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 language | English (US) |
|---|---|
| Pages (from-to) | 225-233 |
| Number of pages | 9 |
| Journal | Antisense and Nucleic Acid Drug Development |
| Volume | 12 |
| Issue number | 4 |
| State | Published - Aug 2002 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Genetics
- Pharmacology
Cite this
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 journal › Article
}
TY - JOUR
T1 - Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon
AU - Vijayanathan, Veena
AU - Thomas, Thresia
AU - Sigal, Leonard H.
AU - Thomas, T. J.
PY - 2002/8
Y1 - 2002/8
N2 - 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.
AB - 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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M3 - Article
C2 - 12238811
AN - SCOPUS:0036696942
VL - 12
SP - 225
EP - 233
JO - Nucleic Acid Therapeutics
JF - Nucleic Acid Therapeutics
SN - 2159-3337
IS - 4
ER -