Unraveling single-molecule reactions via multiplexed in-situ DNA sequencing

Hazra, J. P.; Andrews, R.; Turner, P.; Zhao, Q.; Steuer, H.; El Sagheer, A. H.; Mazumder, A.; El Sayyed, H.; Kummerlin, M.; Brown, T.; Kapanidis, A.

doi:10.1101/2025.11.27.690701

bioRxiv · 10.1101/2025.11.27.690701

Unraveling single-molecule reactions via multiplexed in-situ DNA sequencing

Hazra, J. P.·Andrews, R.·Turner, P.·Zhao, Q.·Steuer, H.·El Sagheer, A. H.·Mazumder, A.·El Sayyed, H.·Kummerlin, M.·Brown, T.·Kapanidis, A.

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Abstract

DNA sequence regulates complex reactions and protein-DNA interactions, yet sequence effects remain poorly understood due to the lack of direct, high-throughput approaches to study sequence-dependence at the single-molecule level. Here, we introduce Single-molecule Phenotyping and In-Situ Sequencing (SPIN-Seq), a DNA-based, protein-free method that links functional and structural properties of a single DNA molecule with the sequence of that same molecule. After performing functional assays on immobilized DNA molecules, SPIN-Seq uses sequencing-by-transient-hybridization on the same surface and instrument to read sequences in each immobilized DNA molecule, directly linking phenotype and genotype. Applying SPIN-Seq, we dissected the interaction of a transcription factor with its target sequence, and revealed the sequence-dependence of RNA polymerase pausing and reaction-path branching during initial transcription. Our method provides powerful, systematic ways to understand complex molecular mechanisms and their sequence dependence.

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Hazra, J. P., Andrews, R., Turner, P., Zhao, Q., Steuer, H., El Sagheer, A. H., Mazumder, A., El Sayyed, H., Kummerlin, M., Brown, T., Kapanidis, A.. 2025-12-02. Unraveling single-molecule reactions via multiplexed in-situ DNA sequencing. https://doi.org/10.1101/2025.11.27.690701

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