An automated Bayesian pipeline for rapid analysis of single-molecule binding data

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein’s binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.Analysis of single-molecule binding assays still requires substantial manual user intervention. Here, the authors present a pipeline for rapid, automated analysis of co-localization single-molecule spectroscopy images, with a modular user interface that can be adjusted to a range of experimental conditions.

[1]  T. Ha,et al.  Single-Molecule Analysis of Lipid-Protein Interactions in Crude Cell Lysates. , 2016, Analytical chemistry.

[2]  Fluorescent Labeling of Proteins in Whole Cell Extracts for Single-Molecule Imaging. , 2016, Methods in enzymology.

[3]  Changbong Hyeon,et al.  Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics , 2013, Nature Communications.

[4]  F Sachs,et al.  A direct optimization approach to hidden Markov modeling for single channel kinetics. , 2000, Biophysical journal.

[5]  Colin H. LaMont,et al.  The Lindley paradox: The loss of resolution in Bayesian inference , 2016, 1610.09433.

[6]  Colin Echeverría Aitken,et al.  An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. , 2008, Biophysical journal.

[7]  A. Gelfand,et al.  Bayesian Model Choice: Asymptotics and Exact Calculations , 1994 .

[8]  I. Young,et al.  Digital Fluorescence Imaging Using Cooled CCD Array Cameras invisible , 1998 .

[9]  David J. C. MacKay,et al.  Information Theory, Inference, and Learning Algorithms , 2004, IEEE Transactions on Information Theory.

[10]  J. Gelles,et al.  Mechanism of Transcription Initiation at an Activator-Dependent Promoter Defined by Single-Molecule Observation , 2012, Cell.

[11]  Hiroshi M Sasaki,et al.  Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex. , 2015, Molecular cell.

[12]  R. T. Cox.  Probability, frequency and reasonable expectation , 1990 .

[13]  Johan Elf,et al.  Variational Algorithms for Analyzing Noisy Multistate Diffusion Trajectories. , 2018, Biophysical journal.

[14]  M. Moore,et al.  Ordered and Dynamic Assembly of Single Spliceosomes , 2011, Science.

[15]  K. Lidke,et al.  supplementary figures , 2018 .

[16]  Dmitri S. Pavlichin,et al.  Single Molecule Analysis Research Tool (SMART): An Integrated Approach for Analyzing Single Molecule Data , 2012, PloS one.

[17]  M. Moore,et al.  Alternative spliceosome assembly pathways revealed by single-molecule fluorescence microscopy. , 2013, Cell reports.

[18]  T. Tuschl,et al.  Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes , 2009, Nature.

[19]  Ji-Joon Song,et al.  Dynamic anchoring of the 3'-end of the guide strand controls the target dissociation of Argonaute-guide complex. , 2013, Journal of the American Chemical Society.

[20]  Rob Phillips,et al.  Multiple LacI-mediated loops revealed by Bayesian statistics and tethered particle motion , 2014, Nucleic acids research.

[21]  Ji-Joon Song,et al.  Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs. , 2015, Molecular cell.

[22]  James B. Munro,et al.  Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging. , 2009, Biophysical journal.

[23]  K. Imahori,et al.  Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a Nonsporulating Thermophilic Bacterium from a Japanese Thermal Spa , 1974 .

[24]  S. McKinney,et al.  Analysis of single-molecule FRET trajectories using hidden Markov modeling. , 2006, Biophysical journal.

[25]  I. MacRae,et al.  A Dynamic Search Process Underlies MicroRNA Targeting , 2015, Cell.

[26]  M. Saxton.  Single-particle tracking: the distribution of diffusion coefficients. , 1997, Biophysical journal.

[27]  J. Gelles,et al.  Multi-wavelength single-molecule fluorescence analysis of transcription mechanisms. , 2015, Methods.

[28]  Yasushi Sako,et al.  Variational Bayes analysis of a photon-based hidden Markov model for single-molecule FRET trajectories. , 2012, Biophysical journal.

[29]  L. Lim,et al.  MicroRNA targeting specificity in mammals: determinants beyond seed pairing. , 2007, Molecular cell.

[30]  Chris H Wiggins,et al.  Empirical Bayes methods enable advanced population-level analyses of single-molecule FRET experiments. , 2014, Biophysical journal.

[31]  T. Tuschl,et al.  Structure of the guide-strand-containing argonaute silencing complex , 2008, Nature.

[32]  D. C. Swarts,et al.  Autonomous Generation a nd Loading of DNA Guides by Bacterial Argonaute Graphical Abstract Highlights , 2017 .

[33]  Chris H Wiggins,et al.  Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data. , 2009, Biophysical journal.

[34]  Matthew J. Beal.  Variational algorithms for approximate Bayesian inference , 2003 .

[35]  Keith A. Lidke,et al.  Fast, single-molecule localization that achieves theoretically minimum uncertainty , 2010, Nature Methods.

[36]  J. Elf,et al.  Extracting intracellular diffusive states and transition rates from single-molecule tracking data , 2013, Nature Methods.

[37]  Bo W. Han,et al.  The initial uridine of primary piRNAs does not create the tenth adenine that Is the hallmark of secondary piRNAs. , 2014, Molecular cell.

[38]  J. Zerubia,et al.  Gaussian approximations of fluorescence microscope point-spread function models. , 2007, Applied optics.

[39]  Sean P Ryder,et al.  Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing. , 2011, RNA.

[40]  T. Ha,et al.  The Single-Molecule Centroid Localization Algorithm Improves the Accuracy of Fluorescence Binding Assays. , 2018, Biochemistry.

[41]  Stephan Preibisch,et al.  Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking , 2015, The Journal of cell biology.

[42]  R. Levy,et al.  Direct Determination of Kinetic Rates from Single-Molecule Photon Arrival Trajectories Using Hidden Markov Models. , 2003, The journal of physical chemistry. A.

[43]  D. C. Swarts,et al.  Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage , 2013, Proceedings of the National Academy of Sciences.

[44]  A. Laederach,et al.  Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics , 2015, Nature Methods.

[45]  M. Moore,et al.  Visualizing the splicing of single pre-mRNA molecules in whole cell extract. , 2007, RNA.

[46]  Claire M Brown,et al.  A Quantitative Measure of Field Illumination. , 2015, Journal of biomolecular techniques : JBT.

[47]  Arkajit Dey,et al.  Inferring transient particle transport dynamics in live cells , 2015, Nature Methods.

[48]  Stan J. J. Brouns,et al.  DNA-guided DNA interference by a prokaryotic Argonaute , 2014, Nature.

[49]  Robert D. Cousins,et al.  The Jeffreys–Lindley paradox and discovery criteria in high energy physics , 2013, Synthese.

[50]  M. Moore,et al.  Single-Molecule Imaging Reveals that Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides , 2015, Cell.

[51]  Hui Jia,et al.  Minimum variance unbiased subpixel centroid estimation of point image limited by photon shot noise. , 2010, Journal of the Optical Society of America. A, Optics, image science, and vision.

[52]  T. Tuschl,et al.  Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex , 2008, Nature.

[53]  D. Grier,et al.  Methods of Digital Video Microscopy for Colloidal Studies , 1996 .

[54]  Yuhong Yang,et al.  Information Theory, Inference, and Learning Algorithms , 2005 .

[55]  J. Lippincott-Schwartz,et al.  High-density mapping of single-molecule trajectories with photoactivated localization microscopy , 2008, Nature Methods.

[56]  I. MacRae,et al.  Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets , 2015, eLife.