Single-chain MspA for nanopore sequencing of DNA

项目摘要-单链MspA用于DNA的纳米孔测序 新的DNA测序技术改变了生物医学研究和临床 应用.由于其成本低，样品处理时间快，样品量少，缺乏 样品扩增，纳米孔测序是一个非常有前途的技术，以进一步减少 增加了DNA测序的阅读长度。这种方法的关键是纳米孔 其可以由无机、有机或生物材料生产。尽管最近 DNA的纳米孔测序仍然存在重大挑战。比如说 MspA孔中每个核碱基的残余电流由大约四个 核苷酸降低了碱基识别的准确性，DNA通过孔的移位也是如此。 快速，和随机活动的DNA加工酶复杂的数据分析。这些 原则上，这些挑战可以通过蛋白质工程来解决，然而， 受到MspA和所有其他目前使用的生物纳米孔的寡聚结构的阻碍。 该提议基于单链MspA的构建，其中所有八个亚基都是 有联系单链MspA使我们能够控制MspA孔的亚基组装， 通过定点诱变确定每个通道残基的化学性质。这一技术 这一突破为进一步提高MspA的测序能力提供了基础。 该提议的目标是表征单链MspA的通道活性及其在细胞内的表达。 DNA测序特性，并通过以下方法系统地改变MspA的孔径： 构建具有不同亚基化学计量的scMspA孔。这将使我们能够首先 时间，以检查孔径对生物标记的核碱基识别的影响。 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.