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，纽约大学的 Tamar Schlick 博士和北卡罗来纳大学的 Alain Laederach 博士将开发先进的计算工具，以预测和控制当细胞机器（人们发现，翻译 mRNA 三联体代码时的这种移码在病毒和人类细胞中已被预先编程，以修改信使 RNA (mRNA) 中的三字母代码。本研究旨在通过计算预测和实验测试向 mRNA 引入突变如何影响其三维结构，从而揭示原型病毒基因组中的程序移码。原型病毒中的研究将促进新型高效移码元件的设计，并有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、化学和工程学方面的跨学科培训，特别强调增强能力。少数人参与STEM 活动中将包括公众宣传活动，以吸引广大受众，并强调对人类健康具有影响的数学、生物学、计算和生物技术的交叉点。改变 mRNA 三联体核苷酸转录物以产生替代基因产物，对于许多病毒（包括 HIV 和 SARS 相关冠状病毒）翻译重叠 mRNA 阅读框来说是必不可少的，PRF 也是内源性人类中的一种机制。由于 PRF 已被证明可以显着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基因表达工程的平台，然而，移码的复杂性和 RNA 移码的结构可塑性。在工程和治疗策略取得成功之前，必须先了解 FSE 元素。在这个协同生物、化学、数学和计算研究项目中，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测量进行评估，并通过适合具有多种构象的 RNA 的技术来分析由此产生的 FSE 结构景观。除了加深对移码机制和预测 FSE 景观改变突变的计算工具的理解外，该项目还将生产新的生物技术、RNA 修饰工具，作为对抗 RNA 病毒或适用于人类和人类的潜在治疗剂。使用移码的其他基因也出现在病毒包装/药物递送中，因为移码是一种存储基因编码信息的紧凑机制，并且可以用来克服基因组大小的限制。该项目由化学部（CHE）共同资助。 、数学科学部 (DMS) 和数学与物理科学理事会 (MPS) 下的物理部 (PHY)。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准。