Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA.
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.
Kim
Sangjin
S
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Blainey
Paul C
PC
Schroeder
Charles M
CM
Xie
X Sunney
XS
D017103:Bacillus Phages / Q000201:enzymology; D004247:DNA / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004279:DNA, Viral / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004548:Elasticity; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D008853:Microscopy / Q000379:methods; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D014764:Viral Proteins; D004247:DNA; D054303:HIV Reverse Transcriptase; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda