Engineering MspA for Nanopore Sequencing

描述(申请人提供)：单链DNA通过纳米孔的电泳法有可能成为一种廉价、超快的DNA测序技术。所有的纳米孔测序研究都是基于蛋白质孔-溶血素或无机材料中的人工孔。我们建议将污垢分枝杆菌孔蛋白A(MSPA)发展成一个新的孔，用于纳米孔测序。MSPA是设计纳米孔测序设备的一个有前途的平台，原因如下：(I)其短、窄的狭窄区域可使其具有更高的测序灵敏度和分辨率，(Ii)MSPA非常健壮，(Iii)稳定的MSPA孔的形成容易和可靠，(Iv)可以容易地设计出一系列稳定的MSPA突变体。在初步研究中，无论是野生型MSPA还是在其狭窄区有突变的MSPA都不允许DNA易位。因此，我们的目标是为高效的DNA易位量身定做MSPA。我们将通过定点突变去除毛孔边缘和前庭的多余负电荷，稳定缩窄区附近的环，并优化DNA通过的缩窄区。转位将在旨在促进转位的条件下用各种单链DNA结构进行测试。一旦实现了易位，进一步的实验将通知后续的突变，以优化MSPA进行纳米孔测序。

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.