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Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA

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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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Figure 1: Single-molecule assay for nucleic-acid enzymes using flow-stretched DNA templates.
Figure 2: Single-molecule traces obtained with the low-noise assay.

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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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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, 02138, Massachusetts, USA

    Sangjin Kim, Paul C Blainey, Charles M Schroeder & X Sunney Xie

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  1. Sangjin Kim
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  2. Paul C Blainey
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  3. Charles M Schroeder
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  4. X Sunney Xie
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Correspondence to X Sunney Xie.

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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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