On the Origin of Broadening of Single-Molecule FRET Efficiency Distributions beyond Shot Noise Limits

2010 ◽  
Vol 114 (18) ◽  
pp. 6197-6206 ◽  
Author(s):  
Stanislav Kalinin ◽  
Evangelos Sisamakis ◽  
Steven W. Magennis ◽  
Suren Felekyan ◽  
Claus A. M. Seidel
Keyword(s):  
Single Molecule ◽  
Shot Noise ◽  
Single Molecule Fret ◽  
Fret Efficiency

scholarly journals High-Resolution Single-Molecule FRET via DNA eXchange (FRET X)

2020 ◽  
Author(s):  
Mike Filius ◽  
Sung Hyun Kim ◽  
Ivo Severins ◽  
Chirlmin Joo
Keyword(s):  
Structural Analysis ◽  
High Resolution ◽  
Single Molecule ◽  
Single Object ◽  
Single Molecule Fret ◽  
Dna Strands ◽  
Versatile Tool ◽  
Fret Efficiency ◽  
Fret Pair

ABSTRACTSingle-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object. The limited number of available FRET pair fluorophores and complicated data analysis makes it challenging to apply single-molecule FRET for structural analysis of biomolecules. Currently, only a couple of FRET pairs can be reliably measured on a single object. Here we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the FRET efficiency and pair distance with sub-nanometer resolution. We determine the distance between other pairs by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. We envision that our FRET X technology will be a tool for the high-resolution structural analysis of biomolecules and other nano-structures.

scholarly journals ABEL-FRET: tether-free single-molecule FRET with hydrodynamic profiling

2019 ◽  
Author(s):  
Hugh Wilson ◽  
Quan Wang
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Biological Processes ◽  
Resolution Limit ◽  
Single Molecule Fret ◽  
Experimental Protocols ◽  
Fret Efficiency ◽  
Nanoscale Metrology ◽  
Observation Times

ABSTRACTSingle-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental protocols often resort to molecule immobilization for long observation times and rarely approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with near shot-noise limited, Angstrom-level resolution in FRET efficiency. Furthermore, ABEL-FRET naturally integrates hydrodynamic profiling, which harnesses single-molecule diffusion coefficient to enhance FRET sensing of biological processes.

scholarly journals Multi-parameter photon-by-photon hidden Markov modeling

2021 ◽  
Author(s):  
Paul David Harris ◽  
Shimon Weiss ◽  
Eitan Lerner
Keyword(s):  
Single Molecule ◽  
Hidden Markov ◽  
Analysis Tool ◽  
Markov Modeling ◽  
Markov Modelling ◽  
Transcription Bubble ◽  
Multiple Parameters ◽  
Single Molecule Fret ◽  
Hidden Markov Modeling ◽  
Fret Efficiency

AbstractSingle molecule FRET (smFRET) is a useful tool for studying biomolecular sub-populations and their dynamics. Advanced smFRET-based techniques often track multiple parameters simultaneously, increasing the information content of the measurement. Photon-by-photon hidden Markov modelling (H2MM) is a smFRET analysis tool that quantifies FRET dynamics of single biomolecules, even if they occur in sub-milliseconds. However, sub-populations can be characterized by additional experimentally-derived parameters other than the FRET efficiency. We introduce multi-parameter H2MM (mpH2MM) that identifies sub-populations and their transition dynamics based on multiple experimentally-derived parameters, simultaneously. We show the use of this tool in deciphering the number of underlying sub-populations, their mean characteristics and the rate constants of their transitions for a DNA hairpin exhibiting milliseconds FRET dynamics, and for the RNA polymerase promoter open complex exhibiting sub-millisecond FRET dynamics of the transcription bubble. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations in smFRET measurements that are otherwise difficult to identify. Finally we provide the means to use mpH2MM in analyzing FRET dynamics in advanced multi-color smFRET-based measurements.

scholarly journals Shot-Noise Limited Single-Molecule FRET Histograms:  Comparison between Theory and Experiments†

2006 ◽  
Vol 110 (44) ◽  
pp. 22103-22124 ◽  
Author(s):  
Eyal Nir ◽  
Xavier Michalet ◽  
Kambiz M. Hamadani ◽  
Ted A. Laurence ◽  
Daniel Neuhauser ◽  
...  
Keyword(s):  
Single Molecule ◽  
Shot Noise ◽  
Single Molecule Fret ◽  
Theory And Experiments

scholarly journals DNA-sequence dependent positioning of the linker histone in a chromatosome: a single-pair FRET study

2020 ◽  
Author(s):  
Madhura De ◽  
Mehmet Ali Oeztuerk ◽  
Katalin Toth ◽  
Rebecca C. Wade
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Binding Mode ◽  
Linker Histone ◽  
Single Pair ◽  
Globular Domain ◽  
Binding Modes ◽  
Single Molecule Fret ◽  
Fret Efficiency ◽  
Order Structures

The linker histone (LH) associates with the nucleosome with its globular domain (gH) binding in an on or off-dyad binding mode. The positioning of the LH may play a role in the compaction of higher-order structures of chromatin. Preference for different binding modes has been attributed to the LHs amino acid sequence. We here study the effect of the linker DNA (L-DNA) sequence on the positioning of a full-length LH, Xenopus laevis H1.0b, by employing single-molecule FRET spectroscopy. Chromatosomes were fluorescently labelled on one of the two 40bp long L-DNA arms, and on the gH. We varied 11bp of DNA flanking the core (non-palindromic Widom 601) of each chromatosome construct, making them either A-tract, purely GC, or mixed, with 64% AT. The gH consistently exhibited higher FRET efficiency with the L-DNA containing the A-tract, than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA, compared to the pure-GC stretch. We explain our observations with a FRET-distance restrained model that shows that the gH binds on-dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove.

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