Determination of RNA orientation during translocation through a biological nanopore
Determination of RNA orientation during translocation through a biological nanopore
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DOI：
10.1529/biophysj.105.068957
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发表时间：
2006-01-01
影响因子：
3.4
通讯作者：
Troll, MA
中科院分区：
文献类型：
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作者：
Butler, TZ;Gundlach, JH;Troll, MA
我们研究了A(50)、C-50、A(25)C(50)和C(50)A(25)RNA分子通过α-溶血素跨膜蛋白孔的单分子电泳性移位。我们观察到在A(25)C(50)和C(50)A(25)分子的移位过程中发生了明显的双能流阻塞。在这些双水平阻断期间观察到的两个电流水平与在A(50)和C-50易位期间观察到的特征电流水平非常相似。从两能级电流阻塞中两个能级的时间顺序，我们推测单个A(25)C(50)和C(50)A(25)分子是否以3‘-gt；5’或5‘->；3’方向通过孔。电流阻塞程度与推测的A(25)C(50)或C(50)A(25)取向之间的相关性表明，Poly C片段的3‘->5’易位导致的电流阻塞比5‘->3’易位明显更深。我们的分析还表明，C-50和A(25)C(50)RNA分子的39个末端比59个末端更有可能启动易位。在许多转位事件之前紧随其后的较小的电流阻断中的方向依赖性差异表明，这种阻断也包含关于转位过程中RNA定位的信息。这些发现强调了多核苷酸分子的方向性是易位的一个重要因素，并展示了离子电流信号中的结构如何为易位过程提供新的见解。
We investigate single-molecule electrophoretic translocation of A(50), C-50, A(25)C(50), and C(50)A(25) RNA molecules through the alpha-hemolysin transmembrane protein pore. We observe pronounced bilevel current blockages during translocation of A(25)C(50) and C(50)A(25) molecules. The two current levels observed during these bilevel blockages are very similar to the characteristic current levels observed during A(50) and C-50 translocation. From the temporal ordering of the two levels within the bilevel current blockages, we infer whether individual A(25)C(50) and C(50)A(25) molecules pass through the pore in a 3'-> 5' or 5'-> 3' orientation. Correlation between the level of current obstruction and the inferred A(25)C(50) or C(50)A(25) orientation indicates that 3'-> 5' translocation of a poly C segment causes a significantly deeper current obstruction than 5'-> 3' translocation. Our analysis also suggests that the 39 ends of C-50 and A(25)C(50) RNA molecules are more likely to initiate translocation than the 59 ends. Orientation dependent differences in a smaller current blockage that immediately precedes many translocation events suggest that this blockage also contains information about RNA orientation during translocation. These findings emphasize that the directionality of polynucleotide molecules is an important factor in translocation and demonstrate how structure within ionic current signals can give new insights into the translocation process.