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.

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 Simulating freely-diffusing single-molecule FRET data with consideration of protein conformational dynamics

2021 ◽  
Author(s):  
James Losey ◽  
Michael Jauch ◽  
David S. Matteson ◽  
Mahmoud Moradi
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Analysis Techniques ◽  
Intrinsically Disordered ◽  
Single Molecule Fret ◽  
Great Progress

AbstractSingle molecule Förster resonance energy transfer experiments have added a great deal to the under-standing of conformational states of biologically important molecules. While great progress has been made, much is still unknown in systems that are highly flexible such as intrinsically disordered proteins because of the high degeneracy of distance states, particularly when freely diffusing smFRET experiments are used. Simulated smFRET data allows for the control of underlying process that generates the data to examine if analytic techniques can detect these underlying differences. We have extended the PyBroMo software that simulates the freely diffusing smFRET data to include a distribution of inter-dye distances generated using Langevin dynamics in order to model proteins with greater flexibility or disorder in structure. Standard analysis techniques for smFRET data compared highlighted the differences observed between data generated with the base software and data that included the distribution of inter-dye distance.

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

scholarly journals Sub-millisecond conformational dynamics of the A2A adenosine receptor revealed by single-molecule FRET

2020 ◽  
Author(s):  
Ivan Maslov ◽  
Oleksandr Volkov ◽  
Polina Khorn ◽  
Philipp Orekhov ◽  
Anastasiia Gusach ◽  
...  
Keyword(s):  
Single Molecule ◽  
Adenosine Receptor ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
A2a Adenosine Receptor ◽  
Single Molecule Fret ◽  
Lipid Nanodiscs ◽  
G Protein Coupled

AbstractThe complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well-suited to quantify dynamics for individual protein molecules, however, its application to GPCRs is challenging; therefore, smFRET has been limited to studies of interreceptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor’s constitutive activity. For the agonist-bound A2AAR, we detected faster (390±80 μs) ligand efficacy-dependent dynamics. This work establishes a general smFRET platform for GPCR investigations that can potentially be used for drug screening and/or mechanism-of-action studies.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

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.

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