An automated Bayesian pipeline for rapid analysis of single-molecule binding data.
Argonaute Proteins
/ metabolism
Bacterial Proteins
/ metabolism
Bayes Theorem
Binding Sites
DNA, Single-Stranded
/ metabolism
Image Processing, Computer-Assisted
/ methods
Kinetics
Microscopy, Fluorescence
/ instrumentation
Protein Binding
Single Molecule Imaging
/ instrumentation
Software
Thermus thermophilus
/ metabolism
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
17 01 2019
17 01 2019
Historique:
received:
18
04
2018
accepted:
13
12
2018
entrez:
19
1
2019
pubmed:
19
1
2019
medline:
21
3
2019
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 30655518
doi: 10.1038/s41467-018-08045-5
pii: 10.1038/s41467-018-08045-5
pmc: PMC6336789
doi:
Substances chimiques
Argonaute Proteins
0
Bacterial Proteins
0
DNA, Single-Stranded
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Validation Study
Langues
eng
Sous-ensembles de citation
IM
Pagination
272Subventions
Organisme : NIGMS NIH HHS
ID : R37 GM062862
Pays : United States
Organisme : NIDA NIH HHS
ID : U01 DA047733
Pays : United States
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