Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase.
Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase.
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DOI：
10.1021/ja1087612
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发表时间：
2010-12-22
影响因子：
15
通讯作者：
Akeson M
中科院分区：
文献类型：
--
作者：
Lieberman KR;Cherf GM;Doody MJ;Olasagasti F;Kolodji Y;Akeson M
将核酸加工酶偶联到纳米级孔允许单个DNA或RNA链的受控运动，其被报告为离子电流时间序列。数百个单独的酶复合物可以在高带宽和空间分辨率下以单个文件顺序进行检查。噬菌体phi 29 DNA聚合酶（phi 29 DNAP）是该技术的有吸引力的候选者，由于其显著的持续合成能力和对DNA底物的高亲和力。在这里，我们表明phi 29 DNAP-DNA复合物在穿过α-溶血素纳米孔的电场中被捕获时是稳定的。通过利用野生型phi 29 DNAP的3′-5′核酸外切酶活性来切除3′-H末端残基，产生引物链3′-OH，从而激活DNA底物以在纳米孔孔口处复制。在脱氧核苷三磷酸存在下，启动DNA合成，允许真实的实时检测仅受DNA模板长度限制的大量连续核苷酸添加。在复制过程中，当模板链被拉过纳米孔腔时，以埃级精度在真实的时间内观察到phi 29 DNAP沿着DNA底物的易位。
Coupling nucleic acid processing enzymes to nanoscale pores allows controlled movement of individual DNA or RNA strands that is reported as an ionic current time series. Hundreds of individual enzyme complexes can be examined in single-file order at high bandwidth and spatial resolution. The bacteriophage phi29 DNA polymerase (phi29 DNAP) is an attractive candidate for this technology, due to its remarkable processivity and high affinity for DNA substrates. Here we show that phi29 DNAP-DNA complexes are stable when captured in an electric field across the α-hemolysin nanopore. DNA substrates were activated for replication at the nanopore orifice by exploiting the 3′-5′ exonuclease activity of wild-type phi29 DNAP to excise a 3′-H terminal residue, yielding a primer strand 3′-OH. In the presence of deoxynucleoside triphosphates, DNA synthesis was initiated, allowing real time detection of numerous sequential nucleotide additions that was limited only by DNA template length. Translocation of phi29 DNAP along DNA substrates was observed in real time at Angstrom scale precision as the template strand was drawn through the nanopore lumen during replication.
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影响因子：
15
作者：
Hurt N;Wang H;Akeson M;Lieberman KR
通讯作者：
Lieberman KR
影响因子：
15
作者：
Cockroft, Scott L.;Chu, John;Ghadiri, M. Reza
通讯作者：
Ghadiri, M. Reza
影响因子：
11.4
作者：
Berman, Andrea J.;Kamtekar, Satwik;Steitz, Thomas A.
通讯作者：
Steitz, Thomas A.
DOI：
10.1073/pnas.93.24.13770
发表时间：
1996-11-26
影响因子：
11.1
作者：
Kasianowicz, JJ;Brandin, E;Deamer, DW
通讯作者：
Deamer, DW
影响因子：
38.3
作者：
Benner, Seico;Chen, Roger J. A.;Akeson, Mark
通讯作者：
Akeson, Mark