Abstract
We report a single-molecule assay for nucleic-acid enzymes on flow-stretched DNA templates. To facilitate the detection of slow or intermittent enzymatic activities, we developed the assay with 15-nm spatial resolution at a frame rate of 1 Hz and ∼10 nm mechanical stability over the timescale of hours. With multiplexed data collection, we applied the assay to φ29 DNA polymerase, HIV-1 reverse transcriptase, λ exonuclease and Escherichia coli RNA polymerase.
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Acknowledgements
We thank A. van Oijen for his initial contribution to the flow-stretching assay and H. Babcock for suggesting a pressure-driven pump. This work is supported by Jane Coffin Childs Memorial Fund for Medical Research Fellowship and a US National Institutes of Health Pathway to Independence Award for C.M.S. and a National Institutes of Health Director's Pioneer Award to X.S.X.
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Supplementary information
Supplementary Fig. 1
Typical dark-field image of ∼180 DNA-tethered beads. (PDF 134 kb)
Supplementary Fig. 2
Schematic of a pressure-driven pump system. (PDF 85 kb)
Supplementary Note
Technical details on the experimental setup. (DOC 599 kb)
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Kim, S., Blainey, P., Schroeder, C. et al. Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA. Nat Methods 4, 397–399 (2007). https://doi.org/10.1038/nmeth1037
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DOI: https://doi.org/10.1038/nmeth1037
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