# Time-Resolved Fluorescence Detection of Nicked DNA via Site-Specific Stacking of Sulfo-Cy3: The Role of Charge, Polarity, Linker, and Sequence Context

## TL;DR

This study explores how sulfo-Cy3 fluorescence changes when it stacks at nicks in DNA, offering a new method to detect and study DNA damage and nanostructures.

## Contribution

The paper introduces a systematic time-resolved fluorescence method to study site-specific stacking of sulfo-Cy3 on nicked DNA.

## Key findings

- sulfo-Cy3 fluorescence increases when it stacks at nicks in dsDNA.
- Labeling polarity, linker type, and nucleotide identity affect fluorescence behavior.
- Switching to positively charged Cy3 alters stacking and fluorescence properties.

## Abstract

Nicked DNA can result from damage or cellular processing
and is
also prevalent in DNA nanostructures, so sensitive methods to probe
the generation and location of nicks are desirable. It was found recently
that sulfo-Cy3 (sCy3), a disulfonated version of Cy3, could stack
in a nick of double-stranded DNA (dsDNA) when attached to a thymidine
on the 3′ end of one strand, resulting in an increase in fluorescence
brightness. Here, we have performed a systematic time-resolved fluorescence
study of the stacking of sCy3 on nicked DNA. We have varied the labeling
polarity (5′ vs 3′), linker (dT vs phosphate), labeled
nucleotide (A, C, T, or G), DNA structure (nicked dsDNA, dsDNA, single-stranded
DNA, and dsDNA with a single-strand overhang), and NaCl concentration.
We also studied the effect of switching the negatively charged sCy3
to the positively charged Cy3. We have shown that the site-specific
stacking and fluorescence modulation of sCy3 on nicked DNA is a general
phenomenon that could find application in time-resolved assays of
DNA processes such as annealing, strand displacement, and enzymatic
nicking.