A molecular beacon approach was developed to directly determine the association constant of RNA-DNA hy-brid formation. The molecular beacon was composed of a 15-nt loop structure containing the antisense sequence that can hybridize with the AUG translational start site of the HER2/neu gene, which is overexpressed in a significant proportion of breast, ovarian, and lung tumors. The equilibrium association constant (Ka) of DNA binding to the RNA oligonucleotide was 6.4 ± 0.14 x 107 M-1 in the presence of 150 mM NaCl at 22°C. The free energy change (AG) associated with RNA-DNA hybrid formation was -10.7 kcal/mole. The melting temperature (Tm) of RNA-DNA hybrid was 64.4°C±1°C in the presence of 150 mM NaCl. The RNA-DNA by-brid was more stable than the corresponding DNA-DNA duplex in 150 mM NaCl, as judged by both Ka and Tm data. We also determined the Ka and Tm values of RNA-DNA and DNA-DNA duplex formation in the presence of three monovalent cations, Li+, K+, and Cs+. The feasibility of this method was also investigated using a phosphorothioate molecular beacon. The information generated through this new approach for thermodynamic measurements might be useful for the design of oligonucleotides for antisense therapeutics.
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