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bioRxiv · 10.1101/376798

Assessing protein dynamics on low complexity single-strand DNA curtains

Schaub, J. M.·Zhang, H.·Soniat, M. M.·Finkelstein, I. J.
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Abstract

Single-stranded DNA (ssDNA) is a critical intermediate in all DNA transactions. As ssDNA is more flexible than double-stranded (ds)DNA, interactions with ssDNA-binding proteins (SSBs) may significantly compact or elongate the ssDNA molecule. Here, we develop and characterize low-complexity ssDNA curtains, a high-throughput single-molecule assay to simultaneously monitor protein binding and correlated ssDNA length changes on supported lipid bilayers. Low-complexity ssDNA is generated via rolling circle replication of short synthetic oligonucleotides, permitting control over the sequence composition and secondary structure-forming propensity. One end of the ssDNA is functionalized with a biotin, while the second is fluorescently labeled to track the overall DNA length. Arrays of ssDNA molecules are organized at microfabricated barriers for high-throughput single-molecule imaging. Using this assay, we demonstrate that E. coli SSB drastically and reversibly compacts ssDNA templates upon changes in NaCl concentration. We also examine the interactions between a phosphomimetic RPA and ssDNA. Our results indicate that RPA-ssDNA interactions are not significantly altered by these modifications. We anticipate low-complexity ssDNA curtains will be broadly useful for single-molecule studies of ssDNA-binding proteins involved in DNA replication, transcription and repair.

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Schaub, J. M., Zhang, H., Soniat, M. M., Finkelstein, I. J.. 2018-07-25. Assessing protein dynamics on low complexity single-strand DNA curtains. https://doi.org/10.1101/376798

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