Engineering MspA for Nanopore Sequencing

描述（由申请人提供）：单链DNA通过纳米孔的电泳通道有可能成为一种廉价、超快速的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隙。我们建议将耻垢分枝杆菌的孔蛋白A （MspA）开发为纳米孔测序的新孔。MspA是一个很有前途的纳米孔测序设备工程平台，原因有很多：(i)它的短而窄的收缩区可以给它更高的测序灵敏度和分辨率，（ii） MspA非常健壮，（iii）形成稳定的MspA孔是容易和可靠的，（iv）稳定的MspA突变体可以很容易地设计。在初步研究中，无论是野生型的MspA还是收缩区突变的MspA都不允许DNA易位。因此，我们的目标是为DNA的有效易位定制MspA。我们将通过位点定向诱变去除孔边缘和前庭的多余负电荷，稳定收缩区附近的环，并优化收缩区以供DNA通过。易位将在旨在促进易位的条件下用各种ssDNA构建体进行测试。一旦易位实现，进一步的实验将告知后续突变，以优化纳米孔测序的MspA。

DESCRIPTION (provided by applicant): The electrophoretic passage of single-strand DNA through 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.