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首页> 美国卫生研究院文献>Nucleic Acids Research >Design and synthesis of RNA miniduplexes via a synthetic linker approach. 2. Generation of covalently closed double-stranded cyclic HIV-1 TAR RNA analogs with high Tat-binding affinity.
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Design and synthesis of RNA miniduplexes via a synthetic linker approach. 2. Generation of covalently closed double-stranded cyclic HIV-1 TAR RNA analogs with high Tat-binding affinity.

机译：通过合成接头方法设计和合成RNA miniduplex。 2.产生具有高Tat结合亲和力的共价闭合双链环状HIV-1 TAR RNA类似物。

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摘要

We recently developed an approach which allows rapid generation of short, double-stranded oligonucleotides whereby one end of the duplex was joined and stabilized by a synthetic linker of specific design (miniduplexes)(6). Model miniduplexes based on the HIV-1 TAR RNA hairpin were shown to be thermodynamically stable and good substrates for binding by the HIV-1 Tat protein which normally bind to natural TAR (6). In this study, we have extended our studies to the design, synthesis and analysis of the binding properties of covalently closed, double-stranded, cyclic RNA miniduplexes. A strategy using automated chemical synthesis and T4 RNA ligase-catalyzed cyclization was employed to generate cyclic oligoribonucleotides. When both ends of a shortened, wild-type TAR RNA stem (9 bp) were covalently linked through either nucleotidic loops (4-6 nt) or synthetic linkers (derivatized from hexaethylene glycol), the resulting cyclic TAR RNA analogs were good substrates for binding by both Tat-derived peptide or full-length Tat protein. Interestingly, the cyclic TAR analogs failed to show any binding if the synthetic linker was reduced in length (e.g. derivatized from triethylene glycol), although such linkers are acceptable in the hairpin-shaped miniduplexes series (6). This implies that RNA conformational changes are required for Tat binding and that these changes are restricted in certain cyclic variants. Our findings suggest that covalently-closed nucleic acid miniduplexes may be useful both to study nucleic acid-protein interactions as well as to provide a basis for therapeutic intervention as transcription decoys.
机译：我们最近开发了一种方法，该方法可以快速生成短的双链寡核苷酸，从而使双链体的一端通过特定设计的合成接头（微型双链体）连接并稳定（6）。已显示基于HIV-1 TAR RNA发夹的模型微型双链体是热力学稳定的，并且是通常与天然TAR结合的HIV-1 Tat蛋白结合的良好底物（6）。在这项研究中，我们将研究扩展到共价闭合，双链，环状RNA微型双链体结合特性的设计，合成和分析。使用自动化学合成和T4 RNA连接酶催化环化的策略来生成环状寡核糖核苷酸。当缩短的野生型TAR RNA茎的两端（9 bp）通过核苷酸环（4-6 nt）或合成接头（衍生自六甘醇）共价连接时，所得环状TAR RNA类似物是Tat衍生肽或全长Tat蛋白结合。有趣的是，如果缩短了合成接头的长度（例如衍生自三甘醇），则环状TAR类似物不能显示任何结合，尽管这种接头在发夹形微型双链体系列中是可接受的（6）。这意味着Tat结合需要RNA构象变化，并且这些变化在某些环状变体中受到限制。我们的发现表明，共价闭合的核酸微型双链体可能对研究核酸-蛋白质相互作用以及为转录诱饵的治疗干预提供基础均有用。

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