Kinetics of photoinduced RNA refolding by real-time NMR spectroscopy
Kinetics of photoinduced RNA refolding by real-time NMR spectroscopy
复制标题
DOI：
10.1002/anie.200462724
复制
发表时间：
2005-01-01
影响因子：
16.6
通讯作者：
Pitsch, S
中科院分区：
文献类型：
--
作者：
Wenter, P;Fürtig, B;Pitsch, S
RNA的催化和调节功能强烈依赖于三级结构的重组和二级结构的重折叠。[1]这种动态多样性通过频繁出现的多个折叠途径[2]以及亚稳态[3]和共存（“双稳态”）构象来说明。[4]RNA从变性的非天然状态的折叠已经通过各种技术进行了广泛的研究，[5]这些技术已经建立了一个分级折叠途径，其中二级结构元件的快速形成先于三级元件的缓慢形成。[6]相比之下，只有少数时间分辨的研究RNA重折叠从一个未扰动的天然状态已被描述。[7]在本文中，我们报告了一种研究结构和时间分辨RNA重折叠的新方法，该方法基于非平衡状态下预选构象的光解生成，然后用实时NMR光谱观察重折叠。这种方法被应用到详细的动力学表征的一个20个碱基的RNA序列，这是已知的采用两个共存的发夹环结构。[8]20个碱基的RNA序列5о-r [GACCGGAAGGUCCGCCUUCC]-3о在温度依赖性平衡中形成两个相互转换的发夹结构（折叠A和B，图1b）。为了选择性地使更稳定的折叠B不稳定，在折叠B中形成碱基对而在折叠A中不形成碱基对的鸟苷基团被O 6-[（S）-1-（2-硝基苯基）乙基]鸟苷（（S）-NPEG，图1a）取代。[9]预计庞大的NPE组会破坏沃森-克里克GC碱基对
The catalytic and regulatory functions of RNA strongly depend on the reorganization of the tertiary structure and the refolding of secondary structures.[1] This dynamic diversity is illustrated by the frequent occurrence of multiple folding pathways,[2] as well as metastable [3] and coexisting (“bistable”) conformations.[4] The folding of RNA from a denatured, nonnative state has been studied extensively by a variety of techniques,[5] which have established a hierarchical folding pathway in which the rapid formation of secondary structural elements precedes the slower formation of tertiary elements.[6] In contrast, only a few time-resolved studies of RNA refolding from an unperturbed native state have been described.[7]Herein we report a new method for the investigation of structure-and time-resolved RNA refolding that is based on the photolytic generation of preselected conformations in a nonequilibrium state, followed by the observation of refolding with real-time NMR spectroscopy. This method was applied to the detailed kinetic characterization of a bistable 20-base RNA sequence, which is known to adopt two coexisting hairpin-loop structures.[8] The 20-base RNA sequence 5о-r [GACCGGAAGGUCCGCCUUCC]-3о forms two interconverting hairpin structures in a temperature-dependent equilibrium (Folds A and B, Figure 1b). To selectively destabilize the more stable Fold B, the guanosine groups that form base pairs in Fold B, but not in Fold A, were replaced by O6-[(S)-1-(2-nitrophenyl) ethyl] guanosine ((S)-NPEG, Figure 1a).[9] The bulky NPE group was expected to disrupt Watson–Crick GC base-pair