Single molecule force spectroscopy at high data acquisition: A Bayesian nonparametric analysis

Sgouralis, Ioannis; Whitmore, Miles; Lapidus, Lisa J.; Comstock, Matthew; Pressé, Steve

doi:10.1063/1.5008842

Cited by 38 publications

(66 citation statements)

References 36 publications

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“…In particular, analyzing data derived from mobile molecules within an illuminated confocal region breaks down the perennial parametric Bayesian paradigm that has been the workhorse of data analysis [4,21,28,39,81,83,84]. We argue here that BNPs-which provide principled extensions of the Bayesian methodology [40,85]-show promise in physics [4,39,44,45,47,86,87] and give us a working solution to fundamental parametric challenges.…”

Section: Discussionmentioning

confidence: 94%

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Pitching Single-Focus Confocal Data Analysis One Photon at a Time with Bayesian Nonparametrics

et al. 2020

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Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.

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Section: Discussionmentioning

confidence: 94%

“…arrivals. The underlying theory, Bayesian nonparametrics (BNPs) [40], is a powerful set of tools still under active development and largely unknown to the physical sciences [4,39,[41][42][43][44][45][46][47][48].…”

Section: Fig 2 Estimates Of Diffusion Coefficients From Photon Arrivalmentioning

confidence: 99%

Pitching Single-Focus Confocal Data Analysis One Photon at a Time with Bayesian Nonparametrics

et al. 2020

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“…In particular, analyzing data derived from mobile biomolecules within an illuminated confocal region breaks down the perennial parametric Bayesian paradigm that has been the workhorse of biophysical data analysis [16,44,55,60,70,92,96]. We argue here that BNPs-which provide principled extensions of Bayesian logic [31,93]-show promise in Biophysics [46,55,87,88,90,92] and give us a working solution to prior parametric challenges.…”

Section: Discussionmentioning

confidence: 99%

“…While BNPs have had a deep impact on Data Sci-ence since their inception, they are relatively new to Biophysics with a handful of papers [18,47,73] published to date using BNPs in Biophysical applications [50,55,[87][88][89][90]92].…”

Section: Introductionmentioning

confidence: 99%

Pitching single-focus confocal data analysis one photon at a time with Bayesian nonparametrics

Tavakoli

Jazani

Sgouralis

et al. 2019

Preprint

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Fluorescence time traces are used to report on dynamical properties of biomolecules. The basic unit of information drawn from these traces is the individual photon. Single photons carry instantaneous information from the biomolecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus from confocal microscope it is theoretically possible to monitor biomolecular dynamics at such timescales. In practice, however, signals are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-fluorescence signals are collected and temporally auto-correlated over several minutes. So far, it has been impossible to analyze dynamical properties of biomolecules on timescales approaching data acquisition as this demands that we estimate the instantaneous number of biomolecules emitting photons and their positions within the confocal volume. The mathematical structure of this problem demands that we abandon the normal ("parametric") Bayesian paradigm. Here, we exploit novel mathematical tools, namely Bayesian nonparametrics, that allow us to deduce in a principled fashion the same information normally deduced from FCS but from the direct analysis of significantly smaller datasets starting from individual photon arrivals. We discuss the implications of this method in helping dramatically reduce phototoxic damage on the sample and the ability to monitor out-of-equilibrium processes.

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“…Data collected at 66.7 kHz was boxcar averaged to a final lower rate, as specified, but generally sufficiently fast to accurately determine telomerase template elongation states and lifetimes, between 1 and 20 ms per data point. Automatic state and step finding in telomerase trajectories was carried out using the code XL-ICON which is an implementation of the infinite Hidden Markov Modelling (iHMM) method which is a Bayesian nonparametric extension of the very commonly used HMM method and allows for state discovery with simultaneous correction for high-resolution tweezers measurement drift and finite response time 38 . Regions of a small number of traces (4 out of 60) were excluded from contributing to the state and step size distributions because they had a very large number of reversing states that would have obscured the contributions from the remainder of traces.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of processive telomerase catalysis revealed by high-resolution optical tweezers

Patrick

Slivka

Payne

et al. 2019

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Telomerase | Telomere | Optical Tweezers | Single-Molecule Fluorescence | G-quadruplex | Cancer | AgingCorrespondence:schmi706@msu.edu (Twitter: @jenscs83) and mjcomsto@msu.eduTelomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells 1 . Telomerase is a reverse transcriptase composed of telomerase reverse transcriptase (TERT) 2 , telomerase RNA (TR) 3 , and several accessory proteins 4 . To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to the singlestranded overhangs at chromosome ends by copying the template region within its RNA subunit 5,6 . Between repeat additions, the RNA template must be recycled, which requires disrupting the base-pairing between TR and the substrate DNA 6 . How telomerase remains associated with the substrate DNA during this critical translocation step remains unknown. Here, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. Using a newly developed single molecule telomerase activity assay utilizing high-resolution optical tweezers, we directly measured stepwise, processive telomerase activity. We found that telomerase tightly associates with its DNA substrate, synthesizing multiple telomeric repeats before releasing them in a single large step. The rate at which product is released from the anchor site closely corresponds to the overall rate of product dissociation from elongating telomerase 7 , suggesting that it is a key parameter controlling telomerase processivity. We observed folding of the released product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into processive telomerase catalysis, a process critical for telomere length maintenance and therefore cancer cell survival 8,9 .To investigate substrate extension by a single telomerase ribonucleoprotein (RNP), we developed a single molecule telomerase activity assay using dual-trap high-resolution optical tweezers (Fig. 1a). Telomerase and its substrate were attached to separate polystyrene beads (Fig. 1b). The connection between the two beads was formed by the association of telomerase with its substrate DNA ( Fig. 1a,b). When applying a low constant force (4.0-4.5 pN) to the tether, substrate elongation by telomerase was measured as an increase in dis-tance between the two beads ( Fig. 1a). To attach telomerase to the bead, we utilized a 3xFLAG-HaloTag on TERT, modified with biotin ( Fig. 1b,c). This tag did not affect telomerase assembly, catalytic activity, processivity, or stimulation by POT1/TPP1 ( Fig. 1c,d, Extended Data Fig. 1a-c), indicating that it is fully functional.Using this experimental approach, we set out to analyze how telomerase processively synthesizes telomeric repeats. If substrate DNA is only bound to telomerase by basepairing to TR we would expect to observe a sequence of single nucleotide (nt) addition events as a result o...

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Single molecule force spectroscopy at high data acquisition: A Bayesian nonparametric analysis

Cited by 38 publications

References 36 publications

Pitching Single-Focus Confocal Data Analysis One Photon at a Time with Bayesian Nonparametrics

Pitching Single-Focus Confocal Data Analysis One Photon at a Time with Bayesian Nonparametrics

Pitching single-focus confocal data analysis one photon at a time with Bayesian nonparametrics

Mechanism of processive telomerase catalysis revealed by high-resolution optical tweezers

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