Sequence-dependence of Cy3 and Cy5 dyes in 3ʹ terminally-labeled single-stranded DNA

Fluorescence is an ideal tool to see and manipulate nucleic acids, and engage in their rich and complex biophysical properties. Labeling is the preferred approach to track and quantify fluorescence with nucleic acids and cyanine dyes are emblematic in this context. The fluorescent properties of cyanine dyes are known to be sequence-dependent, with purines in the immediate vicinity increasing the fluorescence intensity of Cy3 and Cy5 dyes, and the ability of nucleobases to modulate the photophysical properties of common fluorophores may influence fluorescence measurements in critical assays such as FISH, qPCR or high-throughput sequencing. In this paper, we comprehensively map the sequence-dependence of Cy3 and Cy5 dyes in 3ʹ-fluorescently labeled single-stranded DNA by preparing the complete permutation library of the 5 consecutive nucleotides immediately adjacent to the dye, or 1024 sequences. G-rich motifs dominate the high fluorescence range, while C-rich motifs lead to significant quenching, an observation consistent with 5ʹ-labeled systems. We also uncover GCGC patterns in the extreme top range of fluorescence, a feature specific to 3ʹ-Cy3 and Cy5 oligonucleotides. This study represents the final piece in linking nucleotide identity to fluorescence changes for Cy3, Cy5 and fluorescein in all 3ʹ, 5ʹ, single-stranded and double-stranded DNA formats.