scholarly journals Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4–P6 RNA by smFRET

2015 ◽  
Vol 43 (2) ◽  
pp. 172-178 ◽  
Author(s):  
Namita Bisaria ◽  
Daniel Herschlag
Keyword(s):  
Single Molecule ◽  
Specific Binding ◽  
Specific Interaction ◽  
Monovalent Cations ◽  
Folding Kinetics ◽  
Rna Molecules ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Monovalent Ion

Structured RNA molecules play roles in central biological processes and understanding the basic forces and features that govern RNA folding kinetics and thermodynamics can help elucidate principles that underlie biological function. Here we investigate one such feature, the specific interaction of monovalent cations with a structured RNA, the P4–P6 domain of the Tetrahymena ribozyme. We employ single molecule FRET (smFRET) approaches as these allow determination of folding equilibrium and rate constants over a wide range of stabilities and thus allow direct comparisons without the need for extrapolation. These experiments provide additional evidence for specific binding of monovalent cations, Na+ and K+, to the RNA tetraloop–tetraloop receptor (TL–TLR) tertiary motif. These ions facilitate both folding and unfolding, consistent with an ability to help order the TLR for binding and further stabilize the tertiary contact subsequent to attainment of the folding transition state.

Determination of the size of lipid rafts studied through single-molecule FRET simulations

2021 ◽  
Author(s):  
Pablo Luis Hernández-Adame ◽  
Ulises Meza ◽  
Aldo A. Rodríguez-Menchaca ◽  
Sergio Sánchez-Armass ◽  
Jaime Ruiz-García ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Single Molecule ◽  
Single Molecule Fret

scholarly journals Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription

2015 ◽  
Vol 112 (44) ◽  
pp. 13467-13472 ◽  
Author(s):  
Danya J. Martell ◽  
Chandra P. Joshi ◽  
Ahmed Gaballa ◽  
Ace George Santiago ◽  
Tai-Yen Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Structural Changes ◽  
Dynamic Equilibrium ◽  
Specific Binding ◽  
Binding Mode ◽  
Biased Sampling ◽  
Single Molecule Fret ◽  
Dead End ◽  
The Dead

Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ70-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator−DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)—a Cu+-responsive MerR-family metalloregulator—modulates RNAP interactions with CueR’s cognate suboptimal promoter PcopA, and how RNAP affects CueR−PcopAinteractions. We find that RNAP can form two noninterconverting complexes at PcopAin the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a “biased sampling” instead of “dynamic equilibrium shifting” mechanism in regulating transcription initiation; it modulates RNAP’s binding–unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopAinto its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

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.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
Author(s):  
Lasse L. Hildebrandt ◽  
Søren Preus ◽  
Victoria Birkedal
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Complexes ◽  
Distance Information ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

scholarly journals Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins

2021 ◽  
Author(s):  
Joshua C. Sanders ◽  
Erik D. Holmstrom
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Dynamic Properties ◽  
Resonance Energy ◽  
Integrative Approach ◽  
Biological Macromolecules ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Biomolecular Simulations ◽  
Diverse Interactions

Abstract The conformations of biological macromolecules are intimately related to their cellular functions. Conveniently, the well-characterized dipole–dipole distance-dependence of Förster resonance energy transfer (FRET) makes it possible to measure and monitor the nanoscale spatial dimensions of these conformations using fluorescence spectroscopy. For this reason, FRET is often used in conjunction with single-molecule detection to study a wide range of conformationally dynamic biochemical processes. Written for those not yet familiar with the subject, this review aims to introduce biochemists to the methodology associated with single-molecule FRET, with a particular emphasis on how it can be combined with biomolecular simulations to study diverse interactions between nucleic acids and proteins. In the first section, we highlight several conceptual and practical considerations related to this integrative approach. In the second section, we review a few recent research efforts wherein various combinations of single-molecule FRET and biomolecular simulations were used to study the structural and dynamic properties of biochemical systems involving different types of nucleic acids (e.g., DNA and RNA) and proteins (e.g., folded and disordered).

scholarly journals Bayesian machine learning analysis of single-molecule fluorescence colocalization images

