Sequence-Dependent Fluorescence of Cy3- and Cy5-Labeled Double-Stranded DNA

The fluorescent intensity of Cy3
and Cy5 dyes is strongly dependent
on the nucleobase sequence of the labeled oligonucleotides. Sequence-dependent
fluorescence may significantly influence the data obtained from many
common experimental methods based on fluorescence detection of nucleic
acids, such as sequencing, PCR, FRET, and FISH. To quantify sequence
dependent fluorescence, we have measured the fluorescence intensity
of Cy3 and Cy5 bound to the 5′ end of all 1024 possible double-stranded
DNA 5mers. The fluorescence intensity was also determined for these
dyes bound to the 5′ end of fixed-sequence double-stranded
DNA with a variable sequence 3′ overhang adjacent to the dye.
The labeled DNA oligonucleotides were made using light-directed, in
situ microarray synthesis. The results indicate that the fluorescence
intensity of both dyes is sensitive to all five bases or base pairs,
that the sequence dependence is stronger for double- (vs single-)
stranded DNA, and that the dyes are sensitive to both the adjacent
dsDNA sequence and the 3′-ssDNA overhang. Purine-rich sequences
result in higher fluorescence. The results can be used to estimate
measurement error in experiments with fluorescent-labeled DNA, as
well as to optimize the fluorescent signal by considering the nucleobase
environment of the labeling cyanine dye.