Light‐Guided Molecular Patterning for High‐Throughput Single‐Molecule Mechanical Characterization

ABSTRACT Molecular patterning at single‐molecule resolution could significantly advance biomolecular analysis and the engineering of functional surfaces by enabling precise control over the spatial arrangement and mechanical accessibility of individual biomolecules. Such control is particularly valuable for multiplexed single‐molecule assays, which reveal molecular mechanisms, non‐equilibrium behavior, and nanoscale mechanical properties through the application of mechanical force. Standard surface functionalization methods, however, often lack sufficient precision, programmability, or accessibility, resulting in random or sparse biomolecular arrangements. To address this, we have developed a light‐guided surface patterning method that can covalently organize oligonucleotides (oligos) without the need for lithographic equipment. Oligos with 3‐cyanovinylcarbazole (CNVK) nucleoside are crosslinked in precise spatial arrangements defined by UV patterns projected through a digital micromirror device (DMD), with beads arranged accordingly. We demonstrate compatibility with established single‐molecule methods by performing single‐molecule force spectroscopy experiments on patterned coverslips, using magnetic tweezers and hydrodynamic‐based approaches. Our light‐guided method provides a scalable and accessible platform for biomolecular patterning that allows precise control over molecular identity and spatial positioning, enabling high‐throughput single‐molecule manipulation and mechanical characterization.