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

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.