Programmable folding of fusion RNA in vivo and in vitro driven by pRNA 3WJ motif of phi29 DNA packaging motor.
Programmable folding of fusion RNA in vivo and in vitro driven by pRNA 3WJ motif of phi29 DNA packaging motor.
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DOI：
10.1093/nar/gkt885
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发表时间：
2014-01
影响因子：
14.9
通讯作者：
Guo P
中科院分区：
文献类型：
--
作者：
Shu D;Khisamutdinov EF;Zhang L;Guo P
由于能量景观和近邻原则的变化，错误折叠及相关的功能丧失是构建融合RNA复合物时常见的问题。在此我们报道了将phi29 DNA包装马达的pRNA - 3WJ基序整合并应用到具有可控且可预测折叠的融合RNA中。该基序包含三个不连续的约18个核苷酸（nt）片段，呈现出明显较低的折叠能（Shu D等人，《自然·纳米技术》，2011年，6：658 - 667），并自发折叠成一个主导核心，使得融合到RNA复合物中的其他功能部分能够正确折叠。三个单独的片段分散在序列内的任何位置，都能使其他RNA功能模块折叠成具有真实功能的原始结构，这通过乙肝病毒核酶、小干扰RNA以及针对孔雀石绿（MG）、菠菜和链霉亲和素（STV）的适体进行了测试。三个约18 - nt片段中任意两个之间仅存在9个互补核苷酸，但三个9bp的分支非常强大，它们能破坏融合RNA内超过15bp的其他双链。这个系统能够产生包含多种具有正确折叠的RNA功能的融合复合物，在生物技术、纳米医学和纳米技术中具有潜在应用。我们还应用这个系统研究了体内和体外RNA折叠的原理。体内转录过程中RNA序列的瞬时产生导致RNA折叠成不同的构象，这些构象无法用源自体外研究的常规原理进行预测。
Misfolding and associated loss of function are common problems in constructing fusion RNA complexes due to changes in energy landscape and the nearest-neighbor principle. Here we report the incorporation and application of the pRNA-3WJ motif of the phi29 DNA packaging motor into fusion RNA with controllable and predictable folding. The motif included three discontinuous ∼18 nucleotide (nt) fragments, displayed a distinct low folding energy (Shu D et al., Nature Nanotechnology, 2011, 6:658–667), and folded spontaneously into a leading core that enabled the correct folding of other functionalities fused to the RNA complex. Three individual fragments dispersed at any location within the sequence allowed the other RNA functional modules to fold into their original structures with authentic functions, as tested by Hepatitis B virus ribozyme, siRNA, and aptamers for malachite green (MG), spinach, and streptavidin (STV). Only nine complementary nucleotides were present for any two of the three ∼18-nt fragments, but the three 9 bp branches were so powerful that they disrupted other double strands with more than 15 bp within the fusion RNA. This system enabled the production of fusion complexes harboring multiple RNA functionalities with correct folding for potential applications in biotechnology, nanomedicine and nanotechnology. We also applied this system to investigate the principles governing the folding of RNA in vivo and in vitro. Temporal production of RNA sequences during in vivo transcription caused RNA to fold into different conformations that could not be predicted with routine principles derived from in vitro studies.
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