Gene machines

Abstract:

Switchable FRET is the combination of single-molecule Förster resonance energy transfer (smFRET) with photoswitching, the reversible activation and deactivation of fluorophores by light. By photoswitching, multiple donor-acceptor fluorophore pairs can be probed sequentially, thus allowing observation of multiple distances within a single immobilized molecule. Control of the photoinduced switching rates permits adjustment of the temporal resolution of switchable FRET over a wide range of timescales, thereby facilitating application to various dynamical biological systems. We show that fast total internal reflection (TIRF) microscopy can achieve measurements of two FRET pairs with 10 ms temporal resolution within less than 2 s. The concept of switchable FRET is also compatible with confocal microscopy on immobilized molecules, providing better data quality at high temporal resolution. To identify states and extract their transitions from switchable FRET time traces, we also develop linked hidden Markov modeling (HMM) of both FRET and donor-acceptor stoichiometry. Linked HMM successfully identifies transient states in the two-dimensional FRET-stoichiometry space and reconstructs their connectivity network. Improved temporal resolution and novel data analysis make switchable FRET a valuable tool in molecular and structural biology.

Abstract:

Single-molecule FRET (smFRET) has long been used as a molecular ruler for the study of biology on the nanoscale (∼2-10 nm); smFRET in total-internal reflection fluorescence (TIRF) Förster resonance energy transfer (TIRF-FRET) microscopy allows multiple biomolecules to be simultaneously studied with high temporal and spatial resolution. To operate at the limits of resolution of the technique, it is essential to investigate and rigorously quantify the major sources of noise and error; we used theoretical predictions, simulations, advanced image analysis, and detailed characterization of DNA standards to quantify the limits of TIRF-FRET resolution. We present a theoretical description of the major sources of noise, which was in excellent agreement with results for short-timescale smFRET measurements (<200 ms) on individual molecules (as opposed to measurements on an ensemble of single molecules). For longer timescales (>200 ms) on individual molecules, and for FRET distributions obtained from an ensemble of single molecules, we observed significant broadening beyond theoretical predictions; we investigated the causes of this broadening. For measurements on individual molecules, analysis of the experimental noise allows us to predict a maximum resolution of a FRET change of 0.08 with 20-ms temporal resolution, sufficient to directly resolve distance differences equivalent to one DNA basepair separation (0.34 nm). For measurements on ensembles of single molecules, we demonstrate resolution of distance differences of one basepair with 1000-ms temporal resolution, and differences of two basepairs with 80-ms temporal resolution. Our work paves the way for ultra-high-resolution TIRF-FRET studies on many biomolecules, including DNA processing machinery (DNA and RNA polymerases, helicases, etc.), the mechanisms of which are often characterized by distance changes on the scale of one DNA basepair.