Dielectricity of a molecularly crowded solution accelerates NTP misincorporation during RNA-dependent RNA polymerization by T7 RNA polymerase.

In biological systems, the synthesis of nucleic acids, such as DNA and RNA, is catalyzed by enzymes in various aqueous solutions. However, substrate specificity is derived from the chemical properties of the residues, which implies that perturbations of the solution environment may cause changes in the fidelity of the reaction. Here, we investigated non-promoter-based synthesis of RNA using T7 RNA polymerase (T7 RNAP) directed by an RNA template in the presence of polyethylene glycol (PEG) of various molecular weights, which can affect polymerization fidelity by altering the solution properties. We found that the mismatch extensions of RNA propagated downstream polymerization. Furthermore, PEG promoted the polymerization of non-complementary ribonucleoside triphosphates, mainly due to the decrease in the dielectric constant of the solution. These results indicate that the mismatch extension of RNA-dependent RNA polymerization by T7 RNAP is driven by the stacking interaction of bases of the primer end and the incorporated nucleotide triphosphates (NTP) rather than base pairing between them. Thus, proteinaceous RNA polymerase may display different substrate specificity with changes in dielectricity caused by molecular crowding conditions, which can result in increased genetic diversity without proteinaceous modification.

Molecular crowding induces primer extension by RNA polymerase through base stacking beyond Watson-Crick rules.

The polymerisation of nucleic acids is essential for copying genetic information correctly to the next generations, whereas mispolymerisation could promote genetic diversity. It is possible that in the prebiotic era, polymerases might have used mispolymerisation to accelerate the diversification of genetic information. Even in the current era, polymerases of RNA viruses frequently cause mutations. In this study, primer extension under different molecular crowding conditions was measured using T7 RNA polymerase as a model for the reaction in the prebiotic world. Interestingly, molecular crowding using 20 wt% poly(ethylene glycol) 2000 preferentially promoted the primer extensions with ATP and GTP by T7 RNA polymerase, regardless of Watson-Crick base-pairing rules. This indicates that molecular crowding decreases the dielectric constants in solution, resulting in enhancement of stacking interactions between the primer and an incorporated nucleotide. These findings suggest that molecular crowding could accelerate genetic diversity in the prebiotic world and may promote transcription error of RNA viruses in the current era.

Small Molecule Fluorescent Probes for G- Quadruplex Visualization as Potential Cancer Theranostic Agents.

G-quadruplexes have gained prominence over the past two decades for their role in gene regulation, control of anti-tumour activity and ageing. The physiological relevance and significance of these non-canonical structures in the context of cancer has been reviewed several times. Putative roles of G-quadruplexes in cancer prognosis and pathogenesis have spurred the search for small molecule ligands that are capable of binding and modulating the effect of such structures. On a related theme, small molecule fluorescent probes have emerged that are capable of selective recognition of G-quadruplex structures. These have opened up the possibility of direct visualization and tracking of such structures. In this review we outline recent developments on G-quadruplex specific small molecule fluorescent probes for visualizing G-quadruplexes. The molecules represent a variety of structural scaffolds, mechanism of quadruplex-recognition and fluorescence signal transduction. Quadruplex selectivity and in vivo imaging potential of these molecules places them uniquely as quadruplex-theranostic agents in the predominantly cancer therapeutic context of quadruplex-selective ligands.

DNA template-assisted inhibition of tyrosinase activity.

Enzyme-mediated polymerization reactions have been widely studied in the context of DNA template-assisted reactions. We have recently highlighted the ability of DNA templates to modulate enzyme-catalyzed single-step transformations. In this work, we focus on the intramolecular transformation of L-dopa catalyzed by enzyme tyrosinase and report a novel role of DNA templates in inhibition of the enzyme. The kinetics of mushroom tyrosinase monitored by UV-visible spectroscopy reveals significant decrease in the enzyme's efficiency in the presence of short double-stranded DNA molecules. KM of tyrosinase is found to increase by nearly 1.8-fold, implying a lower affinity of the enzyme for L-dopa, whereas Vmax is only marginally affected. The mode of inhibition is assessed to be a mixed mode with kinetic constants of inhibition in the micromolar range. Further, in the presence of cinnamic acid and DNA duplexes, the KM of tyrosinase increases nearly 3.5-fold, whereas Ki and Ki' are lowered by an order of magnitude. These results are a corollary of the known influence of substrate-template interactions and greater local substrate concentrations on enzyme activity and expand the paradigm with respect to use of DNA templates in enzyme-catalyzed reactions.