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

Abstract

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 × 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 Ea for the reaction at physiological temperature (37 °C). Special attention has been paid to assess the value of Ea 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 × 10(3) M(-1) s(-1) at 37 °C with Ea = 28.7 ± 2.3 kcal/mol (120.2 ± 9.6 kJ/mol) for the temperature range of 23 to 55 °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 (tm = 53.7 ± 0.2 °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 Ea, their effect on kinetic transients for target binding is negligible. No toehold preceding steps in the mechanism of target binding were identified.

Optical Biosensing System for the Detection of Survivin mRNA in Colorectal Cancer Cells Using a Graphene Oxide Carrier-Bound Oligonucleotide Molecular Beacon

“…Our calculations—performed using the UNAFold software—indicate that the target oligonucleotide (tDNA), complementary to the loop of SurMB, preferentially forms a stable small-loop hairpin structure instead of relaxing to a linear conformation. Figure 1 A presents the sequence and the most thermodynamically stable hairpin structure of SurMB-Joe, with negative Gibbs free energy of formation ΔG° SurMB-Joe = −4.26 kcal/mol [ 37 ]. The loop sequence of the probe was composed of an antisense oligonucleotide targeting the survivin mRNA region [ 38 ].…”

“…On the other hand, the attachment of a small quenching graphite nanoparticle (NP) to the end of a MB strand was applied by Piao et al [ 36 ]. Although the use of GO is gaining popularity in DNA sensing applications [ 39 ], the exact conformation of DNA strands has rarely been evaluated [ 37 ].…”

Preparation of cationized gelatin nanospheres incorporating molecular beacon to visualize cell apoptosis

“…This finding demonstrates that the physicochemical properties of cGNS incorporating MB are not influenced by the sequence of MB. Considering the biological function of MB, it is important to evaluate the sequence specificity of MB and MB incorporated in cGNS for the hybridization 30 , 31 . The hybridization study indicated that the fluorescent recovery of both MB and MB incorporated in cGNS was specific for the target sequences (Fig.…”

Exosome Proteome of U-87MG Glioblastoma Cells

“…It was also shown that polycystin-1 co-localizes with E-cadherin at the cell-cell contacts, accelerating the recruitment of intracellular E-cadherin to reforming junctions [40]. These exosome proteins could be proposed as targets for the detection of a cancer biomarker [41,42,43]. …”