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Reversible Fluorescence Photoswitching in DNA
journal contribution
posted on 2015-12-16, 21:47 authored by Darren
A. Smith, Philipp Holliger, Cristina FlorsWe describe the engineering of reversible fluorescence
photoswitching
in DNA with high-density substitution, and its applications in advanced
fluorescence microscopy methods. High-density labeling of DNA with
cyanine dyes can be achieved by polymerase chain reaction using a
modified DNA polymerase that has been evolved to efficiently incorporate
Cy3- and Cy5-labeled cytosine base analogues into double-stranded
DNA. The resulting biopolymer, “CyDNA”, displays hundreds
of fluorophores per DNA strand and is strongly colored and highly
fluorescent, although previous observations suggest that fluorescence
quenching at such high density might be a concern, especially for
Cy5. Herein, we first investigate the mechanisms of fluorescence quenching
in CyDNA and we suggest that two different mechanisms, aggregate formation
and resonance energy transfer, are responsible for fluorescence quenching
at high labeling densities. Moreover, we have been able to re-engineer
CyDNA into a reversible fluorescence photoswitchable biopolymer by
using the properties of the Cy3–Cy5 pair. This novel biopolymer
constitutes a new class of photoactive DNA-based nanomaterial and
is of great interest for advanced microscopy applications. We show
that reversible fluorescence photoswitching in CyDNA can be exploited
in optical lock-in detection imaging. It also lays the foundations
for improved and sequence-specific super-resolution fluorescence microscopy
of DNA.