No abstract

A molecular beacon (MB) with stem-loop (hairpin) DNA structure and with attached fluorophore-quencher pair at the ends of the strand has been applied to study the interactions of Hg(2+) ions with a thymine-thymine (T-T) mismatch in Watson-Crick base-pairs and the ligative disassembly of MB·Hg(2+) complex by Hg(2+) sequestration with small biomolecule ligands. In this work, a five base-pair stem with configuration 5'-GGTGG...CCTCC-3' for self-hybridization of MB has been utilized. In this configuration, the four GC base-pair binding energy is not sufficient to hybridize fully at intermediate temperatures and to form a hairpin MB conformation. The T-T mismatch built-in into the stem area can effectively bind Hg(2+) ions creating a bridge, T-Hg-T. We have found that the T-Hg-T bridge strongly enhances the ability of MB to hybridize, as evidenced by an unusually large MB melting temperature shift observed on bridge formation, ΔT(m) = +15.1 ± 0.5 °C, for 100 nM MB in MOPS buffer. The observed ΔT(m) is the largest of the ΔT(m) found for other MBs and dsDNA structures. By fitting the parameters of the proposed model of reversible MB interactions to the experimental data, we have determined the T-Hg-T bridge formation constant at 25 °C, K(1) = 8.92 ± 0.42 × 10(17) M(-1) from mercury(II) titration data and K(1) = 1.04 ± 0.51 × 10(18) M(-1) from the bridge disassembly data; ΔG° = -24.53 ± 0.13 kcal/mol. We have found that the biomarker of oxidative stress and cardiovascular disease, homocysteine (Hcys), can sequester Hg(2+) ions from the T-Hg-T complex and withdraw Hg(2+) ions from MB in the form of stable Hg(Hcys)(2)H(2) complexes. Both the model fitting and independent (1)H NMR results on the thymidine-Hg-Hcys system indicate also the high importance of 1:1 complexes. The high value of K(1) for T-Hg-T bridge formation enables analytical determinations of low concentrations of Hg(2+) (limit of detection LOD = 19 nM or 3.8 ppb, based on 3σ method) and Hcys (LOD = 23 nM, 3σ method). The conditional stability constants for Hg(Hcys)H(2)(2+) and Hg(Hcys)(2)H(2) at 52 °C have been determined, β(112) = 5.37 ± 0.3 × 10(46) M(-3), β(122) = 3.80 ± 0.6 × 10(68) M(-4), respectively.

The resonance energy transfer (RET) from excited fluorescent probe molecules to plasmonic gold nanoparticles (AuNPs) can be gated by modulating the width of channels (gates) in submonolayer protein shells surrounding AuNPs. We have explored the gated-RET (gRET) processes using an antiapoptotic protein survivin (Sur) as the gating material, citrate-capped gold nanoparticles (AuNP@Cit), and fluorescein isothiocyanate as the fluorescent probe. Despite the electrostatic repulsive forces between these components, a strong modulation of RET efficiency by Sur down to 240 pM (S/N = 3) is possible. Using piezometric measurements, we have confirmed the Sur adsorbability on Cit-coated Au surfaces with monolayer coverage: γSur = 5.4 pmol/cm(2) and Langmuirian adsorption constant KL,Sur = 1.09 × 10(9) M(-1). The AuNP@Cit/Sur stability has been corroborated using resonance elastic light scattering. The quantum mechanical calculations indicate that multiple hydrogen bonding between Cit ligands and -NH3(+), =NH2(+), and -NH2 groups of lysines and arginines of Sur have likely facilitated Sur bonding to nanoparticles. A theoretical model of gated-RET has been developed, enabling predictions of the system behavior. In contrast to the positive slope of the Stern-Volmer quenching dependence (F0/F) = f(QA), a negative slope has been obtained for gRET relationship (F0/F) = f(cP), attributed to the dequenching.

A fundamental molecular beacon (MB) consists of a special short nucleic acid strand with a fluorophore-quencher pair attached to its ends. It provides a unique framework that is susceptible to conformational transitions between a hairpin (closed) conformation and an extended (open) conformation. These two conformations are readily discernible because of their differing fluorescence emission characteristics. The broad applicability of the robust MB sensing platform has attracted widespread interest, resulting in extensive research studies ranging from theoretical and bioanalytical chemistry to molecular biology and biomedical applications. In this paper, the principles of MB design and the modes and mechanisms of MB operation are reviewed, including MB modifications based on the utilisation of a thymidine-thymidine mismatch in hybridised MB stem, aptamers, peptides and locked nucleic acid strands in an MB loop, as well as plasmonic quenchers, quantum dots and interactions with graphene and graphene oxide. Specific applications of MBs in the analysis of enzymes, DNA mutation, phosphorylation, methylation and ligation, followed by the detection of pathogens and applications in cancer and other disease diagnostics and therapeutics are also reviewed. Molecular beacon-based sensing platforms are expanding rapidly and offer a promising bioanalytical tool for inexpensive and reliable analysis for research and field diagnostics.

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.