Effect of Thiazole Orange Doubly Labeled Thymidine on DNA Duplex Formation

Nucleic acid oligonucleotides are widely used in hybridization experiments for specific detection of complementary nucleic acid sequences. For design and application of oligonucleotides, an understanding of their thermodynamic properties is essential. Recently, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHOs) were developed as uniquely labeled DNA oligomers containing commonly one thymidine having two covalently linked thiazole orange dye moieties. The fluorescent signal of an ECHO is strictly hybridization-controlled, where the dye moieties have to intercalate into double-stranded DNA for signal generation. Here we analyzed the hybridization thermodynamics of ECHO/DNA duplexes, and thermodynamic parameters were obtained from melting curves of 64 ECHO/DNA duplexes measured by ultraviolet absorbance and fluorescence. Both methods demonstrated a substantial increase in duplex stability (ΔΔ<i>G°</i><sub>37</sub> ∼ −2.6 ± 0.7 kcal mol<sup>–1</sup>) compared to that of DNA/DNA duplexes of the same sequence. With the exception of T·G mismatches, this increased stability was mostly unaffected by other mismatches in the position opposite the labeled nucleotide. A nearest neighbor model was constructed for predicting thermodynamic parameters for duplex stability. Evaluation of the nearest neighbor parameters by cross validation tests showed higher predictive reliability for the fluorescence-based than the absorbance-based parameters. Using our experimental data, a tool for predicting the thermodynamics of formation of ECHO/DNA duplexes was developed that is freely available at http://genome.gsc.riken.jp/echo/thermodynamics/. It provides reliable thermodynamic data for using the unique features of ECHOs in fluorescence-based experiments.