A Molecular Beacon Strategy for the Thermodynamic Characterization of Triplex DNA: Triplex Formation at the Promoter Region of Cyclin D1
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- 作者：Thomas Antony ; Thresia Thomas ; Leonard H. Sigal ; Akira Shirahata ; and T. J. Thomas
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：August 7, 2001
- 年：2001
- 卷：40
- 期：31
- 页码：9387 - 9395
- 全文大小：111K
- 年卷期：v.40,no.31(August 7, 2001)
- ISSN：1520-4995
文摘
We studied the formation of triplex DNA in the purine-pyrimidine-rich promoter site sequenceof cyclin D1, located at -116 to -99 from the transcription initiation site, with a molecular beaconcomprised of a G-rich 18-mer triplex forming oligodeoxyribonucleotide. Formation of triplex DNA wasmonitored by enhanced fluorescence of the beacon, due to the weakening of fluorescence energy transfer,upon its binding to the target duplex. Electrophoretic mobility shift assay confirmed triplex DNA formationby these oligonucleotides. In low salt buffer (10 mM Na+), triplex DNA formation was not observed inthe absence of a ligand such as spermine. At room temperature (22 
C), the equilibrium association constant(Ka) calculated in the presence of 1 
M spermine and 10 mM Na+ was 3.2 × 108 M-1. The Ka value was1.0 × 109 M-1 in the presence of 150 mM Na+, and it increased by 10-fold by the addition of 1 mMspermine. 
H, S, and G of triplex DNA formation, calculated from the temperature dependence of Kain the range of 20-45 C, were -35.9 kcal/mol, -77 cal/(mol·K), and -13 kcal/mol, respectively, in thepresence of 150 mM NaCl. The corresponding values were -52.9 kcal/mol, -132.5 cal/(mol·K), and-13.4 kcal/mol in the presence of 150 mM NaCl and 1 mM spermine. Structurally related polyaminesexerted different degrees of triplex DNA stabilization, as determined by binding constant measurements.Comparison of spermine versus hexamine showed a 17-fold increase in the equilibrium association constant,whereas bis(ethyl) derivatization lead to a 4-fold decrease of this value. In the absence of added duplexand polyamines, the molecular beacon dissociated with a melting temperature of 67 C. Thermodynamicparameters of beacon melting were calculated from the melting curve, and the H, S, and G valueswere 37.8 kcal/mol, 112 cal/(mol·K), and 4.4 kcal/mol, respectively. These results demonstrate thatmolecular beacons can be used for the direct determination of the equilibrium association constants andthermodynamic parameters of triplex DNA formation in the presence of ligands such as polyamines.
C), the equilibrium association constant(Ka) calculated in the presence of 1 M spermine and 10 mM Na+ was 3.2 × 108 M-1. The Ka value was1.0 × 109 M-1 in the presence of 150 mM Na+, and it increased by 10-fold by the addition of 1 mMspermine. H, S, and G of triplex DNA formation, calculated from the temperature dependence of Kain the range of 20-45 C, were -35.9 kcal/mol, -77 cal/(mol·K), and -13 kcal/mol, respectively, in thepresence of 150 mM NaCl. The corresponding values were -52.9 kcal/mol, -132.5 cal/(mol·K), and-13.4 kcal/mol in the presence of 150 mM NaCl and 1 mM spermine. Structurally related polyaminesexerted different degrees of triplex DNA stabilization, as determined by binding constant measurements.Comparison of spermine versus hexamine showed a 17-fold increase in the equilibrium association constant,whereas bis(ethyl) derivatization lead to a 4-fold decrease of this value. In the absence of added duplexand polyamines, the molecular beacon dissociated with a melting temperature of 67 C. Thermodynamicparameters of beacon melting were calculated from the melting curve, and the H, S, and G valueswere 37.8 kcal/mol, 112 cal/(mol·K), and 4.4 kcal/mol, respectively. These results demonstrate thatmolecular beacons can be used for the direct determination of the equilibrium association constants andthermodynamic parameters of triplex DNA formation in the presence of ligands such as polyamines.