Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence spectroscopy

Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence... Fluorescent dyes are broadly used in many biotechnological applications to detect and visualize DNA molecules. However, their binding to DNA alters the structural and nanomechanical properties of DNA and, thus, interferes with associated biological processes. In this work we employed magnetic tweezers and fluorescence spectroscopy to investigate the binding of PicoGreen to DNA at room temperature in a concentration-dependent manner. PicoGreen is an ultrasensitive quinolinium nucleic acid stain exhibiting hardly any background signal from unbound dye molecules. By means of stretching and overwinding single, torsionally constrained, nick-free double-stranded DNA molecules, we acquired force-extension and supercoiling curves which allow quantifying DNA contour length, persistence length and other thermodynamical binding parameters, respectively. The results of our magnetic tweezers single-molecule binding study were well supported through analyzing the fluorescent spectra of stained DNA. On the basis of our work, we could identify a concentration-dependent bimodal binding behavior, where, apparently, PicoGreen associates to DNA as an intercalator and minor-groove binder simultaneously. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Biophysics Journal Springer Journals

Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence spectroscopy

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Publisher
Springer Berlin Heidelberg
Copyright
Copyright © 2017 by European Biophysical Societies' Association
Subject
Life Sciences; Biochemistry, general; Biological and Medical Physics, Biophysics; Cell Biology; Neurobiology; Membrane Biology; Nanotechnology
ISSN
0175-7571
eISSN
1432-1017
D.O.I.
10.1007/s00249-017-1204-z
Publisher site
See Article on Publisher Site

Abstract

Fluorescent dyes are broadly used in many biotechnological applications to detect and visualize DNA molecules. However, their binding to DNA alters the structural and nanomechanical properties of DNA and, thus, interferes with associated biological processes. In this work we employed magnetic tweezers and fluorescence spectroscopy to investigate the binding of PicoGreen to DNA at room temperature in a concentration-dependent manner. PicoGreen is an ultrasensitive quinolinium nucleic acid stain exhibiting hardly any background signal from unbound dye molecules. By means of stretching and overwinding single, torsionally constrained, nick-free double-stranded DNA molecules, we acquired force-extension and supercoiling curves which allow quantifying DNA contour length, persistence length and other thermodynamical binding parameters, respectively. The results of our magnetic tweezers single-molecule binding study were well supported through analyzing the fluorescent spectra of stained DNA. On the basis of our work, we could identify a concentration-dependent bimodal binding behavior, where, apparently, PicoGreen associates to DNA as an intercalator and minor-groove binder simultaneously.

Journal

European Biophysics JournalSpringer Journals

Published: Mar 1, 2017

References

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