Mercury/Homocysteine Ligation-Induced ON/OFF-Switching of a T-T Mismatch-Based Oligonucleotide Molecular Beacon

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 Hg2+ ions with a thymine-thymine (T-T) mismatch in Watson Crick base-pairs and the ligative disassembly of MB center dot Hg2+ complex by Hg2+ sequestration with small biomolecule ligands. In this work, a five base-pair stem with configuration 5'-GGTGG center dot center dot center dot 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 Hg2+ 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, Delta T-m = +15.1 +/- 0.5 degrees C, for 100 nM MB in MOPS buffer. The observed Delta T-m is the largest of the Delta T-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 degrees C, K-1 = 8.92 +/- 0.42 x 10(17) M-1 from mercury(II) titration data and K-1 = 1.04 +/- 0.51 x 10(18) M-1 from the bridge disassembly data; Delta G degrees = -24.53 +/- 0.13 kcal/mol. We have found that the biomarker of oxidative stress and cardiovascular disease, homocysteine (Hcys), can sequester Hg2+ ions from the T-Hg-T complex and withdraw Hg2+ ions from MB in the form of stable Hg(Hcys)(2)H-2 complexes. Both the model fitting and independent H-1 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 Hg2+ (limit of detection LOD = 19 nM or 3.8 ppb, based on 3 sigma method) and Hcys (LOD = 23 nM, 3 sigma method). The conditional stability constants for Hg(Hcys)H-2(2+) and Hg(Hcys)(2)H-2 at 52 degrees C have been determined, beta(112) = 5.37 +/- 0.3 x 10(46) M-3, beta(122) = 3.80 +/- 0.6 x 10(68) M-4, respectively.