Enzymatic Primer-Extension with Glycerol-Nucleoside Triphosphates on DNA Templates

selection. Template-dependent GNA synthesis is essential to any GNA-based selection system.In this study, we investigated the ability of various DNA polymerases to use glycerol-nucleoside triphosphates (gNTPs) as substrates for GNA synthesis on DNA templates. Therminator DNA polymerase catalyzes quantitative primer-extension by the incorporation of two glyceronucleotides, with much less efficient extension up to five glyceronucleotides. Steady-state kinetic experiments suggested that GNA synthesis by Therminator was affected by both decreased catalytic rates and weakened substrate binding, especially for pyrimidines. In an attempt to improve pyrimidine incorporation by providing additional stacking interactions, we synthesized two new gNTP analogs with 5-propynyl substituted pyrimidine nucleobases. This led to more efficient incorporation of gC, but not gT.We suggest that directed evolution of Therminator might lead to mutants with improved substrate binding and catalytic efficiency

Neurostimulation, doping, and the spirit of sport

There is increasing interest in using neuro-stimulation devices to achieve an ergogenic effect in elite athletes. Although the World Anti-Doping Authority (WADA) does not currently prohibit neuro-stimulation techniques, a number of researchers have called on WADA to consider its position on this issue. Focusing on trans-cranial direct current stimulation (tDCS) as a case study of an imminent so-called ‘neuro-doping’ intervention, we argue that the emerging evidence suggests that tDCS may meet WADA’s own criteria (pertaining to safety, performance-enhancing effect, and incompatibility with the ‘spirit of sport’) for a method’s inclusion on its list of prohibited substances and methods. We begin by surveying WADA’s general approach to doping, and highlight important limitations to the current evidence base regarding the performance-enhancing effect of pharmacological doping substances. We then review the current evidence base for the safety and efficacy of tDCS, and argue that despite significant shortcomings, it may be sufficient for WADA to consider prohibiting tDCS, in light of the comparable flaws in the evidence base for pharmacological doping substances. In the second half of the paper, we argue that the question of whether WADA ought to ban tDCS turns significantly on the question of whether it is compatible with the ‘spirit of sport’ criterion. We critique some of the previously published positions on this, and advocate our own sport-specific and application-specific approach. Despite these arguments, we finally conclude by suggesting that tDCS ought to be monitored rather than prohibited due to compelling non-ideal considerations

Solution structure of a DNA double helix with consecutive metal-mediated base pairs

Metal-mediated base pairs represent a powerful tool for the site-specific functionlization of nucleic acids with metal ions. The development of applications of the metal-modified nucleic acids will depend on the availability of structural information on these double helices. We present here the NMR solution structure of a self-complementary DNA oligonucleotide with three consecutive imidazole nucleotides in its centre. In the absence of transition-metal ions, a hairpin structure is adopted with the artifical nucleotides forming the loop. In the presence of Ag(I) ions, a duplex comprising three imidazole-Ag+-imidazole base pairs is formed. Direct proof for the formation of metal-mediated base pairs was obtained from (1)J(N-15,Ag-107/109) couplings upon incorporation of N-15-labelled imidazole. The duplex adopts a B-type conformation with only minor deviations in the region of the artifical bases. This work represents the first structural characterization of a metal-modified nucleic acid with a continuous stretch of metal-mediated base pairs

Functional characterization of missense mutations in severe methylenetetrahydrofolate reductase deficiency using a human expression system

5,10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADPH-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate using FAD as the cofactor. Severe MTHFR deficiency is the most common inborn error of folate metabolism, resulting in hyperhomocysteinemia and homocystinuria. Approximately 70 missense mutations have been described that cause severe MTHFR deficiency, however, in most cases their mechanism of dysfunction remains unclear. Few studies have investigated mutational specific defects; most of these assessing only activity levels from a handful of mutations using heterologous expression. Here, we report the in vitro expression of 22 severe MTHFR missense mutations and two known single nucleotide polymorphisms (p.Ala222Val, p.Thr653Met) in human fibroblasts. Significant reduction of MTHFR activity (<20 % of wild-type) was observed for five mutant proteins that also had highly reduced protein levels on Western blot analysis. The remaining mutations produced a spectrum of enzyme activity levels ranging from 22-122 % of wild-type, while the SNPs retained wild-type-like activity levels. We found increased thermolability for p.Ala222Val and seven disease-causing mutations all located in the catalytic domain, three of which also showed FAD responsiveness in vitro. By contrast, six regulatory domain mutations and two mutations clustering around the linker region showed increased thermostability compared to wild-type protein. Finally, we confirmed decreased affinity for NADPH in individual mutant enzymes, a result previously described in primary patient fibroblasts. Our expression study allows determination of significance of missense mutations in causing deleterious loss of MTHFR protein and activity, and is valuable in detection of aberrant kinetic parameters, but should not replace investigations in native material