ProtSeq: Toward high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes
Jessica M. Hong, Michael C. Gibbons, Ali Bashir, Diana Wu, Shirley Shao, Zachary Cutts, Mariya Chavarha, Ye Chen, Lauren Schiff, Mikelle Foster, Victoria A. Church, Llyke Ching, Sara Ahadi, Anna Hieu-Thao Le, Alexander Tran, Michelle Dimon, Marc Coram, Brian Williams, P. Jess, Marc Berndl, Annalisa Pawlosky
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 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.