No abstract

Quantum-tunneling-based DNA sequencing is a single molecular technology that has great potential for achieving facile and high-throughput DNA sequencing. In principle, the sequence of DNA could be read out by the time trace of the tunnel current that can be changed according to molecular conductance of nucleobases passing through nanosized gap electrodes. However, efficient basecalling of four genetic alphabets has been seriously impeded due to the similarity of molecular conductance among canonical nucleotides. In this article, we demonstrate that replacement of canonical 2′-deoxyadenosine (dA) with a highly conductive dA analogue, 7-deaza dA, could expand the difference of molecular conductance between four genetic alphabets. Additionally, systematic evaluation of molecular conductance using a series of dA and dG analogues revealed that molecular conductance of the nucleotide is highly dependent on the HOMO level. Thus, the present study demonstrating that signal characteristics of the nucleotide can be modulated based on the HOMO level provides a widely applicable chemical approach and insight for facilitation of single molecular sensing as well as DNA sequencing based on quantum tunneling.

Ubiquitylation—the attachment of ubiquitin (Ub) to proteins in eukaryotic cells—involves a vast number of enzymes from three different classes, resulting in heterogeneous attachment sites and ubiquitin chains. Recently, we introduced lysine acylation using conjugating enzymes (LACE) in which ubiquitin or peptide thioester is site-specifically transferred to a short peptide tag by the SUMO E2 conjugating enzyme Ubc9. This process, however, suffers from slow kinetics—due to a rate-limiting thioester loading step—and the requirement for thioesters restricts its use to in vitro reactions. To overcome these challenges, we devised a chimeric E1 containing the Ub fold domain of the SUMO E1 and the remaining domains of the Ub E1, which activates and loads native Ub onto Ubc9 and obviates the need for Ub thioester in LACE. The chimeric E1 was subjected to directed evolution to improve its apparent second-order rate constant ( k cat / K M ) 400-fold. We demonstrate the utility of the chimeric E1 by site-specific transfer of mono- and oligo-Ub to various target proteins in vitro . Additionally, the chimeric E1, Ubc9, Ub, and the target protein can be coexpressed in Escherichia coli for the facile preparation of monoubiquitylated proteins.

The reaction pathways of lithium 2,2,6,6-tetramethylpiperidide (LiTMP)-mediated deprotonative metalation of methoxy-substituted arenes were investigated. Importantly, it was experimentally observed that, whereas TMEDA has no effect on the course of the reactions, the presence of more than the stoichiometric amount of LiCl is deleterious, in particular without an in situ trap. These effects were corroborated by the DFT calculations. The reaction mechanisms, such as the structure of the active species in the deprotonation event, the reaction pathways by each postulated LiTMP complex, the stabilization effects by in situ trapping using zinc species, and some kinetic interpretation, are discussed herein.