Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Thursday Speaker Abstracts

ELUCIDATING SINGLE-MOLECULE REACTION KINETICS USING MULTIPLEXED IN SITU FLUORESCENCE-BASED DNA SEQUENCING Jagadish P. Hazra ; Piers Turner 1 ; Qing Zhao 1 ; Achillefs P Kapanidis 1 ; 1 University of Oxford, Physics, Oxford, United Kingdom Single-molecule FRET has been widely applied to investigate protein–DNA interactions, particularly the kinetics of gene expression. However, no single-platform technique currently enables the simultaneous determination of DNA sequence and sequence-dependent reaction kinetics at the single-molecule level within a single experimental platform. Here, we introduce Single-molecule Phenotyping and In Situ Sequencing (SPIN-Seq), a transient DNA hybridization-based short-read sequencing approach that identifies the sequence of individual DNA molecules while enabling FRET-based kinetic measurements on the same molecules. SPIN-Seq exploits mismatch-induced changes in transient DNA hybridization kinetics to decode DNA sequences. We apply SPIN-Seq to dissect sequence-dependent pausing and kinetic branching during bacterial transcription initiation. Furthermore, by incorporating multicolour fluorescence detection, we accelerate sequencing and increase throughput. Using the same hybridization-based principle, we also developed a DNA barcoding strategy that identifies individual DNA species immobilized on a surface. This approach enables parallel FRET measurements of promoter escape kinetics across multiple natural promoters under identical reaction conditions, providing a scalable platform for investigating sequence-dependent mechanisms of DNA–protein interactions at the single-molecule level.

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