Title: Development of simulation techniques to study co-transcriptional RNA nanostructure folding

该项目的目的是利用多尺度模拟方法探索共转录RNA折叠的动力学，从而产生RNA纳米结构设计的新方法。了解生物分子折叠的物理学是理论生物物理学的关键挑战。DNA纳米结构是通过用可预测的碱基配对相互作用对多个寡核苷酸进行退火来组装的：通过合理的设计可以创建包含&gt；104组件的3D结构。相比之下，谷歌DeepMind的Alphafold2团队最近在预测蛋白质折叠方面的进展需要生物物理学、生物信息学和机器学习的投入。RNA纳米结构的共转录折叠结合了蛋白质的单链结构和相对可预测的核苷酸碱基之间的相互作用，因此是一个有趣的模型系统。共转录折叠具有丰富的非平衡物理学：在亚稳定亚结构中生产聚合物锁的过程中组装。例如，双螺旋的形成需要两条链相互缠绕，除非非常短，否则只有在自由端可用(通常是多链组装的情况)或整个部分能够自由旋转的情况下才有可能。防止缠绕的三级接触可能会导致装配的拓扑障碍。因此，单链折叠成功的关键是控制子结构的组装顺序。当使用随时间变化的退火温度时，顺序由不同元素的相对稳定性控制，这在DNA纳米结构组装中很常见。在共转录组装的等温条件下，人们必须利用合成的顺序，通过设计自由能垒和非平衡组装路径来控制组装。为了设计纳米结构组装的非平衡组装路径，我们需要更好地理解共转录折叠的物理基础上的设计工具。到目前为止，相对简单的工具已经被用来理解共转录组装。我们将使用粗粒度oxRNA模型为组装提供更多自由能景观的细节，以及如何对这些景观进行雕刻以优化组装路径。OxRNA将每个核苷酸表示为单个刚体，并根据经验将相互作用参数化，以重现单链和双链RNA的结构、机械和热力学性质。这种有效的表示使研究大型结构(&gt；104碱基)以及罕见的过程，如碱基对的形成或断裂和链移位成为可能。OxRNA成功地复制了RNA纳米技术中使用的一些常见基序，例如接吻环和PX交叉。在设计合成RNA纳米结构时，我们将专注于正则碱基配对，避免在生物RNA中发现许多非正则结构基序，这极大地简化了折叠的建模和研究。使用折叠模拟获得的基本见解将被用于创建和改进设计工具，以提高产量和扩大共转录RNA折纸纳米结构可靠形成的尺寸范围。该项目将为细胞内RNA纳米结构制备的发展奠定基础。细胞内自组装纳米结构的潜在应用包括：报告细胞过程的细胞功能探针；原位药物合成；干预自然基因控制系统或基因编辑；以及作为新一代自主医疗设备，将诊断和治疗与亚细胞分辨相结合。该项目属于跨理事会优先领域合成生物学。它融合了(EPSRC)物理科学-生物物理学和软物质物理学的研究。该项目由教授指导。安德鲁·特伯菲尔德教授和Ard Louis教授(牛津物理学)将密切合作，Jonathan Doye教授(牛津化学)和Petr Sulc教授(亚利桑那州立大学)将密切合作。

The aim of the project is to use multi-scale simulation methods to explore the kinetics of co-transcriptional RNA folding, leading to new methods of RNA nanostructure design.Understanding the physics of biomolecular folding is a key challenge of theoretical biophysics. DNA nanostructures are assembled by annealing multiple oligonucleotides with predictable base-pairing interactions: 3D structures with >104 components can be created by rational design. In contrast, recent progress in predicting protein folding by the Alphafold2 team from Google DeepMind required input from biophysics, bioinformatics and machine learning. Co-transcriptional folding of RNA nanostructures combines the single-strand architecture of proteins with relatively predictable interactions between nucleobases and is thus a fascinating model system.Co-transcriptional folding has rich non-equilibrium physics: assembly during production of the polymer locks in metastable sub-structures. For example, formation of a double helix requires the wrapping of two strands around each other which, unless very short, is only possible if a free end is available (normally the case in multi-stranded assembly) or if the entire section is able to rotate freely. Tertiary contacts which prevent wrapping can lead to topological barriers to assembly. Key to the success of single-stranded folding is therefore control of the order of assembly of sub-structures. When a time-dependent annealing temperature is used, as is typical in DNA nanostructure assembly, order is controlled by the relative stabilities of the different elements. Under the isothermal conditions of co-transcriptional assembly one must instead exploit the order of synthesis to control assembly through design of free-energy barriers and non-equilibrium assembly pathways. To design non-equilibrium assembly pathways for nanostructure assembly we need design tools based on better understanding of the physics of co-transcriptional folding.So far, relatively simple tools have been used to understand co-transcriptional assembly. We will use the coarse-grained oxRNA model to provide more detail of free-energy landscapes for assembly and how these can be sculpted to optimize assembly pathways. oxRNA represents each nucleotide as a single rigid body with interactions parametrized empirically to reproduce structural, mechanical and thermodynamic properties of single- and double-stranded RNA. This efficient representation enables study of large structures (>104 bases) as well as rare processes such as the formation or breaking of base pairs and strand displacement. oxRNA successfully reproduces some common motifs used in RNA nanotechnology e.g. kissing loops and PX crossovers. In designing synthetic RNA nanostructures we will focus on canonical base pairing, avoiding the many non-canonical structural motifs found in biological RNA, which hugely simplifies modelling and study of folding.Fundamental insights obtained using folding simulations will be used to create and refine design tools to improve yield and to extend the size range over which co-transcriptional RNA origami nanostructures form reliably. This project will lay the foundations for the development of intracellular RNA nanostructure fabrication. Potential applications of self-assembled nanostructures within the cell include: probes of cellular function to report on cellular processes; in situ drug synthesis; intervention in natural genetic control systems or gene editing; and as a new generation of autonomous medical device, combining diagnosis and treatment with sub-cellular resolution.This project lies within cross-council priority area Synthetic Biology. It integrates research in (EPSRC) Physical Sciences - Biophysics and Soft Matter Physics.The project is supervised byProf. Andrew Turberfield and Prof. Ard Louis (Oxford Physics) and will involve close collaboration Prof. Jonathan Doye (Oxford Chemistry) and Prof. Petr Sulc (ASU).