Transient DNA binding to gapped DNA substrates links DNA sequence to the single-molecule kinetics of protein-DNA interactions

Rebecca Andrews, Horst Steuer, Afaf H El-Sagheer, Abhishek Mazumder, Hafez el Sayyed, Arun Shivalingam, Tom Brown, Achillefs N Kapanidis. bioRxiv 0 (0), 2022.

Protein interactions with nucleic acids are central to all genetic processes and many biotechnological applications. While many sequence-dependent protein-DNA interactions have been studied in detail using single-molecule methods, there is no standard high-throughput way to link the complex single-molecule kinetics of protein-DNA interactions with the DNA sequence of a single molecule. Here we provide the missing link by introducing a single-molecule imaging method (Gap-Seq) that interrogates DNA sequences via transient binding of short fluorescent DNA to a single DNA molecule previously used to characterise a protein-DNA interaction. In Gap-Seq, we identify a base by the degree of binding of 6-9 nt-long DNAs to surface-immobilised DNA substrates featuring a short single-stranded gap. To facilitate detection, we also developed a fluorescence quenching strategy that allows single-molecule detection at up to 500 nM of unbound fluorescent DNA. We link single-base differences on single DNA molecules to the kinetics of protein-DNA interactions by studying the interaction of a transcription activator with its cognate site. Finally, we show that our assay can address mixed sequences by distinguishing between two different sequences immobilised on the same field of view, paving the way for interrogation of sequence libraries for both mechanistic work and biotechnological applications

Abstract

Protein interactions with nucleic acids are central to all genetic processes and many biotechnological applications. While many sequence-dependent protein-DNA interactions have been studied in detail using single-molecule methods, there is no standard high-throughput way to link the complex single-molecule kinetics of protein-DNA interactions with the DNA sequence of a single molecule. Here we provide the missing link by introducing a single-molecule imaging method (Gap-Seq) that interrogates DNA sequences via transient binding of short fluorescent DNA to a single DNA molecule previously used to characterise a protein-DNA interaction. In Gap-Seq, we identify a base by the degree of binding of 6-9 nt-long DNAs to surface-immobilised DNA substrates featuring a short single-stranded gap. To facilitate detection, we also developed a fluorescence quenching strategy that allows single-molecule detection at up to 500 nM of unbound fluorescent DNA. We link single-base differences on single DNA molecules to the kinetics of protein-DNA interactions by studying the interaction of a transcription activator with its cognate site. Finally, we show that our assay can address mixed sequences by distinguishing between two different sequences immobilised on the same field of view, paving the way for interrogation of sequence libraries for both mechanistic work and biotechnological applications