Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Monday Speaker Abstracts

CONFORMATIONAL CONTROL: FROM SINGLE MOLECULE FRET TO NOVEL BIOTECHNOLOGIES Taekjip Ha ;

1 Boston Children's Hospital, Boston, MA, USA 2 Harvard Medical School, Boston, MA, USA

After an introduction to single-molecule FRET from my own personal perspective, I will discuss how it can be used to uncover functional regulation via conformational control and how we leverage these fundamental insights to engineer powerful biotechnologies. For example, our single-molecule investigations into CRISPR-Cas9 DNA unwinding and conformational dynamics have yielded a suite of tools. By exploiting the differential sequence requirements for Cas9 binding versus cleavage, we engineered a light-controlled, very fast CRISPR (vfCRISPR) system to synchronously generate double-strand breaks (DSBs) across cell populations. Tracking these repair kinetics in real time has provided fundamental insights into nonhomologous end joining and homologous recombination. Concurrently, we developed GOLDFISH for high resolution, non-denaturing genome imaging and successfully integrated it with vfCRISPR. Beyond CRISPR, I will introduce SHARP, a novel isothermal DNA amplification technology for repetitive sequences, which emerged directly from our foundational studies on helicase regulation and the subsequent engineering of superhelicases. Finally, I will present single molecule FRET investigations of prime-editing mechanisms, in which we dissect the individual steps of this gene-editing method without generating a DSB. FROM SCALING LAWS OF ENERGY TRANSFER TO FRET SENSING OF BROWNIAN DNA COMPUTING Philip Tinnefeld 1 ; 1 Ludwig-Maximilians-Universität München, Chemistry, Munich, Germany Förster resonance energy transfer obeys the famous r -6 dependence for the interaction of two dipoles in the near-field. Using DNA nanotechnology, we arranged dye molecules relative to different materials such as gold nanoparticles, carbon nanotubes and graphene and studied the scaling laws experimentally. Among the different options, graphene energy transfer (GET) in combination with the discovery of a vertical orientation of dsDNA on graphene turned out to be most useful and has emerged into a versatile technique for studying DNA-protein interactions with nanometer precise superresolution microscopy. Here, the axial information is deduced from GET-efficiency and xy-information is obtained by photon-efficient pMINFLUX revealing simultaneous bending and torsion of enzyme induced DNA conformations. Finally, we discuss how mutli-color smFRET with dark quenchers probes states of DNA nanorobots and Brownian DNA computers that work close to the thermodynamic optimum.

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