Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data

Greenfeld, Max; Pavlichin, Dmitri S.; Mabuchi, Hideo; Herschlag, Daniel

doi:10.1371/journal.pone.0030024

Cited by 89 publications

(115 citation statements)

References 66 publications

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“…Depending on the folding equilibrium and change in FRET for each molecule, smFRET data were collected at an acquisition rate between 50 and 310 frames per second. Data were analyzed using the SMART data analysis package (92). See Dataset S1 for the imaging parameters and buffer conditions for each measurement reported herein.…”

Section: Methodsmentioning

confidence: 99%

“…Thermodynamic and kinetic parameters were inferred from single-molecule FRET traces that were fit with a hidden Markov model (HMM)-based algorithm to a two-state model with a single unfolded (low FRET = 0.16) and single folded (high FRET = 0.45 or 0.8) state to extract transition probabilities, taking into account the noise observed in each intensity channel (92).…”

Section: Methodsmentioning

confidence: 99%

“…In each case, the data were found to fit better to the two-state model than to a three-state model based on the difference in the Bayesian information criterion (BIC) (92), which includes a statistical penalty for including additional states, with the following exceptions that do not affect the conclusions of this work: WT P4-P6 folding with a Cy3b/Atto674N dye pair at 1 mM Mg, and MC/MCR iso at 5 and 10 mM Mg 2+ . (Example traces can be found in Dataset S1.)…”

Section: Methodsmentioning

confidence: 99%

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Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

Bisaria

Greenfeld

Limouse

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.RNA folding | single-molecule FRET | kinetics | folding pathways | RNA tertiary motifs S tructured RNAs play central roles in biology, in the splicing and alternative splicing of eukaryotic pre-mRNAs, in the synthesis of proteins and their transport, and in chromosome maintenance (1-4). Beyond the RNAs and RNA-protein machines involved in these processes, it has been increasingly recognized that Nature has taken extensive advantage of RNA at multiple levels of gene regulation, and considerable current efforts are focused on uncovering the pathways and molecular mechanisms that underlie the functions of small RNAs and long noncoding RNAs (2, 5-8). The pervasive functions of RNA in biology underscore the importance of understanding RNA's fundamental physical properties and, ultimately, of using this understanding to predict the kinetics and thermodynamics of folding and conformational transitions responsible for RNA function.Decades of characterization of RNA folding and structure have led to generalizable principles and provided biological insights. The observations that RNA encodes genetic information, forms highly stable local structures, and can catalyze reactions provided support for the possibility that early life evolved using functional RNAs (9-12). This high stability was also re...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

Bisaria

Greenfeld

Limouse

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…1) The local background mean estimator (5,(12)(13)(14)(15)20) was calculated as the mean value of the pixel distribution within a local background aperture:…”

Section: Background Estimatorsmentioning

confidence: 99%

“…A common background estimator is the mean pixel intensity within a local background aperture surrounding the molecule of interest (Fig. 1 b, inset) (5,(12)(13)(14)(15)20). The advantage of the local aperture mean is that it is simple, fast, and robust when the peak of interest in the image frame is well isolated from neighboring peaks (17).…”

Section: Performance Of Traditional Background Estimatorsmentioning

confidence: 99%

Optimal Background Estimators in Single-Molecule FRET Microscopy

Preus

Hildebrandt

Birkedal

2016

Biophysical Journal

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Single-molecule total internal reflection fluorescence (TIRF) microscopy constitutes an umbrella of powerful tools that facilitate direct observation of the biophysical properties, population heterogeneities, and interactions of single biomolecules without the need for ensemble synchronization. Due to the low signal/noise ratio in single-molecule TIRF microscopy experiments, it is important to determine the local background intensity, especially when the fluorescence intensity of the molecule is used quantitatively. Here we compare and evaluate the performance of different aperture-based background estimators used particularly in single-molecule Fö rster resonance energy transfer. We introduce the general concept of multiaperture signatures and use this technique to demonstrate how the choice of background can affect the measured fluorescence signal considerably. A new, to our knowledge, and simple background estimator is proposed, called the local statistical percentile (LSP). We show that the LSP background estimator performs as well as current background estimators at low molecular densities and significantly better in regions of high molecular densities. The LSP background estimator is thus suited for single-particle TIRF microscopy of dense biological samples in which the intensity itself is an observable of the technique.

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Single‐molecule Fluorescence Imaging Techniques

Reid

Rothenberg

2015

Encyclopedia of Analytical Chemistry

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The past decade has been witnessed to exciting developments in advanced fluorescence microscopy techniques that rely on visualizing single emitting fluorophores. The proliferation of single‐molecule fluorescent imaging techniques and their application in biological research have the potential to revolutionize how research is performed and greatly increase our understanding of biological systems. Presently, these techniques are still relatively niche owing to technological barriers, but it is foreseeable that they will become an increasingly common way in which insights are sought in biology. Here, we review the basic principles of key single‐molecule techniques and their recent biological applications.

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Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data

Cited by 89 publications

References 66 publications

Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

Optimal Background Estimators in Single-Molecule FRET Microscopy

Single‐molecule Fluorescence Imaging Techniques

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