Fluorescence intensity of cyanine-labeled single-stranded DNA.

<p>(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.</p

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

<p>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.</p

Comparison of the Sequence-Dependent Fluorescence of the Cyanine Dyes Cy3, Cy5, DyLight DY547 and DyLight DY647 on Single-Stranded DNA

<div><p>Cyanine dyes are commonly used for fluorescent labeling of DNA and RNA oligonucleotides in applications including qPCR, sequencing, fluorescence <i>in situ</i> 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.</p></div

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

Optimized Light-Directed Synthesis of Aptamer Microarrays

Aptamer microarrays are a promising high-throughput method for ultrasensitive detection of multiple analytes, but although much is known about the optimal synthesis of oligonucleotide microarrays used in hybridization-based genomics applications, the bioaffinity interactions between aptamers and their targets is qualitatively different and requires significant changes to synthesis parameters. Focusing on streptavidin-binding DNA aptamers, we employed light-directed in situ synthesis of microarrays to analyze the effects of sequence fidelity, linker length, surface probe density, and substrate functionalization on detection sensitivity. Direct comparison with oligonucleotide hybridization experiments indicates that aptamer microarrays are significantly more sensitive to sequence fidelity and substrate functionalization and have different optimal linker length and surface probe density requirements. Whereas microarray hybridization probes generate maximum signal with multiple deletions, aptamer sequences with the same deletion rate result in a 3-fold binding signal reduction compared with the same sequences synthesized for maximized sequence fidelity. The highest hybridization signal was obtained with dT 5mer linkers, and the highest aptamer signal was obtained with dT 11mers, with shorter aptamer linkers significantly reducing the binding signal. The probe hybridization signal was found to be more sensitive to molecular crowding, whereas the aptamer probe signal does not appear to be constrained within the density of functional surface groups commonly used to synthesize microarrays