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

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.

Figure 1

Interaction modes of dyes (red) on DNA. (A) 5′ dye with adjacent nucleobases (blue) in ssDNA. (B) 5′ dye with base-paired nucleobases (orange) in dsDNA. (C) 5′ dye with nucleobases of ssDNA (green) adjacent to a terminal dye on dsDNA.

Figure 2

Double-stranded DNA labeling with Cy3 and Cy5 (Figure 1B). (A) Relative fluorescence intensity of Cy3 and Cy5 end-labeled 5mers, ranked from most to least intense. The intensity falls by 55% for Cy3 and almost 70% for Cy5. The horizontal lines show the fluorescence intensity of single-stranded reference sequences on the same arrays. Fluorescence intensity consensus sequences of all 1024 dsDNA 5mers 5′-end-labeled using (B) Cy3 and (C) Cy5. The fluorescent range was equally divided into eight bins of equal intensity ranges, and the consensus sequence for all the 5mers is plotted for each such octile.

Scheme 1. Sequence Design for the 5′-Dye Self-Hybridizing DNA Strands

Sequence (A) is used to measure the interaction of the dyes with dsDNA and sequence (B) is used to measure the interactions of the dyes with the ssDNA overhang of dsDNA.

Figure 3

5′-Cy3-dsDNA with a permuted 3′ overhang (Figure 1C). The dsDNA strand to which the Cy3 is attached has one of two sequences: GAAAA (bright) or CGTGG (dark). (A) Relative fluorescence of Cy3-GAAAA and Cy3-CGTGG ranked from most to least intense over the range of all ssDNA 3′ overhang 5mers. The intensity falls by ∼35% for both Cy3-GAAAA and Cy3-CGTGG. Fluorescence intensity consensus sequences of all 1024 5mers on the 3′-overhang of (B) Cy3-dsGAAAA and (C) Cy3-dsCGTGG. The fluorescent was equally divided into eight bins of equal intensity ranges. The consensus sequence is plotted for each bin.

Figure 4

5′-Cy5-dsDNA with a permuted 3′ overhang (Figure 1C). The dsDNA strand to which the Cy5 is attached has one of two sequences: GAAAA (bright) or CGTGG (dark). (A) Relative fluorescence of Cy5-GAAAA and Cy5-CGTGG, ranked from most to least intense over the range of all ssDNA 3′ overhang 5mers. The intensity falls by ∼40% for both Cy5-GAAAA and Cy5-CGTGG. Fluorescence intensity consensus sequences of all 1024 5mers on the 3′-overhang of (B) Cy5-dsGAAAA and (C) Cy5-dsCGTGG. The fluorescent was equally divided into eight bins of equal intensity ranges. The consensus sequence is plotted for each bin.

Figure 5

Molecular structures of the Cy3 and Cy5 cyanine dye phosphoramidites used in this study. After the end of the synthesis and the chemical deprotection step, the dyes are linked to the 5′ DNA nucleoside via a phosphodiester bond.