A molecular beacon strategy for the thermodynamic characterization of triplex DNA: triplex formation at the promoter region of cyclin D1.
We studied the formation of triplex DNA in the purine-pyrimidine-rich promoter site sequence of cyclin D1, located at -116 to -99 from the transcription initiation site, with a molecular beacon comprised of a G-rich 18-mer triplex forming oligodeoxyribonucleotide. Formation of triplex DNA was monitored 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 formation by these oligonucleotides. In low salt buffer (10 mM Na(+)), triplex DNA formation was not observed in the absence of a ligand such as spermine. At room temperature (22 degrees C), the equilibrium association constant (K(a)) calculated in the presence of 1 microM spermine and 10 mM Na(+) was 3.2 x 10(8) M(-1). The K(a) value was 1.0 x 10(9) M(-1) in the presence of 150 mM Na(+), and it increased by 10-fold by the addition of 1 mM spermine. Delta H, Delta S, and Delta G of triplex DNA formation, calculated from the temperature dependence of K(a) in the range of 20--45 degrees C, were -35.9 kcal/mol, -77 cal/(mol.K), and -13 kcal/mol, respectively, in the presence 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 polyamines exerted 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 duplex and polyamines, the molecular beacon dissociated with a melting temperature of 67 degrees C. Thermodynamic parameters of beacon melting were calculated from the melting curve, and the Delta H, Delta S, and Delta G values were 37.8 kcal/mol, 112 cal/(mol.K), and 4.4 kcal/mol, respectively. These results demonstrate that molecular beacons can be used for the direct determination of the equilibrium association constants and thermodynamic parameters of triplex DNA formation in the presence of ligands such as polyamines.
Antony
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T
Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903, USA.
Thomas
T
T
Sigal
L H
LH
Shirahata
A
A
Thomas
T J
TJ
D019938:Cyclin D1 / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes / Q000138:chemical synthesis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D012965:Sodium Chloride; D013050:Spectrometry, Fluorescence; D013096:Spermine / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D015342:DNA Probes; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; C051802:triplex DNA; D019938:Cyclin D1; D013096:Spermine; D012965:Sodium Chloride; D004247:DNA