Comparison of the sequence-dependent fluorescence of the cyanine dyes Cy3, Cy5, DyLight DY547 and DyLight DY647 on single-stranded DNA

Abstract

Cyanine dyes are commonly used for fluorescent labeling of DNA and RNA oligonucleotides in applications including qPCR, sequencing, fluorescence in situ hybridization, Förster resonance energy transfer, and labeling for microarray hybridization. Previous research has shown that the fluorescence efficiency of Cy3 and Cy5, covalently attached to the 5' end of single-stranded DNA, is strongly sequence dependent. Here, we show that DY547 and DY647, two alternative cyanine dyes that are becoming widely used for nucleic acid labeling, have a similar pattern of sequence-dependence, with adjacent purines resulting in higher intensity and adjacent cytosines resulting in lower intensity. Investigated over the range of all 1024 possible DNA 5mers, the intensities of Cy3 and Cy5 drop by ∼ 50% and ∼ 65% with respect to their maxima, respectively, whereas the intensities of DY547 and DY647 fall by ∼ 45% and ∼ 40%, respectively. The reduced magnitude of change of the fluorescence intensity of the DyLight dyes, particularly of DY647 in comparison with Cy5, suggests that these dyes are less likely to introduce sequence-dependent bias into experiments based on fluorescent labeling of nucleic acids.

Figure 1. Molecular structures of the cyanine dye phosphoramidites used in this study: Cy3, Cy5, DY547 and DY647.

Monomethoxytrityl (MMT) groups are present on the Cy-dyes; the 2-cyanoethyl group (CNEt) is the standard phosphate protecting group in oligonucleotide synthesis, and the diisopropyl group (N(iPr)₂) is displaced during the coupling reaction by the 5′-hydroxyl group to form a phosphate linkage between the terminal nucleoside and the dye.

Figure 2. Fluorescence intensity of cyanine-labeled single-stranded DNA.

(A) Fluorescence intensity consensus sequence logos of all 1024 ssDNA 5-mers labeled with a 5′ Cy3 phosphoramidite. Each consensus logo corresponds to those sequences spanning one eighth of the intensity range. (B) Fluorescence intensity of Cy3 and DY547 end-labeled 5-mers, ranked from most to least intense. The Cy3 curve drops by about 50% of the maximum intensity, while the DY547 curve drops about 45%. (C) Consensus sequence logos of all 1024 ssDNA 5-mers labeled with a 5′ DY547 phosphoramidite. (D) Consensus sequence logos of all 1024 ssDNA 5-mers labeled with a 5′ Cy5 phosphoramidite. (E) Fluorescence intensity of Cy5 and DY647 end-labeled 5-mers, ranked from most to least intense. The Cy5 curve drops by about 65% of the maximum intensity, while the DY647 curve drops about 40%. (F) Consensus sequence logos of all 1024 ssDNA 5-mers labeled with a 5′ DY647 phosphoramidite. The single error bars (SEM) on each curve are representative. The z-axis height measures the information content at each site in units of bits.