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Single-Molecule Kinetic Studies of Nucleic Acids by Förster Resonance Energy Transfer


Hadzic, Mélodie C A S; Sigel, Roland K O; Börner, Richard (2022). Single-Molecule Kinetic Studies of Nucleic Acids by Förster Resonance Energy Transfer. In: Steger, Gerhard; Rosenbach, Hannah; Span, Ingrid. DNAzymes : Methods and Protocols. New York: Springer, 173-190.

Abstract

Single-molecule microscopy is often used to observe and characterize the conformational dynamics of nucleic acids (NA). Due to the large variety of NA structures and the challenges specific to single-molecule observation techniques, the data recorded in such experiments must be processed via multiple statistical treatments to finally yield a reliable mechanistic view of the NA dynamics. In this chapter, we propose a comprehensive protocol to analyze single-molecule trajectories in the scope of single-molecule Förster resonance energy transfer (FRET) microscopy. The suggested protocol yields the conformational states common to all molecules in the investigated sample, together with the associated conformational transition kinetics. The given model resolves states that are indistinguishable by their observed FRET signals and is estimated with 95% confidence using error calculations on FRET states and transition rate constants. In the end, a step-by-step user guide is given to reproduce the protocol with the Multifunctional Analysis Software to Handle single-molecule FRET data (MASH-FRET).

Abstract

Single-molecule microscopy is often used to observe and characterize the conformational dynamics of nucleic acids (NA). Due to the large variety of NA structures and the challenges specific to single-molecule observation techniques, the data recorded in such experiments must be processed via multiple statistical treatments to finally yield a reliable mechanistic view of the NA dynamics. In this chapter, we propose a comprehensive protocol to analyze single-molecule trajectories in the scope of single-molecule Förster resonance energy transfer (FRET) microscopy. The suggested protocol yields the conformational states common to all molecules in the investigated sample, together with the associated conformational transition kinetics. The given model resolves states that are indistinguishable by their observed FRET signals and is estimated with 95% confidence using error calculations on FRET states and transition rate constants. In the end, a step-by-step user guide is given to reproduce the protocol with the Multifunctional Analysis Software to Handle single-molecule FRET data (MASH-FRET).

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Additional indexing

Item Type:Book Section, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Scopus Subject Areas:Life Sciences > Molecular Biology
Life Sciences > Genetics
Language:English
Date:2022
Deposited On:10 Jan 2023 16:39
Last Modified:23 Jun 2024 03:30
Publisher:Springer
Series Name:Methods in Molecular Biology
ISSN:1064-3745
ISBN:978-1-0716-2046-5
OA Status:Closed
Publisher DOI:https://doi.org/10.1007/978-1-0716-2047-2_12
Other Identification Number:eBook ISBN 978-1-0716-2047-2
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