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

Author(s)

Nicole Kretschy, Matej Sack, Mark M. Somoza

Abstract

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.

Organisation(s)

Department of Inorganic Chemistry

Journal

Bioconjugate Chemistry

Volume

27

Pages

840-848

No. of pages

9

ISSN

1043-1802

DOI

https://doi.org/10.1021/acs.bioconjchem.6b00053

Publication date

03-2016

Peer reviewed

Yes

Austrian Fields of Science 2012

104004 Chemical biology

Keywords

ASJC Scopus subject areas

Bioengineering, Biotechnology, Biomedical Engineering, Pharmacology, Pharmaceutical Science, Organic Chemistry

Portal url

https://ucrisportal.univie.ac.at/en/publications/3f45f7ea-19fd-445c-8c24-9433581331b7