Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon.
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 (Ka) of DNA binding to the RNA oligonucleotide was 6.4 +/- 0.14 x 10(7) M(-1) in the presence of 150 mM NaCl at 22 degrees 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 degrees C +/- 1 degree 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 Ka and Tm data. We also determined the Ka, deltaG, 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.
Vijayanathan
Veena
V
Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick 08903, USA.
Thomas
Thresia
T
Sigal
Leonard H
LH
Thomas
T J
TJ
D002586:Cesium / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D016373:DNA, Antisense / Q000378:metabolism*; D018734:Genes, erbB-2; D005819:Genetic Markers; D007700:Kinetics; D018021:Lithium Chloride / Q000378:metabolism; D011189:Potassium Chloride / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D002712:Chlorides; D016373:DNA, Antisense; D005819:Genetic Markers; D012333:RNA, Messenger; D002586:Cesium; D011189:Potassium Chloride; D018021:Lithium Chloride; C028019:cesium chloride