Manara Richard M A, Tomasio Susana, Khalid Syma
School of Chemistry, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK.
Nanomaterials (Basel). 2015 Jan 27;5(1):144-153. doi: 10.3390/nano5010144.
Nanopore technology for DNA sequencing is constantly being refined and improved. In strand sequencing a single strand of DNA is fed through a nanopore and subsequent fluctuations in the current are measured. A major hurdle is that the DNA is translocated through the pore at a rate that is too fast for the current measurement systems. An alternative approach is "exonuclease sequencing", in which an exonuclease is attached to the nanopore that is able to process the strand, cleaving off one base at a time. The bases then flow through the nanopore and the current is measured. This method has the advantage of potentially solving the translocation rate problem, as the speed is controlled by the exonuclease. Here we consider the practical details of exonuclease attachment to the protein alpha hemolysin. We employ molecular dynamics simulations to determine the ideal (a) distance from alpha-hemolysin, and (b) the orientation of the monophosphate nucleotides upon release from the exonuclease such that they will enter the protein. Our results indicate an almost linear decrease in the probability of entry into the protein with increasing distance of nucleotide release. The nucleotide orientation is less significant for entry into the protein.
用于DNA测序的纳米孔技术正在不断完善和改进。在链测序中，单链DNA被送入纳米孔，并测量电流的后续波动。一个主要障碍是DNA穿过孔的速度对于当前的测量系统来说太快了。另一种方法是“核酸外切酶测序”，其中核酸外切酶附着在能够处理链的纳米孔上，每次切割一个碱基。然后碱基流过纳米孔并测量电流。这种方法的优点是有可能解决转运速率问题，因为速度由核酸外切酶控制。在这里，我们考虑将核酸外切酶附着到α-溶血素蛋白上的实际细节。我们采用分子动力学模拟来确定（a）与α-溶血素的理想距离，以及（b）单磷酸核苷酸从核酸外切酶释放时的取向，以便它们能够进入蛋白质。我们的结果表明，随着核苷酸释放距离的增加，进入蛋白质的概率几乎呈线性下降。核苷酸取向对于进入蛋白质的影响较小。
