DNA Strand Replacement Mechanism in Molecular Beacons Encoded for the Detection of Cancer Biomarkers

Signaling properties of a fluorescent hairpin oligonucleotide molecular beacon (MB) encoded to recognize protein survivin (Sur) mRNA have been investigated. The process of complementary target binding to SurMB with 20-mer loop sequence is spontaneous, as expected, and characterized by a high affinity constant (K = 2.51 X 10(16) M-1). However, the slow kinetics at room temperature makes it highly irreversible. To understand the intricacies of target binding to MB, a detailed kinetic study has been performed to determine the rate constants and activation energy E-a for the reaction at physiological temperature (37 degrees C). Special attention has been paid to assess the value of E-a in view of reports of negative activation enthalpy for some nucleic acid reactions that would make the target binding even slower at increasing temperatures in a non-Arrhenius process. The target-binding rate constant determined is k = 3.99 X 10(3) M(-1)s(-1) at 37 degrees C with E-a = 28.7 +/- 2.3 kcal/mol (120.2 +/- 9.6 kJ/mol) for the temperature range of 23 to 55 degrees C. The positive high value of Ea is consistent with a kinetically controlled classical Arrhenius process. We hypothesize that the likely contribution to the activation energy barrier comes from the SurMB stem melting (t(m) = 53.7 +/- 0.2 degrees C), which is a necessary step in the completion of target strand hybridization with the SurMB loop. A low limit of detection (LOD = 2 nM) for target tDNA has been achieved. Small effects of conformational polymorphs of SurMB have been observed on melting curves. Although these polymorphs could potentially cause a negative E-a, their effect on kinetic transients for target binding is negligible. No toehold preceding steps in the mechanism of target binding were identified.