Single-chain MspA for nanopore sequencing of DNA

项目摘要-用于DNA纳米孔测序的单链MSPA 新的DNA测序技术改变了生物医学研究和临床 申请。由于其成本低、样品处理时间快、样本量少且缺乏 在样品放大方面，纳米孔测序是一项非常有前途的技术，可以进一步减少 成本和增加DNA测序的读取长度。这种方法的关键是纳米孔 可以从无机、有机或生物材料中生产。尽管最近 进展对DNA纳米孔测序仍然存在重大挑战。例如， MSPA孔中每个核酸基的剩余电流由大约四个 核苷酸降低了碱基调用的准确性，DNA通过毛孔的移位也是如此 速度快，DNA处理酶的随机活动使数据分析变得复杂。这些 原则上，挑战可以通过蛋白质工程来解决，然而，这是 受到MSPA和所有其他目前使用的生物纳米孔低聚结构的阻碍。 这一建议是基于构建单链MSPA，其中所有八个亚基都是 已链接。单链MSPA使我们能够控制MSPA孔的亚基组装和 定点诱变每个通道残基的化学性质。这项技术 这一突破为进一步提高MSPA的测序能力奠定了基础。 该方案的目标是表征单链MSPA的通道活性及其 DNA测序性质和系统改变MSPA的孔径 构建具有不同亚基化学计量比的scMspA孔。这将使我们能够第一次 时间，以考察孔径对生物识别核苷酸碱基的影响 纳米孔。

Project Summary - Single-chain MspA for nanopore sequencing of DNA New DNA sequencing technologies have transformed biomedical research and clinical applications. Due to its low cost, fast sample processing time, minimal sample amount, and lack of sample amplification, nanopore sequencing is a very promising technology to further reduce costs and increase read length of the DNA sequencing. Essential to this method is a nanopore which can be produced from inorganic, organic or biological materials. Despite recent advancements significant challenges remain for nanopore sequencing of DNA. For example, the residual current of each nucleobase in the MspA pore is determined by approximately four nucleotides reducing the accuracy of base calling, DNA translocation through the pore is too fast, and the stochastic activity of DNA-processing enzymes complicates data analysis. These challenges could, in principle, be addressed by protein engineering, which, however, is hampered by the oligomeric structure of MspA and all other currently used biological nanopores. This proposal is based on the construction of single-chain MspA in which all eight subunits are linked. Single-chain MspA enables us to control the subunit assembly of the MspA pore and the chemical properties of each channel residue by site-directed mutagenesis. This technological breakthrough provides the basis for further improvement of the sequencing capabilities of MspA. The goals of this proposal are to characterize the channel activity of single-chain MspA and its DNA sequencing properties and to systematically alter the pore diameter of MspA by constructing scMspA pores with different subunit stoichiometries. This will enable us, for the first time, to examine the influence of the pore diameter on nucleobase recognition by a biological nanopore.