title: Mercury/homocysteine ligation-induced ON/OFF-switching of a T-T mismatch-based oligonucleotide molecular beacon.
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.
authors.affiliation: Department of Chemistry, State University of New York at Potsdam, Potsdam, New York 13676, United States.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Stobiecka
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Molinero
authors.forename: Agata
authors.initials: A
authors.lastname: Chałupa
authors.forename: Maria
authors.initials: M
authors.lastname: Hepel
mesh_terms: D020029:Base Pairing; D005456:Fluorescent Dyes; D006710:Homocysteine / Q000032:analysis* / Q000737:chemistry; D055029:Inverted Repeat Sequences; D007700:Kinetics; D057230:Limit of Detection; D009682:Magnetic Resonance Spectroscopy; D008628:Mercury / Q000032:analysis* / Q000737:chemistry; D015335:Molecular Probes / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics; D013941:Thymine / Q000032:analysis* / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D015335:Molecular Probes; D009841:Oligonucleotides; D006710:Homocysteine; D008628:Mercury; D013941:Thymine