PDF(Pubmed)
Abstract：
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 kon) by the presence of the upstream oligonucleotide, with the thermodynamic stabilization coming entirely from an extended lifetime (lower koff) 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.
摘要：
多个RNA链可以在溶液中相互作用，并呈现由它们的碱基配对潜力决定的多种构型。尽管已经详细研究了由两个互补寡核苷酸形成的双链体,我们仍然缺乏对高阶配合物行为的系统表征。这里,我们专注于上游寡核苷酸对下游寡核苷酸与共同模板结合的热力学和动力学效应，因为我们改变了接触界面的顺序和结构。我们表明，在类似的完整双螺旋步骤中，RNA中的同轴堆叠与螺旋传播密切相关，但比螺旋传播更稳定（中位ΔG°37°C≈1.7kcal/mol）。因此,用螺旋传播项近似RNA中的同轴堆叠会导致预测与我们实验确定的解链温度之间的巨大差异，偏移约10°C。我们的动力学研究表明，下游探针寡核苷酸的杂交被上游寡核苷酸的存在损害（较低的kon），热力学稳定完全来自于结合的下游寡核苷酸的延长的寿命（较低的koff），可以从几秒钟增加到几个月。令人惊讶的是,我们表明缺口的作用取决于堆叠寡核苷酸的长度，我们讨论了与生命起源相关的超短（1-4nt）寡核苷酸的结合。在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。
