Simulations of camera-based single-molecule fluorescence experiments

Börner, Richard; Kowerko, Danny; Hadzic, Mélodie C. A. S.; König, S.; Ritter, Marc; Sigel, Roland K. O.

doi:10.1371/journal.pone.0195277

Cited by 20 publications

(30 citation statements)

References 60 publications

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“…Kinetic models with different connectivities and degeneracies were tested and evaluated based on a BIC and biochemical significance (see Supplementary Discussion). To validate the obtained rate system, degenerate FRET traces were re-simulated in a subroutine of MASH-FRET 77…”

Section: Methodsmentioning

confidence: 99%

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts

et al. 2020

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The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons. By single-molecule FRET, we explore the binding kinetics of the most important, structurally conserved contact, the exon and intron binding site 1 (EBS1/IBS1). A comparison of RNA-RNA and RNA-DNA hybrid contacts identifies transient metal ion binding as a major source of kinetic heterogeneity which typically appears in the form of degenerate FRET states. Molecular dynamics simulations suggest a structural link between heterogeneity and the sugar conformation at the exonintron binding interface. While Mg 2+ ions lock the exon in place and give rise to long dwell times in the exon bound FRET state, sugar puckering alleviates this structural rigidity and likely promotes exon release. The interplay of sugar puckering and metal ion coordination may be an important mechanism to balance binding affinities of RNA and DNA interactions in general.

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Section: Methodsmentioning

confidence: 99%

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts

et al. 2020

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“…However, the influence of additional noise sources needs to be considered. For the electron-multiplying CCD (EMCCD) in our setup, the variance in the fluorescence intensity is increased by a factor of two over the shot noise, as has been discussed in detail elsewhere (51,52). This uncertainty can be included in the variance of the FRET efficiency, which can still be approximated, in this case, by a Gaussian distribution (see Supporting Materials and Methods).…”

Section: Observables In Single-molecule Fret Datamentioning

confidence: 99%

“…In addition to the shot noise, which follows a Poisson distribution, other noise factors from the camera need to be incorporated. A detailed description of noise coming from EMCCD cameras can be found in (51,52). The most important source of additional noise comes from the on-chip gain of the EMCCD, which broadens the variance of the intensity by a factor of two (or the SD by ffiffi ffi 2 p ).…”

Section: Emccd Shot-noise Corrections For Hmmmentioning

confidence: 99%

“…Mean total fluorescence intensities and average FRET efficiencies were introduced, and donor and acceptor fluorescence intensity trajectories were determined. Every point of this trajectory pair was finally replaced by a stretched Poissonian random number, taking the original value as its mean and a stretch factor of 2 to incorporate the additional noise component generated by the gain of an EMCCD camera (51,52).…”

Section: Monte Carlo Test Of the New Estimatorsmentioning

confidence: 99%

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Analyzing the Dynamics of Single TBP-DNA-NC2 Complexes Using Hidden Markov Models

Zarrabi

Schluesche

Meisterernst

et al. 2018

Biophysical Journal

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Single-pair Fö rster resonance energy transfer (spFRET) has become an important tool for investigating conformational dynamics in biological systems. To extract dynamic information from the spFRET traces measured with total internal reflection fluorescence microscopy, we extended the hidden Markov model (HMM) approach. In our extended HMM analysis, we incorporated the photon-shot noise from camera-based systems into the HMM. Thus, the variance in Fö rster resonance energy transfer (FRET) efficiency of the various states, which is typically a fitted parameter, is explicitly included in the analysis estimated from the number of detected photons. It is also possible to include an additional broadening of the FRET state, which would then only reflect the inherent flexibility of the dynamic biological systems. This approach is useful when comparing the dynamics of individual molecules for which the total intensities vary significantly. We used spFRET with the extended HMM analysis to investigate the dynamics of TATA-box-binding protein (TBP) on promoter DNA in the presence of negative cofactor 2 (NC2). We compared the dynamics of two promoters as well as DNAs of different length and labeling location. For the adenovirus major late promoter, four FRET states were observed; three states correspond to different conformations of the DNA in the TBP-DNA-NC2 complex and a four-state model in which the complex has shifted along the DNA. The HMM analysis revealed that the states are connected via a linear, four-well model. For the H2B promoter, more complex dynamics were observed. By clustering the FRET states detected with the HMM analysis, we could compare the general dynamics observed for the two promoter sequences. We observed that the dynamics from a stretched DNA conformation to a bent conformation for the two promoters were similar, whereas the bent conformation of the TBP-DNA-NC2 complex for the H2B promoter is approximately three times more stable than for the adenovirus major late promoter.

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“…However, a number of mandatory steps is described herein. We recommend to perform data analysis of smFRET movies by using our Multifunctional Analysis Software for heterogeneous FRET data (MASH-FRET) (https://github.com/RNA-FRETools/MASH-FRET.git) [10,39] or iSMS [40], both Matlab-based home-written software packages:…”

Section: Single Molecule Video (Smv) Processing and Data Analysismentioning

confidence: 99%

Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy

Zelger-Paulus

Hadzic

Sigel

et al. 2020

Methods in Molecular Biology

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Imaging fluorescently labeled biomolecules on a single-molecule level is a well-established technique to follow intra-and intermolecular processes in time, usually hidden in the ensemble average. The classical approach comprises surface immobilization of the molecule of interest, which increases the risk of restricting the natural behavior due to surface interactions. Encapsulation of such biomolecules into surface-tethered phospholipid vesicles enables to follow one molecule at a time, freely diffusing and without disturbing surface interactions. Further, the encapsulation allows to keep reaction partners (reactants and products) in close proximity and enables higher temperatures otherwise leading to desorption of the direct immobilized biomolecules. Here, we describe a detailed protocol for the encapsulation of a catalytically active RNA starting from surface passivation over RNA encapsulation to data evaluation of single-molecule FRET experiments in TIRF microscopy. We present an optimized procedure that preserves RNA functionality and applies to investigations of, e.g., large ribozymes and RNAs, where direct immobilization is structurally not possible.

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Simulations of camera-based single-molecule fluorescence experiments

Cited by 20 publications

References 60 publications

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts

Metal ions and sugar puckering balance single-molecule kinetic heterogeneity in RNA and DNA tertiary contacts

Analyzing the Dynamics of Single TBP-DNA-NC2 Complexes Using Hidden Markov Models

Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy

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