Cross-Talk-Free Dual-Color Fluorescence Cross-Correlation Spectroscopy for the Study of Enzyme Activity

摘要

We have developed an instrument for spectral cross-talkfreendual-color fluorescence cross-correlation spectroscopyn(FCCS), which provides a readout modality for thenstudy of enzyme activity in application areas such as highthroughputnscreening. Two spectrally distinct (∼250 nm)nfluorophores, Cy3 and IRD800, were excited simultaneouslynusing two different excitation sources: one poisednat 532 nm and the other at 780 nm. The fluorescenceninformation was processed on two different color channelsnmonitored with single-photon avalanche diodes (SPADs)nthat could transduce events at the single-molecule level.nThe system provided no color cross-talk (cross-excitationnand/or cross-emission) and/or fluorescence resonancenenergy transfer (FRET), significantly improving data quality.nTo provide evidence of cross-talk-free operation, thensystem was evaluated using bright microspheres (λabs )n532 nm, λem ) 560 nm) and quantum dots (λabs ) 532nnm, λem ) 810 nm). Experimental results indicatednthat no color leakage from the microspheres or quantumndots into inappropriate color channels was observed.nTo demonstrate the utility of the system, thenenzymatic activity of APE1, which is responsible fornnicking the phosphodiester backbone in DNA on then5′ side of an apurinic/apyrimidinic site, was monitorednby FCCS using a double-stranded DNA substrate dualnlabeled with Cy3 and IRD800. Activity of APE1 wasnalso monitored in the presence of an inhibitor (7-nnitroindole-2-carboxylic acid) of the enzyme using thisncross-talk-free FCCS platform. In all cases, no spectralnleakage from single-molecule events into inappropriatencolor channels was observed.