Sequence-dependent fluorescence of cyanine dyes on microarrays

Abstract

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.

Figure 1. Oligonucleotide labeling with Cy3 and Cy5 phosphoramidites.

(A) Fluorescence intensity of Cy3 and Cy5 end-labeled 5-mers, ranked from most to least intense. The Cy3 curve drops by about half of the maximum intensity, while the Cy5 curve drops by about two-thirds. The shape of the curves suggests that the intensity range for both dyes will increase with additional sequence length. However the insets, which each plot the four N _xT_5-x subsets of the same data, preserve the inverse sigmoidal shape, and the trend can be extrapolated to indicate a small additional range for Cy3. Fluorescence intensity consensus sequences of all 1024 DNA 5-mers 5′-end-labeled using (B) Cy3 and (C) Cy5, synthesized simultaneously on a microarray. The fluorescent range was equally divided into eight bins of equal intensity range, and the consensus sequence for all the 5-mers in each bin is plotted for each octile.

Figure 2. Oligonucleotide labeling with biotin-streptavidin-Cy3 and -Cy5.

(A) Fluorescence intensity of Cy3 and Cy5 end-labeled 5-mers, ranked from most to least intense. The fluorescence intensity of the Cy3-streptavidin-biotin conjugate drops by about 75% of maximum intensity over the range of sequences; in the case of Cy5, the intensity drops by about 95%. Fluorescence intensity consensus sequences of all 1024 DNA 5-mers with 5′-biotin and labeled using (B) streptavidin-Cy3 conjugation and (C) streptavidin-Cy5 conjugation. The fluorescent range was equally divided into eight bins of equal intensity range, and the consensus sequence for all the 5-mers in each bin is plotted for each octile.

Figure 3. Intensity distribution of oligonucleotides labeled with Cy3 and Cy5 phosphoramidites.

(A) Intensity distribution for Cy3 and Cy5 end-labeled 5-mers divided into 16 bins of equal intensity range. The histograms have been fitted with Gaussian functions (Cy3: A = 119, x ₀ = 8.0, σ = 3.5; Cy5: A = 138, x ₀ = 9.4, σ = 3.0). (B) Effect of purine vs. pyrimidine content of 5-mers on the normalized intensity of Cy3- and Cy5-labels. The horizontal lines represent the intensity of the least fluorescent Cy3 and Cy5 end-labeled 5-mers.