Detailed analysis of complex single molecule FRET data with the software MASH

Hadzic, Mélodie C. A. S.; Kowerko, Danny; Börner, Richard; Zelger-Paulus, Susann; Sigel, Roland K. O.

doi:10.1117/12.2211191

Cited by 9 publications

(8 citation statements)

References 13 publications

(6 reference statements)

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“…Upon addition of the cofactor the relative populations of the states shift in favor of the high transfer efficiencies. FRET time traces are analyzed with a home-written Matlab software ( 40 ) to build cumulative histograms (Figure 4C ). In addition to the high and low FRET states, we find a substantial contribution of intermediate FRET states originating from static molecules ( Supplementary Figure S14 , static molecule 2).…”

Section: Resultsmentioning

confidence: 99%

Site-specific dual-color labeling of long RNAs for single-molecule spectroscopy

Zhao

Steffen

Börner

et al. 2017

Nucleic Acids Research

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Labeling of long RNA molecules in a site-specific yet generally applicable manner is integral to many spectroscopic applications. Here we present a novel covalent labeling approach that is site-specific and scalable to long intricately folded RNAs. In this approach, a custom-designed DNA strand that hybridizes to the RNA guides a reactive group to target a preselected adenine residue. The functionalized nucleotide along with the concomitantly oxidized 3′-terminus can subsequently be conjugated to two different fluorophores via bio-orthogonal chemistry. We validate this modular labeling platform using a regulatory RNA of 275 nucleotides, the btuB riboswitch of Escherichia coli, demonstrate its general applicability by modifying a base within a duplex, and show its site-selectivity in targeting a pair of adjacent adenines. Native folding and function of the RNA is confirmed on the single-molecule level by using FRET as a sensor to visualize and characterize the conformational equilibrium of the riboswitch upon binding of its cofactor adenosylcobalamin. The presented labeling strategy overcomes size and site constraints that have hampered routine production of labeled RNA that are beyond 200 nt in length.

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Section: Resultsmentioning

confidence: 99%

Site-specific dual-color labeling of long RNAs for single-molecule spectroscopy

Zhao

Steffen

Börner

et al. 2017

Nucleic Acids Research

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“…20˚C) on a custom-built TIRF microscope with a Sapphire, green (532 nm) laser that has been previously described (57). Images were acquired every 200 msec and singlemolecule fluorescence intensity time trajectories from multiple fields of view were generated and analysed using a Matlab-based software (MASH-FRET) (58). Donor leakage into the acceptor channel was corrected during image analysis.…”

Section: Smfret Sample Preparation Instrument Setup and Data Analysismentioning

confidence: 99%

Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function

Johnston

Marzano

Paudel

et al. 2021

Journal of Biological Chemistry

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Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones; some sHsp family members are upregulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation; however, fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. The dynamic and polydisperse nature of sHsp oligomers has made studying them challenging using traditional biochemical approaches. Therefore, we have utilized a single-molecule fluorescence-based approach to observe the chaperone action of human alphaB-crystallin (αBc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between αBc and a model client protein, chloride intracellular channel 1. By examining the dispersity and stoichiometries of these complexes over time, and in response to different concentrations of αBc, we have uncovered unique and important insights into a two-step mechanism by which αBc interacts with misfolded client proteins to prevent their aggregation.

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“…SMVs are characterized by a low net signal and a low signal-to-noise ratio ( SNR ), a high molecule surface density (ρ), short intermolecular distances ( IMD ), and inhomogeneous background profiles. Much effort has been geared towards SMV data analysis in recent years [ 11 – 13 ], but their analysis is at present not standardized. Instead, individual researchers face a host of different data analysis strategies developed by different groups [ 12 , 14 – 17 ].…”

Section: Introductionmentioning

confidence: 99%

“…We present the concepts for performance evaluation of algorithms addressing multiple aspects of SMV data analysis, including SM localization, background correction, intensity time trace generation, trajectory discretization, as well as kinetic and thermodynamic modeling. We use our MATLAB-based SMV simulation tool within our M ultifunctional A nalysis S oftware for H andling single molecule FRET data ( MASH-FRET ) [ 13 ], which is easy-to-use and permits rapid and integrated analysis of camera-based smFRET experiments, to simulate well-annotated SMV test data sets. We apply them to optimize method parameters (MP) of different SM localization algorithms commonly used in the field of camera-based SM fluorescence and compare their performance as a function of the total emitted intensity of single molecules.…”

Section: Introductionmentioning

confidence: 99%

Simulations of camera-based single-molecule fluorescence experiments

et al. 2018

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Single-molecule microscopy has become a widely used technique in (bio)physics and (bio)chemistry. A popular implementation is single-molecule Förster Resonance Energy Transfer (smFRET), for which total internal reflection fluorescence microscopy is frequently combined with camera-based detection of surface-immobilized molecules. Camera-based smFRET experiments generate large and complex datasets and several methods for video processing and analysis have been reported. As these algorithms often address similar aspects in video analysis, there is a growing need for standardized comparison. Here, we present a Matlab-based software (MASH-FRET) that allows for the simulation of camera-based smFRET videos, yielding standardized data sets suitable for benchmarking video processing algorithms. The software permits to vary parameters that are relevant in cameras-based smFRET, such as video quality, and the properties of the system under study. Experimental noise is modeled taking into account photon statistics and camera noise. Finally, we survey how video test sets should be designed to evaluate currently available data analysis strategies in camera-based sm fluorescence experiments. We complement our study by pre-optimizing and evaluating spot detection algorithms using our simulated video test sets.

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Detailed analysis of complex single molecule FRET data with the software MASH

Cited by 9 publications

References 13 publications

Site-specific dual-color labeling of long RNAs for single-molecule spectroscopy

Site-specific dual-color labeling of long RNAs for single-molecule spectroscopy

Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function

Simulations of camera-based single-molecule fluorescence experiments

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