2021 ◽  
Author(s):  
Yerdos A. Ordabayev ◽  
Larry J. Friedman ◽  
Jeff Gelles ◽  
Douglas L. Theobald
Keyword(s):  
Machine Learning ◽  
Single Molecule ◽  
Specific Binding ◽  
Image Data ◽  
Automated Analysis ◽  
Surface Binding ◽  
Single Molecule Fluorescence ◽  
Binding Characteristics ◽  
Wide Range ◽  
Downstream Analysis

AbstractMulti-wavelength single-molecule fluorescence colocalization (CoSMoS) methods allow elucidation of complex biochemical reaction mechanisms. However, analysis of CoSMoS data is intrinsically challenging because of low image signal-to-noise ratios, non-specific surface binding of the fluorescent molecules, and analysis methods that require subjective inputs to achieve accurate results. Here, we use Bayesian probabilistic programming to implement Tapqir, an unsupervised machine learning method based on a holistic, physics-based causal model of CoSMoS data. This method accounts for uncertainties in image analysis due to photon and camera noise, optical non-uniformities, non-specific binding, and spot detection. Rather than merely producing a binary “spot/no spot” classification of unspecified reliability, Tapqir objectively assigns spot classification probabilities that allow accurate downstream analysis of molecular dynamics, thermodynamics, and kinetics. We both quantitatively validate Tapqir performance against simulated CoSMoS image data with known properties and also demonstrate that it implements fully objective, automated analysis of experiment-derived data sets with a wide range of signal, noise, and non-specific binding characteristics.

scholarly journals Multiplexed imaging of high density libraries of RNAs with MERFISH and expansion microscopy

2017 ◽  
Author(s):  
Guiping Wang ◽  
Jeffrey R. Moffitt ◽  
Xiaowei Zhuang
Keyword(s):  
Single Molecule ◽  
Detection Efficiency ◽  
High Abundance ◽  
Total Density ◽  
Accurate Identification ◽  
Rna Molecules ◽  
Wide Range ◽  
Multiplexed Imaging ◽  
Labeling Approach

AbstractAs an image-based single-cell transcriptomics approach, multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows hundreds to thousands of RNA species to be identified, counted and localized in individual cells while preserving the native spatial context of RNAs. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule FISH (smFISH) to read out these barcodes. The accuracy of RNA identification relies on spatially separated signals from individual RNA molecules, which limits the density of RNAs that can be measured and makes the multiplexed imaging of a large number of high-abundance RNAs challenging. Here we report an approach that combines MERFISH and expansion microscopy to substantially increase the total density of RNAs that can be measured. Using this approach, we demonstrate accurate identification and counting of RNAs, with a near 100% detection efficiency, in a ~130-RNA library composed of many high-abundance RNAs, the total density of which is more than 10 fold higher than previously reported. In parallel, we demonstrate the combination of MERFISH with immunofluorescence. These advances increase the versatility of MERFISH and will facilitate its application of a wide range of biological problems.

scholarly journals Complex RNA Folding Kinetics Revealed by Single-Molecule FRET and Hidden Markov Models

2014 ◽  
Vol 136 (12) ◽  
pp. 4534-4543 ◽  
Author(s):  
Bettina G. Keller ◽  
Andrei Kobitski ◽  
Andres Jäschke ◽  
G. Ulrich Nienhaus ◽  
Frank Noé
Keyword(s):  
Hidden Markov Models ◽  
Single Molecule ◽  
Rna Folding ◽  
Markov Models ◽  
Hidden Markov ◽  
Folding Kinetics ◽  
Single Molecule Fret

scholarly journals Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

2019 ◽  
Author(s):  
Ikenna C Okafor ◽  
Digvijay Singh ◽  
Yanbo Wang ◽  
Minhee Jung ◽  
Haobo Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Engineering ◽  
Dna Unwinding ◽  
Dependent Manner ◽  
Guide Rnas ◽  
Single Molecule Fret ◽  
Wide Range ◽  
The Cost ◽  
Single Molecule Analysis ◽  
Target Activity

Abstract Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5′ end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5′ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

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