Reliable State Identification and State Transition Detection in Fluorescence Intensity-Based Single-Molecule Förster Resonance Energy-Transfer Data

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique to probe biomolecular structure and dynamics. A popular implementation of smFRET consists of recording fluorescence intensity time traces of surface-immobilized, chromophore-tagged molecules. This approach generates large and complex data sets, the analysis of which is to date not standardized. Here, we address a key challenge in smFRET data analysis: the generation of thermodynamic and kinetic models that describe with statistical rigor the behavior of FRET trajectories recorded from surface-tethered biomolecules in terms of the number of FRET states, the corresponding mean FRET values, and the kinetic rates at which they interconvert. For this purpose, we first perform Monte Carlo simulations to generate smFRET trajectories, in which a relevant space of experimental parameters is explored. Then, we provide an account on current strategies to achieve such model selection, as well as a quantitative assessment of their performances. Specifically, we evaluate the performance of each algorithm (change-point analysis, STaSI, HaMMy, vbFRET, and ebFRET) with respect to accuracy, reproducibility, and computing time, which yields a range of algorithm-specific referential benchmarks for various data qualities. Data simulation and analysis were performed with our MATLAB-based multifunctional analysis software for handling smFRET data (MASH-FRET)