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.