Folding of 8-17 deoxyribozyme studied by three-color alternating-laser excitation of single molecules
Folding of 8-17 deoxyribozyme studied by three-color alternating-laser excitation of single molecules
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DOI：
10.1021/ja0725145
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发表时间：
2007-12-19
影响因子：
15
通讯作者：
Kim, Seong Keun
中科院分区：
文献类型：
--
作者：
Lee, Nam Ki;Koh, Hye Ran;Kim, Seong Keun
采用三色交替激光激发（3c-ALEX）技术研究了8-17脱氧核酶的折叠。由于3c-ALEX具有同时分选荧光分子的标记状态的基础上，并通过采用三色FRET监测生物分子的三个探针间距离的能力，它是一个理想的工具，研究折叠的多分支分子。8-17脱氧核酶是一种切割特定RNA底物的DNA酶，是多分支分子的良好模型系统，因为它具有带凸起的三向DNA连接的结构。用不同的荧光探针标记8-17的所有三个分支，我们研究了其在Na+缓冲溶液中的[Mg 2 +]依赖性折叠。利用3c-ALEX的化学计量分选能力，我们首先在所有异质混合物的溶液中仅选择三重标记的8-17，然后同时测量所选物种的所有三个探针间距离。我们的研究结果表明，8-17折叠成金字塔的形式增加[Mg 2 +]]，以类似的方式与[Zn 2 +]在早期的研究中发现在系综水平。Mg ~（2+）的表观解离常数是Zn ~（2+）的100倍以上，且随缓冲液浓度的变化而变化。没有明显的迹象，观察到两步折叠的Mg 2+，在相反的情况下，Zn 2+。与锤头状核酶相比，发现8-17需要高10倍的[Mg 2 +]进行折叠。通过与几种失活的8-17类似物的折叠比较，我们发现最短分支的三联环的两个保守序列（A和G）是折叠的关键元件，特别是在低[Mg ~（2+）]下的折叠。我们的研究结果表明，茎环的作用是提供一个支架的两个碱基被正确定位的必要的相互作用，这两个碱基直接参与的相互作用，在折叠中起着关键作用。这项工作表明，3c-ALEX是一个强大的单分子方法来研究复杂的和多分支的生物分子的结构和折叠。
The folding of 8-17 deoxyribozyme was investigated by three-color alternating-laser excitation (3c-ALEX), a new single-molecule fluorescence resonance energy transfer (FRET) method we recently developed. Since 3c-ALEX has the capability of simultaneously sorting fluorescent molecules based on their labeling status and monitoring three interprobe distances of a biomolecule by employing three-color FRET, it is an ideal tool to study folding of multibranched molecules. The 8-17 deoxyribozyme, a DNA enzyme that cleaves a specific RNA substrate, is a good model system for a multibranched molecule, since it. has the structure of a three-way DNA junction with a bulge. Labeling all three branches of the 8-17 with different fluorescent probes, we studied its [Mg2+]-dependent folding in a Na+ buffer solution. With the stoichiometric sorting capability of 3c-ALEX, we first selected only the triply labeled 8-17 in a solution of all heterogeneous mixtures and then simultaneously measured all three interprobe distances of the selected species. Our results show that the 8-17 folds into a pyramidal form upon increasing [Mg2+]], in a similar way with [Zn2+] as found in an earlier study conducted at the ensemble level. The apparent dissociation constant of Mg2+ was more than 100 times larger than that of Zn2+ and showed considerable variance with buffer concentration. No clear sign of two-step folding was observed for Mg2+, in contrast to the case of Zn2+. Compared with the hammerhead ribozyme, the 8-17 was found to require 10 times higher [Mg2+] to undergo folding. By comparison with the folding of several inactive 8-17 analogues, we found that the two conserved sequences (A and G) of the triad loop of the shortest branch are critical elements for folding, especially for the folding at low [Mg2+]. Our results suggest that the role of the stem loop is to provide a scaffold for the two bases to be properly positioned for the necessary interaction and that the two bases are directly involved in the interaction that plays a critical role in folding. This work demonstrates that 3c-ALEX is a powerful single-molecule method to study the structure and folding of complex and multibranched biomolecules.