SOLVING SINGLE BIOMOLECULES BY ADVANCED FRET-BASED SINGLE-MOLECULE FLUORESCENCE TECHNIQUES

2013 ◽  
Vol 08 (03n04) ◽  
pp. 161-190 ◽  
Author(s):  
M. J. RUEDAS-RAMA ◽  
J. M. ALVAREZ-PEZ ◽  
A. ORTE
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Coincidence Detection ◽  
Special Issue ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Technical Developments ◽  
Orthogonal Dimension ◽  
Alternating Laser Excitation

The use of Förster resonance energy transfer (FRET) has undergone a renaissance in the last two decades, especially in the study of structure of biomolecules, biomolecular interactions, and dynamics. Thanks to powerful advances in single-molecule fluorescence (SMF) techniques, seeing molecules at work is a reality, which has helped to build up the mindset of molecular machines. In the last few years, many technical developments have broadened the applications of SMF-FRET, expanding the amount of information that can be recovered from individual molecules. Here, we focus on the non-standard SMF-FRET techniques, such as two-color coincidence detection (TCCD), alternating laser excitation (ALEX), multiparameter fluorescence detection (MFD); the addition of fluorescence lifetime as an orthogonal dimension in single-molecule experiments; or the development of novel and improved methods of analysis constituting to a set of advanced methodologies that may become routine tools in a close future. [Formula: see text]Special Issue Comment: This review about advanced single-molecule FRET techniques is specially related to the review by Jørgensen and Hatzakis,6 who detail experimetal strategies to solve the activity of single enzymes. The advanced techniques described in our paper may serve as interesting alternatives when applied to enzyme studies. Our manuscript is also related to the reviews in this Special Issue that deal with model solving.22,130

Single molecule fluorescence resonance energy transfer scanning near-field optical microscopy: potentials and challenges

2015 ◽  
Vol 184 ◽  
pp. 51-69 ◽  
Author(s):  
S. K. Sekatskii ◽  
K. Dukenbayev ◽  
M. Mensi ◽  
A. G. Mikhaylov ◽  
E. Rostova ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Near Field ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nitrogen Vacancy ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Fluorescence Resonance

A few years ago, single molecule Fluorescence Resonance Energy Transfer Scanning Near-Field Optical Microscope (FRET SNOM) images were demonstrated using CdSe semiconductor nanocrystal–dye molecules as donor–acceptor pairs. Corresponding experiments reveal the necessity to exploit much more photostable fluorescent centers for such an imaging technique to become a practically used tool. Here we report the results of our experiments attempting to use nitrogen vacancy (NV) color centers in nanodiamond (ND) crystals, which are claimed to be extremely photostable, for FRET SNOM. All attempts were unsuccessful, and as a plausible explanation we propose the absence (instability) of NV centers lying close enough to the ND border. We also report improvements in SNOM construction that are necessary for single molecule FRET SNOM imaging. In particular, we present the first topographical images of single strand DNA molecules obtained with fiber-based SNOM. The prospects of using rare earth ions in crystals, which are known to be extremely photostable, for single molecule FRET SNOM at room temperature and quantum informatics at liquid helium temperatures, where FRET is a coherent process, are also discussed.

scholarly journals Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

2017 ◽  
Author(s):  
Mengyi Yang ◽  
Sijia Peng ◽  
Ruirui Sun ◽  
Jingdi Lin ◽  
Nan Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclease Activity ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Allosteric Communication ◽  
Target Binding

SummaryOff-target binding and cleavage by Cas9 pose as major challenges in its applications. How conformational dynamics of Cas9 governs its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms all spontaneously transits between three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We furthermore uncovered a surprising long-range allosteric communication between the HNH domain and RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

Red light, green light: probing single molecules using alternating-laser excitation

2008 ◽  
Vol 36 (4) ◽  
pp. 738-744 ◽  
Author(s):  
Yusdi Santoso ◽  
Ling Chin Hwang ◽  
Ludovic Le Reste ◽  
Achillefs N. Kapanidis
Keyword(s):  
Single Molecule ◽  
Laser Excitation ◽  
Dynamic Range ◽  
Red Light ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Fret ◽  
Alternating Laser Excitation

Single-molecule fluorescence methods, particularly single-molecule FRET (fluorescence resonance energy transfer), have provided novel insights into the structure, interactions and dynamics of biological systems. ALEX (alternating-laser excitation) spectroscopy is a new method that extends single-molecule FRET by providing simultaneous information about structure and stoichiometry; this new information allows the detection of interactions in the absence of FRET and extends the dynamic range of distance measurements that are accessible through FRET. In the present article, we discuss combinations of ALEX with confocal microscopy for studying in-solution and in-gel molecules; we also discuss combining ALEX with TIRF (total internal reflection fluorescence) for studying surface-immobilized molecules. We also highlight applications of ALEX to the study of protein–nucleic acid interactions.

