Journal Article

The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics

Roijmans, Roel F. H., Hoekstra, Tjalle P., Wuite, Gijs J. L., Heller, Iddo, Peterman, Erwin J. G., Biebricher, Andreas S.

2015

Overview

DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro . It is well known that they perturb DNA structure and stability, which can in turn influence DNA-processing by proteins. Here we elucidate this perturbation by combining single-dye fluorescence microscopy with force spectroscopy and measuring the kinetics of DNA intercalation by the mono- and bis-intercalating cyanine dyes SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3. We show that their DNA-binding affinity is mainly governed by a strongly tension-dependent dissociation rate. These rates can be tuned over a range of seven orders of magnitude by changing DNA tension, intercalating species and ionic strength. We show that optimizing these rates minimizes the impact of intercalators on strand separation and enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and maximal efficacy. DNA intercalators, a type of fluorescent probes widely used to visualize DNA, can perturb DNA structure and stability. Here, the authors show how DNA-binding affinity can be tuned using DNA tension, ionic strength and dye species, and how this can be used to minimize DNA structural perturbations.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 14/34 / 14/63 / 631/1647/1888/1889 / 631/337 / 631/45/147 / 631/45/173 / Deoxyribonucleic acid / DNA / DNA - chemistry / DNA-Directed DNA Polymerase / Dyes / Enzymatic activity / Enzymes / Fluorescence microscopy / Humanities and Social Sciences / Intercalating Agents - chemistry / Kinetics / Kymography / Microscopy / multidisciplinary / Probes / Science / Science (multidisciplinary)