a binder discovery pipeline that ultimately could merge with the chip assay into a technology called ProtSeq, for future high-throughput, single-molecule protein sequencing.