Protseq: An Investigation of High-Throughput, Single-Molecule Protein Sequencing via Amino Acid Conversion into DNA Barcodes

Despite a need for high-throughput protein sequencing, existing methods are not capable of sequencing the entire human proteome. We demonstrate early progress toward constructing the components of a protein sequencing technology that requires sequential capture and recording of binding over many cycles. The Barcode Cycle Sequencing (BCS) assay uses DNA-barcoded binders to construct a chain of barcodes directly onto a DNA sequencing chip that are used to identify a target. We demonstrated successful barcode capture and DNA-barcode chain sequencing for DNA-DNA and nanobody-protein binding pairs. For DNA-DNA binding, we showed assembly and sequencing of barcodes over 6 consecutive binding cycles. We additionally introduce a binder discovery pipeline called Target-Switch SELEX to discover aptamer binders. Ultimately, we envision merging BCS and Target-Switch SELEX into an end-to-end technology called ProtSeq, a high-throughput, single-molecule protein sequencing method in which DNA-barcoded aptamers read a protein’s amino acid sequence over multiple cycles of terminal amino acid degradation.