Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon

Veena Vijayanathan, Thresia Thomas, Leonard H. Sigal, T. J. Thomas

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)225-233
Number of pages9
JournalAntisense and Nucleic Acid Drug Development
Volume12
Issue number4
StatePublished - Aug 2002
Externally publishedYes

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Antisense Oligonucleotides
Association reactions
RNA
DNA
erbB-2 Genes
Temperature
Monovalent Cations
Thermodynamics
Oligonucleotides
Freezing
Free energy
Melting point
Tumors
Breast
Genes
Lung

ASJC Scopus subject areas

  • Genetics
  • Pharmacology

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Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon. / Vijayanathan, Veena; Thomas, Thresia; Sigal, Leonard H.; Thomas, T. J.

In: Antisense and Nucleic Acid Drug Development, Vol. 12, No. 4, 08.2002, p. 225-233.

Research output: Contribution to journalArticle

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