Bipyridines mediate electron transfer from an electrode to nicotinamide adenine dinucleotide phosphate.

Biocatalysts are widely used in industry, but few examples of the use of oxidoreductases, in which enzymatic function often requires electrons, have been reported. NADPH is a cofactor that supplies an electron to oxidoreductases, but is consequently inactivated and no longer able to act as an electron donor. NADP+ can not receive electrons from electrodes through straightforward electrochemistry owing to its complicated three-dimensional structure. This study reports that bipyridines effectively mediate electron transfer between an electrode and NADP+, allowing them to serve as electron mediators for NADPH production. Using bipyridines, quinones, and anilines, which have negative oxidation-reduction potentials, an electrochemical investigation was conducted into whether electrons were transferred to NADP+. Only bipyridines with a reduction potential near -1.0 V exhibited electron transfer. Furthermore, the NADPH production level was measured using spectroscopy. NADPH was efficiently produced using bipyridines, such as methyl viologen and ethyl viologen, in which the bipyridyl 1- and 1'-positions bear small substituents. However, methyl viologen caused a dehydrogenation reaction of NADPH, making it unsuitable as an electron mediator for NADPH production. The dehydrogenation reaction did not occur using ethyl viologen. These results indicated that NADP+ can be reduced more effectively using substituents that prevent a dehydrogenation reaction at the bipyridyl 1- and 1'-positions while maintaining the reducing power.

Binding and Structural Properties of DNA Aptamers with VEGF-A-Mimic Activity.

Vascular endothelial growth factors (VEGFs) are hypoxia-inducible secreted proteins to promote angiogenesis, in which VEGF-A is an important molecule that binds and activates VEGF receptor-1 (VEGFR-1) and VEGFR-2. In this study, two DNA aptamers, Apt01 and Apt02, were successfully isolated by alternating consecutive systematic evolution of ligands by exponential enrichment (SELEX) against VEGFR-1 and -2 using deep sequencing analysis in an early selection round. Their binding affinities for VEGFR-2 were lower than that of VEGFR-1, which is similar to that of VEGF-A. Structural analyses with the measurements of circular dichroism spectra and ultraviolet melting curve showed that Apt01 possessed the stem-loop structure in the molecule, whereas Apt02 formed G-quadruplex structures. In addition, Apt02 accelerated a tube formation of human umbilical vein endothelial cells faster than Apt01, which was affected by difference of binding affinity and nuclease resistance due to G-quadruplex structures. These results demonstrated that Apt02 might have a potential to function as an alternative to VEGF-A.

Screening of DNA Signaling Aptamer from Multiple Candidates Obtained from SELEX with Next-generation Sequencing.

Here, we demonstrated a strategy for developing signaling aptamers, based on screening of signaling aptamers from multiple aptamer candidates obtained by SELEX with next generation sequencing. Among aptamer candidates labelled by 6-carboxyfluorescein and quencher at both end termini, there is the possibility of discovering a potent signaling aptamer. In this study, we discovered DNA signaling aptamers against VEGFR-1. This strategy has the potential for signaling aptamer discovery without the extremely laborious task of optimization of oligodeoxynucleotide modifications.