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 approximately 10 nm mechanical stability over the timescale of hours. With multiplexed data collection, we applied the assay to phi29 DNA polymerase, HIV-1 reverse transcriptase, lambda exonuclease and Escherichia coli RNA polymerase.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Bacillus Phages / enzymology
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DNA / chemistry
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DNA, Single-Stranded / chemistry
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DNA, Viral / metabolism*
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DNA-Directed DNA Polymerase / metabolism*
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DNA-Directed RNA Polymerases / metabolism*
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Elasticity
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Escherichia coli / enzymology
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Exodeoxyribonucleases / metabolism*
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HIV Reverse Transcriptase / metabolism*
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Microscopy / methods
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Viral Proteins / metabolism*
Substances
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DNA, Single-Stranded
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DNA, Viral
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Viral Proteins
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DNA
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HIV Reverse Transcriptase
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DNA-Directed RNA Polymerases
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DNA-Directed DNA Polymerase
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Exodeoxyribonucleases
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exo protein, Bacteriophage lambda