Engineering MspA for Nanopore Sequencing

单链DMA通过纳米孔的电泳通道有可能成为一种新的分子。 廉价的超快DNA测序技术。纳米孔测序的所有研究都是基于 蛋白质孔<$-溶血素或涉及无机材料中的人工孔。我们建议发展 将耻垢分枝杆菌孔蛋白A（MspA）插入新孔中用于纳米孔测序。MspA是一个有前途的 由于许多原因，纳米孔测序装置的工程化平台是不可能的： 缩窄区可以给予其更高的测序灵敏度和分辨率，（ii）MspA是极其稳健的，（iii） 稳定的MspA孔的形成是容易且可靠的。（iv）可以制备宽范围的稳定的MspA突变体。 易于设计。在初步研究中，野生型MspA和在其基因组中具有突变的MspA都不能表达。 收缩区允许DNA移位。因此，我们的目标是定制MspA， DNA易位。我们将通过以下方法从毛孔的边缘和前庭中去除多余的负电荷： 定点诱变，稳定收缩区附近的环，并优化收缩区 用于DNA传代。易位将在设计用于以下的条件下用各种ssDNA构建体进行测试： 促进易位。一旦易位实现，进一步的实验将告知随后的突变 来优化MspA用于纳米孔测序。

The electrophoretic passage of single-strand DMAthrough a nanopore has the potential to become an inexpensive, ultrafast DNA sequencing technique. All research in nanopore sequencing is based on the protein pore ¿-Hemolysin or involves artificial pores in inorganic materials. We propose to develop the Mycobacterium smegmatis porin A (MspA) into a new pore for nanopore sequencing. MspA is a promising platform for engineering a nanopore sequencing device for a number of reasons: (i) Its short, narrow constriction zone may give it higher sequencing sensitivity and resolution, (ii)MspA is extremely robust,(iii) Formation of stable MspA pores is easy and reliable, (iv) A wide range of stable MspA mutants can be readily engineered. In preliminary studies neither wild-type MspA nor MspA with a mutation in its constriction zone allowed translocation of DNA. Therefore, our goal is to tailor MspA for efficient translocation of DNA. We will remove excess negative charges from the rim and vestibule of the pore by site-directed mutagenesis, stabilize the loops near the constriction zone, and optimize the constriction zone for DNA passage. Translocation will be tested with a variety of ssDNA constructs in conditions designed to facilitate translocation. Once translocation is realized, further experiments will inform subsequent mutations to optimize MspA for nanopore sequencing.