Single-molecule FRET for Ultrasensitive Detection of Biomolecules

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Kun Yang ◽  
Yong Yang ◽  
Chun-yang Zhang
Keyword(s):  
Single Molecule ◽  
New Technologies ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Probes ◽  
Ultrasensitive Detection ◽  
Single Molecule Fret ◽  
Biological Reaction ◽  
Spatio Temporal ◽  
Detection Of Biomolecules

AbstractSingle-molecule Förster resonance energy transfer (sm- FRET) has been widely employed to detect biomarkers and to probe the structure and dynamics of biomolecules. By monitoring the biological reaction in a spatio-temporal manner, smFRET can reveal the transient intermediates of biological processes that cannot be obtained by conventional ensemble measurements. This review provides an overview of singlemolecule FRET and its applications in ultrasensitive detection of biomolecules, including the major techniques and the molecular probes used for smFRET as well as the biomedical applications of smFRET. Especially, the combination of sm- FRET with new technologies might expand its applications in clinical diagnosis and biomedical research

scholarly journals Closing and opening of the RNA polymerase trigger loop

2019 ◽  
Author(s):  
Abhishek Mazumder ◽  
Miaoxin Lin ◽  
Achillefs N. Kapanidis ◽  
Richard H. Ebright
Keyword(s):  
Rna Polymerase ◽  
Fluorescent Probe ◽  
Active Center ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fluorescence ◽  
Structural Element ◽  
Trigger Loop ◽  
Nucleotide Addition

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an “unfolded”/“open” state that allows an NTP substrate to enter the active center and a “folded”/“closed” state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.

scholarly journals Single-molecule FRET monitors CLC transporter conformation and subunit independence

2020 ◽  
Author(s):  
Ricky C. Cheng ◽  
Ayush Krishnamoorti ◽  
Vladimir Berka ◽  
Ryan J Durham ◽  
Vasanthi Jayaraman ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Chloride Ions ◽  
Conformational State ◽  
Triple Mutant ◽  
Transport Pathways ◽  
Single Molecule Fret ◽  
Extracellular Side

Abstract“CLC” transporters catalyze the exchange of chloride ions for protons across cellular membranes. As secondary active transporters, CLCs must alternately allow ion access to and from the extracellular and intracellular sides of the membrane, adopting outward-facing and inward-facing conformational states. Here, we use single-molecule Förster resonance energy transfer (smFRET) to monitor the conformational state of CLC-ec1, an E. coli homolog for which high-resolution structures of occluded and outward-facing states are known. Since each subunit within the CLC homodimer contains its own transport pathways for chloride and protons, we developed a labeling strategy to follow conformational change within a subunit, without crosstalk from the second subunit of the dimer. Using this strategy, we evaluated smFRET efficiencies for labels positioned on the extracellular side of the protein, to monitor the status of the outer permeation pathway. When [H+] is increased to enrich the outward-facing state, the smFRET efficiencies for this pair decrease. In a triple-mutant CLC-ec1 that mimics the protonated state of the protein and is known to favor the outward-facing conformation, the lower smFRET efficiency is observed at both low and high [H+]. These results confirm that the smFRET assay is following the transition to the outward-facing state and demonstrate the feasibility of using smFRET to monitor the relatively small (~1 Å) motions involved in CLC transporter conformational change. Using the smFRET assay, we show that the conformation of the partner subunit does not influence the conformation of the subunit being monitored by smFRET, thus providing evidence for the independence of the two subunits in the transport process.SUMMARYCheng, Krishnamoorti et al. use single-molecule Förster energy resonance transfer measurements to monitor the conformation of a CLC transporter and to show that the conformational state is not influenced by the neighboring subunit.

scholarly journals Automatic classification and segmentation of single-molecule fluorescence time traces with deep learning

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jieming Li ◽  
Leyou Zhang ◽  
Alexander Johnson-Buck ◽  
Nils G. Walter
Keyword(s):  
Deep Learning ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Markov Modeling ◽  
Single Molecule Fluorescence ◽  
Hidden Markov Modeling ◽  
Improved Performance ◽  
Automated Screening ◽  
Expectation Bias

AbstractTraces from single-molecule fluorescence microscopy (SMFM) experiments exhibit photophysical artifacts that typically necessitate human expert screening, which is time-consuming and introduces potential for user-dependent expectation bias. Here, we use deep learning to develop a rapid, automatic SMFM trace selector, termed AutoSiM, that improves the sensitivity and specificity of an assay for a DNA point mutation based on single-molecule recognition through equilibrium Poisson sampling (SiMREPS). The improved performance of AutoSiM is based on accepting both more true positives and fewer false positives than the conventional approach of hidden Markov modeling (HMM) followed by hard thresholding. As a second application, the selector is used for automated screening of single-molecule Förster resonance energy transfer (smFRET) data to identify high-quality traces for further analysis, and achieves ~90% concordance with manual selection while requiring less processing time. Finally, we show that AutoSiM can be adapted readily to novel datasets, requiring only modest Transfer Learning.

Sign in / Sign up

Export Citation Format

Share Document