A Comparison of Donor-Acceptor Distance Distributions Derived from Ensemble and Single-Pair FRET Measurements

Abstract

We engineered a FRET system consisting of a DNA oligonucleotide with FRET acceptor AlexaFluor647 at the 3′ end and a downstream DNA oligonucleotide with FRET donor Cy3 at the 5′ end. These were annealed to antisense DNA oligonucleotide that provided a four nucleotide gap between donor and acceptor residues. Molecular modeling placed the mean Forster radius-normalized inter-probe distance r/R0 between 0.5 and 1.Transfer efficiency was estimated from steady state fluorescence (ssFRET), ensemble time-resolved fluorescence (en-trFRET), and single-pair time resolved fluorescence (sp-trFRET). In our analysis, we sought internal consistency in the estimated FRET efficiency across the measurements. A distribution of inter-probe distances was obtained from the en-trFRET data after including contributions from scattering and unquenched donor in the analysis. Similarly, a distribution of inter-probe distance was obtained from the sp-trFRET data after accounting for scattering by passing the time-tagged time-resolved (TTTR) data through a time-correlated single photon counting (TCSPC)-based time gated filter and accounting for the population of unquenched donor by collecting data using pulsed interleaved excitation (PIE). We compare the recovered distance distributions from en-trFRET and sp-trFRET.