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 promo ter site sequence of cyclin Dl, located at -116 to -99 from the transcripti on initiation site, with a molecular beacon comprised of a G-rich 18-mer tr iplex forming oligodeoxyribonucleotide. Formation of triplex DNA was monito red by enhanced fluorescence of the beacon, due to the weakening of fluores cence energy transfer, upon its binding to the target duplex. Electrophoret ic mobility shift assay confirmed triplex DNA formation by these oligonucle otides. In low salt buffer (10 mM Na+), triplex DNA formation was not obser ved in the absence of a ligand such as spermine. At room temperature (22 de greesC), the equilibrium association constant (Ka) calculated in the presen ce of 1 muM 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 b y the addition of 1 mM spermine. DeltaH, DeltaS, and DeltaG of triplex DNA formation, calculated from the temperature dependence of K-a in the range o f 20-45 degreesC, were -35.9 kcal/mol, -77 cal/(mol.K), and -13 kcal/mol, r espectively, 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 1 50 mM NaCl and 1 mM spermine. Structurally related polyamines exerted diffe rent degrees of triplex DNA stabilization, as determined by binding constan t measurements. Comparison of spermine versus hexamine showed a 17-fold inc rease in the equilibrium association constant, whereas bis(ethyl) derivatiz ation lead to a 4-fold decrease of this value. In the absence of added dupl ex and polyamines, the molecular beacon dissociated with a melting temperat ure of 67 degreesC. Thermodynamic parameters of beacon melting were calcula ted from the melting curve, and the DeltaH, DeltaS, and DeltaG values were 37.8 kcal/mol, 112 cal/(mol.K), and 4.4 kcal/mol, respectively. These resul ts demonstrate that molecular beacons can be used for the direct determinat ion of the equilibrium association constants and thermodynamic parameters o f triplex DNA formation in the presence of ligands such as polyamines.