Direct Measurement of the Association Constant of HER2/ neu Antisense Oligonucleotide to Its Target RNA Sequence Using a Molecular Beacon

Abstract

A molecular beacon approach was developed to directly determine the association constant of RNA-DNA hybrid 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 (K_a) of DNA binding to the RNA oligonucleotide was 6.4 ± 0.14 × 10⁷ M^-1 in the presence of 150 mM NaCl at 22°C. The free energy change ( Δ G) associated with RNA-DNA hybrid formation was - 10.7 kcal/mole. The melting temperature (T_m) of RNA-DNA hybrid was 64.4°C ± 1°C in the presence of 150 mM NaCl. The RNA-DNA hybrid was more stable than the corresponding DNA-DNA duplex in 150 mM NaCl, as judged by both K_a and T_m data. We also determined the K_a, ΔG, and T_m 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.