Aposhian Jordan, Deopa Surya Pratap S, Horowitz Scott, Yesselman Joseph D
Department of Chemistry & Biochemistry and the Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, 80231, USA.
JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO, 80309, USA.
bioRxiv. 2025 Jul 3:2025.07.02.662786. doi: 10.1101/2025.07.02.662786.
Recent developments in RNA nanotechnology have led to the rise in designing specific higher order RNA structures with functional goals in mind, such as drug delivery and immunomodulation. As researchers create RNA nanostructures with the goal of becoming common in the molecular biologists' toolkit, more investigation is required on the robustness of RNA designs. Primarily, what different molecular contexts are the designed and intended nanostructures stable in? In this work we show that by using second-order right-angle light scattering, that RNA nanostructure self-assembly is highly sensitive to environmental conditions. While a test RNA hexagonal grid nanostructure that forms correctly though 120° kissing loops under ideal conditions, small variations in salt conditions and annealing times cause the nanostructure to form less structured variants. Tertiary contacts for self-assembly require magnesium and break over a broad range of low temperatures, melting at 42 °C. In contrast this was found to be considerably lower than the secondary structure melting which occurred at 75 °C. This work underscores the importance of quantitative and thermodynamic characterization of self-assembling nanostructures as they begin to be deployed for engineering and therapeutic applications.
RNA纳米技术的最新进展促使人们在设计特定的高阶RNA结构时考虑功能目标，如药物递送和免疫调节。随着研究人员创建RNA纳米结构并希望其在分子生物学家的工具包中变得常见，需要对RNA设计的稳健性进行更多研究。主要问题是，设计的和预期的纳米结构在哪些不同的分子环境中是稳定的？在这项工作中，我们表明，通过使用二阶直角光散射，RNA纳米结构的自组装对环境条件高度敏感。虽然一种测试RNA六边形网格纳米结构在理想条件下通过120°亲吻环正确形成，但盐条件和退火时间的微小变化会导致纳米结构形成结构较少的变体。自组装的三级接触需要镁，并且在很宽的低温范围内会断裂，在42°C时熔化。相比之下，这被发现远低于在75°C时发生的二级结构熔化温度。这项工作强调了在自组装纳米结构开始用于工程和治疗应用时，对其进行定量和热力学表征的重要性。
