Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: Predictive thermodynamic model

Abstract

Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5' end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2 kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine-purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications.

Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

“…Therefore, the observed change in T m for ON1+ON2 was caused by modification with Cy3/Cy5 dyes. This is in agreement with the previous literature on cyanines and dangling ends that stabilise DNA helices through interactions with their environment and with terminal nucleotides …”

“…Simultaneously, the difference between the T m values obtained in the UV and fluorescenceassisted melting experiments was larger for the decamer systems. Overall, for both systems, our data fit with existing reports on the stabilising effect of terminally attached cyanines and dangling ends on DNA helices . In addition, an increased stabilising effect was observed at low concentrations (3.5–4 vs. 1.5 °C at 0.125 vs. 0.5 μ m ).…”

“…Organic fluorophores are hydrophobic moieties that upon attachment to oligonucleotides affect their T m values. Given that they are covalently linked to the terminal positions of DNA probes, fluorophores interact with both the environment and the nucleic acid helix, which results in enthalpy‐driven stabilisation of up to 12 °C . In particular, terminally attached cyanine dyes have a strong stabilisation of the DNA helix that has been studied by UV melting at a concentration of 1 μ m .…”

Nuclear and Cytoplasmatic Quantification of Unconjugated, Label-Free Locked Nucleic Acid Oligonucleotides

“…These studies involving fluorescently labeled oligonucleotides are beneficial to understanding the distribution, and some constructs mimic very well the situation of unconjugated AON [44]. However, chemically modified AONs will not, or cannot, act exactly in vitro and in vivo as the unconjugated AON, and some reports have shown that certain modifications or fluorescent tags alter the properties of an oligonucleotide significantly [45][46][47][48].…”

Novel carbocyanine and bis-carbocyanine dyes: synthesis, visible spectra studies, solvatochromism and halochromism

“…Cyanine dyes are a large class of complex organic compounds which have been extensively used in a wide range of applications, from lasers and photography to diagnostic by fluorescent detection of analytics and sensitizers for photodynamic therapy, optical communication technology, laser disks materials and in textile industry (Ehret et al, 2001, Ferreira, et al, 2015, Moreira, et al, 2015, Upadhyayula, et al, 2015, Solomon, 2014, Keisar, et al, 2014, Parvathy, et al, 2015, Yi, et al, 2014, Owens, et al, 2014, Ansari, et al, 2014, Shindy, et al, 2012. In addition cyanine dyes also used in clinical chemistry, fluorescent labels in DNA, sensitizers in solar cells, anti-tumor and anti-cancer agents, lithographic printing plate precursors, in pharmaceutical industry, as probe sensors, in bio-analytical applications, as pH indicators and as probes for determining solvent polarity , Gabbutt, et al, 2012, Li, et al, 2012, Xiang, et al, 2012, Panigrahi, et al, 2012, Lynch, et al, 2013Zhao, et al, 2013.…”