MFB: RNA modifications of frameshifting stimulators: cellular platforms to engineer gene expression by computational mutation predictions and functional experiments

MFB：移码刺激器的RNA修饰：通过计算突变预测和功能实验来设计基因表达的细胞平台

• 批准号: 2330628
• 负责人: Tamar Schlick
• 金额: $ 150万
• 依托单位: New York University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2330628&HistoricalAwards=false
• 关键词: MFB; RNA; modifications; frameshifting; stimulators

In this Molecular Foundations for Biotechnology (MFB) project, Dr. Tamar Schlick from New York University and Dr. Alain Laederach from the University of North Carolina will develop advanced computational tools to predict and control how viral protein synthesis is affected when the cell’s machinery (the ribosome) shifts and thus changes how the three-letter code in messenger RNA (mRNA) is read. This frameshifting in translating the mRNA triplet code has been found to be preprogrammed in viruses and human cells to modify the expression of gene products and to regulate biochemical processes. This study aims to computationally predict and experimentally test how introducing mutations to mRNA affects its three-dimensional structure and, consequently, programmed frameshifting in prototypical viral genomes. Revealing the specific structural and sequence requirements for frameshifting in prototype viruses will facilitate the design of novel efficient frameshifting elements, with potential applications to viral packaging of genes. This project will provide interdisciplinary training to students in mathematics, computer science, biology, physics, chemistry, and engineering, with particular emphasis on enhancing minority participation in STEM activities. Public outreach efforts will be included to reach general audiences and highlight the intersection of mathematics, biology, computing, and biotechnologies that have implications in human health. Programmed ribosomal frameshifting (PRF) is a widespread mechanism for modifying the gene expressed by altering the mRNA triplet-nucleotide transcript to generate an alternate gene product. Indispensable to many viruses including HIV and SARS-associated coronaviruses for translating overlapping mRNA reading frames, PRF is also a mechanism in endogenous human, eukaryotic and prokaryotic genes. Because PRF has been shown to dramatically influence viral viability or the biochemical regulation of human processes, the modulation of frameshifting defines a platform for engineering gene expression. However, the complex aspects of frameshifting and the structural plasticity of the RNA frameshifting element (FSE) must be understood before engineering and therapeutic strategies can succeed. In this synergistic biological, chemical, mathematical, and computational research program, graph-theory-based tools will be developed to predict FSE mutations for prototype viral systems aimed at substantially lowering frameshifting efficiency as a novel biotechnological strategy against viral infections and related human diseases associated with PRF. The effect of these mutations will be assessed by Luciferase assay measurements, and the resulting FSE structural landscapes analyzed by techniques suitable for RNAs with multiple conformations. Besides an improved understanding of the mechanisms of frameshifting and computational tools for predicting FSE-landscape-altering mutations, this project will produce new biotechnological, RNA modifying tools as potential therapeutic agents against RNA viruses or applicable to human and other genes that employ frameshifting. Applications to viral packaging/drug delivery also arise, as frameshifting is a compact mechanism to store gene coding information and can be exploited to overcome genomic size limitations.This project is jointly funded by the Division of Chemistry (CHE), the Division of Mathematical Sciences (DMS), and the Division of Physics (PHY) in the Directorate for Mathematical and Physical Sciences (MPS).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在这个生物技术分子基础（MFB）项目中，来自纽约大学的Tamar Schlick博士和来自北卡罗来纳州大学的Alain Laederach博士将开发先进的计算工具，以预测和控制当细胞的机器（核糖体）发生变化时病毒蛋白质合成如何受到影响，从而改变信使RNA（mRNA）中的三个字母代码的读取方式。已经发现在翻译mRNA三联体密码时的这种移码在病毒和人类细胞中被预编程以修改基因产物的表达并调节生物化学过程。这项研究的目的是计算预测和实验测试如何引入突变的mRNA影响其三维结构，因此，在原型病毒基因组编程移码。揭示原型病毒中移码的特定结构和序列要求将有助于设计新的高效移码元件，并可能应用于基因的病毒包装。该项目将为学生提供数学、计算机科学、生物学、物理学、化学和工程学方面的跨学科培训，特别强调加强少数群体参与STEM活动。将包括公共宣传工作，以接触普通受众，并突出数学，生物学，计算和生物技术的交叉对人类健康的影响。程序性核糖体移码（PRF）是通过改变mRNA三联核苷酸转录物以产生替代基因产物来修饰表达的基因的广泛机制。与包括HIV和SARS相关的冠状病毒在内的许多病毒翻译重叠的mRNA阅读框架不同，PRF也是内源性人类、真核和原核基因中的一种机制。由于PRF已被证明可以显著影响病毒活力或人类过程的生化调节，因此移码的调节定义了工程基因表达的平台。然而，移码的复杂方面和RNA移码元件（FSE）的结构可塑性必须在工程和治疗策略成功之前得到理解。在这个协同生物，化学，数学和计算研究计划中，将开发基于图论的工具来预测原型病毒系统的FSE突变，旨在大幅降低移码效率，作为对抗病毒感染和与PRF相关的相关人类疾病的新型生物技术策略。这些突变的影响将通过荧光素酶测定法测量来评估，并且通过适用于具有多种构象的RNA的技术来分析所得FSE结构景观。除了提高对移码机制的理解和预测FSE景观改变突变的计算工具外，该项目还将产生新的生物技术，RNA修饰工具，作为对抗RNA病毒的潜在治疗剂或适用于人类和其他采用移码的基因。病毒包装/药物递送的应用也出现了，因为移码是一种存储基因编码信息的紧凑机制，可以用来克服基因组大小的限制。以及数学和物理科学理事会（MPS）的物理部（PHY）该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。