Dual-channel single-molecule fluorescence resonance energy transfer to establish distance parameters for RNA nanoparticles.
Dual-channel single-molecule fluorescence resonance energy transfer to establish distance parameters for RNA nanoparticles.
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DOI：
10.1021/nn1014853
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发表时间：
2010-11-23
期刊：
影响因子：
17.1
通讯作者：
Guo P
中科院分区：
文献类型：
--
作者：
Shu D;Zhang H;Petrenko R;Meller J;Guo P
人们对 RNA 纳米技术的兴趣日益浓厚，以及使用 RNA 纳米颗粒作为治疗药物的可行性已得到证实，这促使人们需要具有纳米级分辨率的成像系统来进行 RNA 研究。 Phi29 二聚体 pRNA 可以作为组装成纳米马达六聚环的构建块、RNA 纳米颗粒的模块以及将治疗药物特异性递送至癌症或病毒感染细胞的载体。了解这种新型 RNA 二聚体颗粒的 3D 结构具有根本性和实际意义。尽管基于生化分析提出了 pRNA 二聚体的 3D 模型，但尚未报道距离测量或 X 射线衍射数据。在这里，我们评估了我们定制的单分子双观察系统在 pRNA 二聚体中使用单分子荧光共振能量转移 (smFRET) 进行距离测量的应用。构建了在不同位置用单个供体或受体荧光团标记的十个 pRNA 单体，并组装了八个二聚体。检测到六个二聚体的 smFRET 信号。荧光团的束缚臂大小是根据双标记 RNA/DNA 标准品凭经验估计的。计算供体和受体之间的距离并将其用作距离参数来评估和完善先前报道的 pRNA 二聚体的 3D 模型。发现pRNA二聚体中核苷酸之间的距离与结合到衣壳原的二聚体的核苷酸距离不同，这表明pRNA二聚体在结合衣壳原时发生了构象变化。
The increasing interest in RNA nanotechnology and the demonstrated feasibility of using RNA nanoparticles as therapeutics have prompted the need for imaging systems with nanometer-scale resolution for RNA studies. Phi29 dimeric pRNAs can serve as building blocks in assembly into the hexameric ring of the nanomotors, as modules of RNA nanoparciles, and as vehicles for specific delivery of therapeutics to cancers or viral infected cells. The understanding of the 3D structure of this novel RNA dimeric particle is fundamentally and practically important. Although a 3D model of pRNA dimer has been proposed based on biochemical analysis, no distance measurements or X-ray diffraction data have been reported. Here we evaluated the application of our customized single-molecule dual-viewing system for distance measurement within pRNA dimers using single-molecule Fluorescence Resonance Energy Transfer (smFRET). Ten pRNA monomers labeled with single donor or acceptor fluorophores at various locations were constructed and eight dimers were assembled. smFRET signals were detected for six dimers. The tethered arm sizes of the fluorophores were estimated empirically from dual-labeled RNA/DNA standards. The distances between donor and acceptor were calculated and used as distance parameters to assess and refine the previously reported 3D model of the pRNA dimer. Distances between nucleotides in pRNA dimers were found to be different from those of the dimers bound to procapsid, suggesting a conformational change of the pRNA dimer upon binding to the procapsid.
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DOI：
10.1007/s00249-008-0383-z
发表时间：
2009-04-01
影响因子：
2
作者：
Cherny, Dmitry I.;Eperon, Ian C.;Bagshaw, Clive R.
通讯作者：
Bagshaw, Clive R.
DOI：
10.1073/pnas.0401638101
发表时间：
2004-04-27
影响因子：
11.1
作者：
Gordon, MP;Ha, T;Selvin, PR
通讯作者：
Selvin, PR
影响因子：
5.6
作者：
Horton, NC;Finzel, BC
通讯作者：
Finzel, BC
影响因子：
4.8
作者：
Chen, CP;Sheng, ST;Guo, PX
通讯作者：
Guo, PX
影响因子：
--
作者：
FORSTER, T
通讯作者：
FORSTER, T