Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs.
Circular RNA oligonucleotides. Synthesis, nucleic acid binding properties, and a comparison with circular DNAs.
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环状RNA寡核苷酸。
DOI：
10.1093/nar/22.12.2326
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发表时间：
1994
影响因子：
14.9
通讯作者：
Kool,ET
中科院分区：
文献类型：
--
作者：
Wang,S;Kool,ET
我们报告了两种环状RNA寡核苷酸的合成和核酸结合特性，这两种寡核苷酸设计用于通过pyr·pur·pyr型三螺旋形成结合单链核酸。环状 RNA 的大小为 34 个核苷酸，并使用模板引导的非酶连接进行环化。为了确保连接反应中异构体 3'-5' 的纯度，连接位点的一个核苷酸是 2'-脱氧核糖。第一个圆 (1) 与序列 5'-A12 互补，第二个圆 (2) 与 5'-AAGAAAGAAAAG 互补。热变性实验和混合研究的结果表明，两个环通过三螺旋形成结合互补的单链DNA或RNA底物，其中富含嘧啶的环中的两个结构域夹着中央富含嘌呤的底物。这些环与其嘌呤互补体的亲和力远高于线性前体或简单的 Watson-Crick DNA 互补体的亲和力。例如，圆 1 与 rA12（pH 7.0、10 mM MgCl2、100 mM NaCl）结合，Tmof 48°C，Kd(37°C) 4.1 × 10−9M，而圆的线性前体结合 Tmof 34°C，Kdof 1.2 × 10−6M。环 2 的复合物是 pH 依赖性的，正如涉及 C(+)G·C 三联体的三螺旋复合物所预期的那样，并且两个环的混合图揭示了与 RNA 或 DNA 底物结合的一对一化学计量。将环状RNA与先前合成的相同序列的环状DNA寡核苷酸进行比较，揭示了DNA结合的相似行为，但RNA结合的行为却截然不同。环状DNA表现出高DNA结合选择性，与互补RNA的双链体结合相对较弱。发现这里研究的四种类型的三链体的热力学稳定性的相对顺序是DDD>>RRR>RDR>>DRD。最近关于 RNA 与 DNA 主链对三链体的强烈依赖的报道背景下讨论了这些结果。三链体形成的环状 RNA 代表了一种新颖且潜在有用的 RNA 高亲和力结合策略。
We report the synthesis and nucleic acid binding properties of two cyclic RNA oligonucleotides designed to bind single-stranded nucleic acids by pyr·pur·pyr-type triple helix formation. The circular RNAs are 34 nucleotides in size and were cyclized using a template-directed nonenzymatic ligation. To ensure isomeric 3′-5′ purity in the ligation reaction, one nucleotide at the ligation site is a 2′-deoxyribose. One circle (1) is complementary to the sequence 5′-A12, and the second (2) is complementary to 5′-AAGAAAGAAAAG. Results of thermal denaturation experiments and mixing studies show that both circles bind complementary single-stranded DNA or RNA substrates by triple helix formation, in which two domains in a pyrimidine-rich circle sandwich a central purine-rich substrate. The affinities of these circles with their purine complements are much higher than the affinities of either the linear precursors or simple Watson-Crick DNA complements. For example, circle 1 binds rA12(pH 7.0, 10 mM MgCl2, 100 mM NaCl) with a Tmof 48°C and a Kd(37°C) of 4.1 × 10−9M, while the linear precursor of the circle binds with a Tmof 34°C and a Kdof 1.2 × 10−6M. The complexes of circle 2 are pH-dependent, as expected for triple helical complexes involving C(+)G·C triads, and mixing plots for both circles reveal one-to-one stoichiometry of binding either to RNA or DNA substrates. Comparison of circular RNAs with previously synthesized circular DNA oligonucleotides of the same sequence reveals similar behavior in the binding of DNA, but strikingly different behavior in the binding of RNA. The cyclic DNAs show high DNA-binding selectivity, giving relatively weaker duplex-type binding with complementary RNAs. The relative order of thermodynamic stability for the four types of triplex studied here is found to be DDD > > RRR > RDR > > DRD. The results are discussed in the context of recent reports of strong triplex dependence on RNA versus DNA backbones. Triplex-forming circular RNAs represent a novel and potentially useful strategy for high-affinity binding of RNA.