Reed, B. D. | bioRxiv Science

bioRxiv2025

Parallelization of single-molecule binding kinetic measurements via protein barcode sequencing

Screening protein variants for desired functions has long relied on coupling of genotype (gene sequence) to phenotype (protein function), limiting the use of powerful single-molecule (SM) techniques. Here, we introduce a scalable SM screening method that bypasses this constraint by linking SM functional analysis to protein identity through SM protein sequencing. Protein variants are tagged with unique C-terminal peptide barcodes and loaded onto a semiconductor chip containing millions of nanowells. Protein-ligand interactions are monitored in real time at the SM level, and a dye-cycling strategy extends the measurable dynamic range, enabling quantification of slow dissociation rates typical of high-affinity interactions. After functional analysis, each protein molecule is identified by sequencing its barcode. We apply this method to 20 barcoded nanobodies spanning over 1,000-fold in affinity, yielding results consistent with published values and individual SM measurements. Our approach should accelerate protein engineering by enabling rapid, multiplexed SM screening of protein libraries.

biochemistry↗

bioRxiv2022

Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

biophysics↗