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造孔。我们建议将耻垢分枝杆菌孔蛋白A（MspA）开发成用于纳米孔测序的新孔。MspA是用于工程化纳米孔测序装置的有前景的平台，原因有很多：（i）其短、窄的收缩区可以赋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孔的形成是容易和可靠的，（iv）可以容易地工程化广泛的稳定的MspA突变体。在初步研究中，无论是野生型MspA还是在其收缩区具有突变的MspA都不允许DNA易位。因此，我们的目标是定制MspA用于DNA的有效易位。我们将通过定点诱变从孔的边缘和前庭去除多余的负电荷，稳定收缩区附近的环，并优化DNA通过的收缩区。将在设计用于促进易位的条件下用各种ssDNA构建体测试易位。一旦实现易位，进一步的实验将告知后续突变以优化MspA用于纳米孔测序。

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.