Tunable blinking kinetics of cy5 for precise DNA quantification and single-nucleotide difference detection.

Biophysical Journal
Hsin-Chih YehTza-Huei Wang

Abstract

Fluorescence correlation spectroscopy (FCS) can resolve the intrinsic fast-blinking kinetics (FBKs) of fluorescent molecules that occur on the order of microseconds. These FBKs can be heavily influenced by the microenvironments in which the fluorescent molecules are contained. In this work, FCS is used to monitor the dynamics of fluorescence emission from Cy5 labeled on DNA probes. We found that the FBKs of Cy5 can be tuned by having more or less unpaired guanines (upG) and thymines (upT) around the Cy5 dye. The observed FBKs of Cy5 are found to predominantly originate from the isomerization and back-isomerization processes of Cy5, and Cy5-nucleobase interactions are shown to slow down these processes. These findings lead to a more precise quantification of DNA hybridization using FCS analysis, in which the FBKs play a major role rather than the diffusion kinetics. We further show that the alterations of the FBKs of Cy5 on probe hybridization can be used to differentiate DNA targets with single-nucleotide differences. This discrimination relies on the design of a probe-target-probe DNA three-way-junction, whose basepairing configuration can be altered as a consequence of a single-nucleotide substitution on the target. Reconfigu...Continue Reading

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Citations

Apr 20, 2010·Journal of the American Chemical Society·Chris I RichardsRobert M Dickson
Jul 11, 2012·Journal of the American Chemical Society·Hsin-Chih YehJames H Werner
Nov 12, 2009·The Journal of Physical Chemistry. B·Chris I RichardsRobert M Dickson
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Feb 26, 2020·Chemistry : a European Journal·Kiyohiko Kawai, Atsushi Maruyama
Jul 6, 2013·Nanoscale·Judy M OblioscaHsin-Chih Yeh
Apr 20, 2010·The Journal of Physical Chemistry. B·Stanislav KalininClaus A M Seidel
Aug 12, 2010·Nano Letters·Hsin-Chih YehJames H Werner
Dec 11, 2019·The Journal of Physical Chemistry. C, Nanomaterials and Interfaces·Ferdinand F E KohleUlrich B Wiesner
Jan 17, 2020·ACS Sensors·Nicholas E LarkeySean M Burrows
Sep 11, 2021·The Journal of Physical Chemistry Letters·Natalie A PacePeter M Goodwin

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