Multiple RNA strands can interact in solution and assume a large variety of configurations dictated by their potential for base pairing. Although duplex formation from two complementary oligonucleotides has been studied in detail, we still lack a systematic characterization of the behavior of higher order complexes. Here, we focus on the thermodynamic and kinetic effects of an upstream oligonucleotide on the binding of a downstream oligonucleotide to a common template, as we vary the sequence and structure of the contact interface. We show that coaxial stacking in RNA is well correlated with but much more stabilizing than helix propagation over an analogous intact double helix step (median ΔΔG°_37 °C ≈ 1.7 kcal/mol). Consequently, approximating coaxial stacking in RNA with the helix propagation term leads to large discrepancies between predictions and our experimentally determined melting temperatures, with an offset of ≈10 °C. Our kinetic study reveals that the hybridization of the downstream probe oligonucleotide is impaired (lower k_on) by the presence of the upstream oligonucleotide, with the thermodynamic stabilization coming entirely from an extended lifetime (lower k_off) of the bound downstream oligonucleotide, which can increase from seconds to months. Surprisingly, we show that the effect of nicks is dependent on the length of the stacking oligonucleotides, and we discuss the binding of ultrashort (1–4 nt) oligonucleotides that are relevant in the context of the origin of life. The thermodynamic and kinetic data obtained in this work allow for the prediction of the formation and stability of higher-order multistranded complexes.

中文翻译：

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具有缺口和间隙的 RNA 复合物：同轴堆叠和悬挂末端的热力学和动力学效应


多条 RNA 链可以在溶液中相互作用，并根据其碱基配对的潜力呈现多种构型。尽管已经详细研究了两个互补寡核苷酸的双链体形成，但我们仍然缺乏对高级复合物行为的系统表征。在这里，当我们改变接触界面的序列和结构时，我们重点关注上游寡核苷酸对下游寡核苷酸与公共模板结合的热力学和动力学影响。我们表明，RNA 中的同轴堆积与类似完整双螺旋步骤上的螺旋传播密切相关，但比螺旋传播更稳定（中值 ΔΔ G ° _{37 °C} ≈ 1.7 kcal/mol）。因此，用螺旋传播项近似 RNA 中的同轴堆积会导致预测与我们实验确定的熔解温度之间存在巨大差异，偏移约为 10 °C。我们的动力学研究表明，下游探针寡核苷酸的杂交会因上游寡核苷酸的存在而受到损害（较低的k _on ），而热力学稳定性完全来自于结合的下游寡核苷酸的延长寿命（较低的k _off ），这可以从几秒增加到几个月。令人惊讶的是，我们发现切口的影响取决于堆叠寡核苷酸的长度，并且我们讨论了与生命起源相关的超短（1-4 nt）寡核苷酸的结合。这项工作中获得的热力学和动力学数据可以预测高阶多链复合物的形成和稳定性。