摘要：SummaryWe 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 six 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.Graphical abstractDisplay OmittedHighlights•Designed ProtSeq protein sequencing method compatible with widely used NGS technology•Built Target-Switch SELEX to isolate aptamers specific to N-terminal amino acids (AAs)•Showed binding, ligation, cleavage, and NGS of six DNA binders in ordered barcode chain•Developed pipeline to deconvolve AAs from DNA barcodes to identify putative proteinsBiochemistry; Biochemistry applications; Sequence analysis; Proteomics; Transcriptomics
