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

Abstract

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).

The structural arrangement at intersubunit interfaces in homomeric kainate receptors

“…HAMMY creates a model of the step transitions and states that best describe a data set using hidden Markov modeling. A comparison of the strengths and weakness of the two software has been preformed by Sigel and coworkers, and they showed that STaSI is more accurate in predicting number of states and state efficiencies 41 . However STaSI uses denoised data while HAMMY is performed on observed data, hence we have used both to ensure that denoising is accurate.…”

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts

“…Molecules were imaged on a custom-built TIRF microscope using a water-immersion objective (UPlanSApo 60×/1.2-W, Olympus) at a camera frame rate of 5 or 10 Hz (Andor iXon DU-897). Single molecules with anti-correlated donor/acceptor emission were selected and corrected for background and donor bleedthrough into the acceptor channel using the freely available software package MASH-FRET (https://github.com/ RNA-FRETools/MASH-FRET.git) 16,73 . The FRET efficiency was then calculated from the corrected donor and acceptor intensities after donor excitation according to…”

Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy

“…This method allows to follow the folding and reaction kinetics of hundreds of single molecules in parallel over time, only limited by the frame rate (> 100 Hz) of the camera and the limited observation time due to fluorophores photobleaching [8,9]. The identification of distinct states (model selection) and their interconversion (state transition detection) allows to develop a mechanistic view of the investigated biomolecular system [10].…”