Revealing and Resolving the Restrained Enzymatic Cleavage of DNA Self-Assembled Monolayers on Gold: Electrochemical Quantitation and ESI-MS Confirmation

Herein we report a combined electrochemical and ESI-MS study of the enzymatic hydrolysis efficiency of DNA self-assembled monolayers (SAMs) on gold, platform systems for understanding nucleic acid surface chemistry and for constructing DNA-based biosensors. Our electrochemical approach is based on the comparison of the amounts of surface-tethered DNA nucleotides before and after Exonuclease I (Exo I) incubation using electrostatically bound [Ru(NH3)6]3+ as redox indicators. It is surprising to reveal that the hydrolysis efficiency of ssDNA SAMs does not depend on the packing density and base sequence, and that the cleavage ends with surface-bound shorter strands (9-13 mers). The ex-situ ESI-MS observations confirmed that the hydrolysis products for ssDNA SAMs (from 24 to 56 mers) are dominated with 10-15 mer fragments, in contrast to the complete digestion in solution. Such surface-restrained hydrolysis behavior is due to the steric hindrance of the underneath electrode to the Exo I/DNA binding, which is essential for the occurrence of Exo I-catalyzed processive cleavage. More importantly, we have shown that the hydrolysis efficiency of ssDNA SAMs can be remarkably improved by adopting long alkyl linkers (locating DNA strands further away from the substrates)

Overexpression of peach NAC25 promotes anthocyanin biosynthesis in poplar shoots

Anthocyanins, a categories of metabolites derived from flavonoid biosynthesis pathway, are responsible for the red color of peach. Anthocyanin metabolism is modulated by a complicated regulatory network. In this study, our results demonstrated that PpNAC25 was a component of anthocyanin regulatory network in peach. PpNAC25 showed a closed relationship with the well-known ripening-related gene NOR (no-ripening) and was highly expressed in peach fruit. Overexpression of PpNAC25 in poplar resulted in a redder shoot tips compared with EV control. PpNAC25 overexpression upregulated the anthocyanin biosynthetic and transportation genes in transcript levels and then increased anthocyanin contents. In Y1H and Luc/Ren assay, PpNAC25 bound to the promoter of the anthocyanin-activator PpMYB10.1 and PpMYB10.2 and activated its transcript. Moreover, PpNAC25 formed a heterodimer with PpNAC1, an anthocyanin-regulator. Our researches provide evidence that PpNAC25 may be a positive regulator of anthocyanin biosynthesis in peach fruit