Channel: Communication http://journal15.magtechjournal.com/Jwk3_hxs_new/hxxb EN-US http://journal15.magtechjournal.com/Jwk3_hxs_new/hxxb/EN/0567-7351/current.shtml http://journal15.magtechjournal.com/Jwk3_hxs_new/hxxb 0567-7351 http://journal15.magtechjournal.com/Jwk3_hxs_new/hxxb/EN/10.6023/A19060202 Nanopore technology are being developed for large areas in life science, not only in DNA sequencing and protein sequencing, but also in biomolecule detection, bio-interaction measurement and drug screening. Aerolysin is regarded as new powerful tool for oligonucleotide sensing and peptide sensing due to its high charged pore lumen. Applied a transmembrane potential with a pair of Ag/AgCl electrodes, the negatively charged oligonucleotides are driven into the aerolysin nanopore, inducing a series of ionic current blockages, which could distinguish the oligonucleotides with different length or single base variation. However, due to the lack of high-resolution structure of aerolysin nanopore, the mechanism of its high sensing capability is not clear, limiting the further applications of aerolysin. Recently, we presented two sensing regions inside aerolysin, R1 (near R220) and R2 (near K238), having huge influences on oligonucleotide sensing. Especially, the R1 is responsible for distinguished all 4 bases and 2 modified based in the mixture. However, the detailed mechanism of synergistic effect for these two regions in detection of single nucleotides is still unclear. Here, we use dA₁₄-4-X, dA₁₄-11-X, dA₁₄-4-X-11-X (X=C, T, G) as probes to investigate the effects of base types on the sensing ability of R1 and R2. The results show that the A, C or T in R2 region did not change the sensing ability of R1 region, while G in R2 would hinder the base discrimination in R1 region. This may be caused by the large volume of G that would nearly fully occupy the R2 region and the stronger non-covalent interaction between G and R2 region, resulting in determining the residual current of the whole nanopore. Moreover, we evaluated the interaction between different bases with the sensing region. The results show that the interaction is independent with the volume of the bases, which is ordered by A>G>C>T, suggesting the interaction inside the aerolysin lumen is a considerable factor for its sensing capability. These results would guide us to directly design the mutant Aerolysin nanopore that aims for DNA sequencing and peptide sequencing.

]]> http://journal15.magtechjournal.com/Jwk3_hxs_new/hxxb/EN/10.6023/A19060230 Cyclic di-AMP (c-di-AMP) is a ubiquitous second messenger in prokaryotic cells. c-di-AMP can not only effectively regulate various physiological processes such as cell growth, ion transport and cell wall metabolism balance, but also trigger type I interferon response to inspire the body's immune response. Nanopore-based single molecule detection technology is an emerging single molecule detection method which is currently applied to various fields since it has many advantages such as high speed, label-free, high sensitivity and low cost. Aerolysin is a robust biological nanopore with high temporal resolution and high current resolution, which has achieved single oligonucleotide detection, polysaccharide analysis and the studies of enzymolysis kinetics. Aerolysin nanopore is negatively-charged protein nanopore which has numerous negatively charged amino acid residues around its cis entrances. The electrostatic repulsion between the negatively charged c-di-AMP and negatively charged amino acid residues around the cis entrances prevents c-di-AMP entering the nanopore. In this study, 1.0 mol/L LiCl was used as electrolyte solution to facilitate aerolysin analysis of single c-di-AMP molecule. Each event can be characterized by two parameters, the current blockade, I/I₀, and the blockade time, τ_off. The blockades are classified into two populations as PI and PII. The PI events are assigned to c-di-AMP that bump into the pore and then diffuse away. PII events are assigned to traversing of c-di-AMP through the nanopore. Compared with potassium ions, lithium ion can be more effectively to associate with the negative charges on the aerolysin nanopore surface and reduce the electrostatic repulsion between the c-di-AMP molecule and the Aerolysin. The results showed that number of PI events in per minute was significantly increased in 1.0 mol/L LiCl. The number of PI events in per minute in LiCl is 30 times than that in KCl at 90 mV. Hence, Aerolysin nanopore can be used as an ultrasensitive single molecule sensor for cyclic dinucleotides.

]]> http://journal15.magtechjournal.com/Jwk3_hxs_new/hxxb/EN/10.6023/A19060210 Functionalized N-carbonylmethylene-2-pyridones are some of the most important structural motifs and exist in many natural products and bioactive compounds. Thus, the efficient construction of such skeletons has attracted much attention. Generally, the synthesis of N-carbonylmethylene-2-pyridones is realized via an intermolecular nucleophilic substitution of 2-hydroxypyridines and appropriate electrophiles. However, the above reactions often suffer from low yields caused by poor O/N chemoselectivities due to the dual nucleophilicity of the 2-hydroxypyridines. As far as the structure is concerned, N-carbonylmethylene-2-pyridones can be divided into three sections: a pyridone, a carbonylmethyl group and a side chain. When the side chain is a H atom, the N-substituted pyridones can be constructed conveniently via a reaction of 2-hydroxypyridines and primary α-bromocarbonyl compounds in high yields with excellent chemoselectivities. However, when the side chain is not a H atom, for example an alkyl group, only limited examples have been reported and only moderate yields of the desired N-substituted pyridine products are obtained by a combination of 2-hydroxypyridines and bulky secondary α-bromocarbonyl compounds, mainly due to the poor O/N chemoselectivities. To achieve a general synthetic pathway for the latter, the following practical strategy was designed. 2-Hydroxypyridines were first treated with primary α-bromocarbonyl compounds to generate the unique N-substituted intermediates in situ, which then reacted with the side chain electrophiles to give only the N-alkylated final products. Thus, a Pd-catalyzed three-component chemospecific allylic substitution cascade has been developed for the synthesis of N-carbonylmethylene-2-pyridone derivatives, with the desired products being obtained in up to 98% yield. No O-alkylated by-product was observed. The results suggested that the N-carbonylmethylene-2-pyridones are constructed via a cascade reaction consisting of a nucleophilic substitution followed by an allylic alkylation. The reaction was performed on a gram scale and the corresponding alkylated product was conveniently converted to a pyridone-containing unnatural amino acid. This methodology allows for the highly chemoselective synthesis of biologically important N-carbonylmethylene-2-pyridone derivatives.

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