Yan Shuanghong, Wang Liying, Du Xiaoyu, Zhang Shanyu, Wang Sha, Cao Jiao, Zhang Jinyue, Jia Wendong, Wang Yuqin, Zhang Panke, Chen Hong-Yuan, Huang Shuo
State Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University 210023 Nanjing China
Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University 210023 Nanjing China.
Chem Sci. 2021 Jun 8;12(27):9339-9346. doi: 10.1039/d1sc01399h. eCollection 2021 Jul 14.
Acknowledging its unique conical lumen structure, porin A (MspA) was the first type of nanopore that has successfully sequenced DNA. Recent developments of nanopore single molecule chemistry have also suggested MspA to be an optimum single molecule reactor. However, further investigations with this approach require heavy mutagenesis which is labor intensive and requires high end instruments for purifications. We here demonstrate an efficient and economic protocol which performs rapid and multiplex preparation of a variety of MspA mutants. The prepared MspA mutants were demonstrated in operations such as nanopore insertion, sequencing, optical single channel recording (oSCR), nanopore single molecule chemistry and nanopore rectification. The performance is no different from that of pores however prepared by other means. The time of all human operations and the cost for a single batch of preparation have been minimized to 40 min and 0.4$, respectively. This method is extremely useful in the screening of new MspA mutants, which has an urgent requirement in further investigations of new MspA nanoreactors. Its low cost and simplicity also enable efficient preparations of MspA nanopores for both industrial manufacturing and academic research.
鉴于其独特的锥形内腔结构，孔蛋白A（MspA）是第一种成功用于DNA测序的纳米孔。纳米孔单分子化学的最新进展也表明MspA是一种理想的单分子反应器。然而，采用这种方法进行进一步研究需要大量的诱变操作，这既耗费人力，又需要高端仪器进行纯化。我们在此展示了一种高效且经济的方案，该方案能够快速且多重地制备多种MspA突变体。所制备的MspA突变体在纳米孔插入、测序、光学单通道记录（oSCR）、纳米孔单分子化学和纳米孔整流等操作中得到了验证。其性能与通过其他方式制备的孔无异。所有人工操作的时间和单批制备成本分别降至了40分钟和0.4美元。该方法在筛选新型MspA突变体方面极为有用，这在进一步研究新型MspA纳米反应器中有着迫切需求。其低成本和简便性也使得为工业制造和学术研究高效制备MspA纳米孔成为可能。
