Establishing a generalizable model for predictable antisense RNA repression

建立可预测反义 RNA 抑制的通用模型

• 批准号: 1714352
• 负责人: Tae Seok Moon
• 金额: $ 42.5万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1714352&HistoricalAwards=false
• 关键词: Establishing; generalizable; model; predictable; antisense

RNA is mostly known as a messenger to convey genetic information, while small RNA plays important roles in gene regulation. Such regulatory RNAs have been found to exist naturally in many organisms, but they have been used in a relatively limited number of bacteria as a tool to regulate gene expression. In this project, the investigators will develop generalizable RNA regulators that can be used to understand and engineer diverse biotechnologically important bacteria. Specifically, design principles for antisense RNA (asRNA), one of the common regulatory RNAs in bacteria, will be determined to provide biologists and bioengineers with predictable RNA tools for gene regulation. Additionally, this project is broadly impactful by cultivating the next generation. Multiple graduate students will be trained to perform the interdisciplinary project. Moreover, our outreach program will introduce state-of-the-art synthetic biology RNA tools to high school teachers, and provide them with teaching kits that will be used in their classroom, broadening student participation in science and engineering.The long-term goal of this project is to determine quantitative design principles for asRNAs and provide a generalizable model that enables predictably tunable asRNA-mediated gene repression in diverse organisms. asRNA is one of several types of small regulatory RNAs, and it represses gene expression by binding to its target messenger RNA. Although this binding occurs following the simple RNA base-pairing rule, predictably tunable asRNA-mediated repression is still challenging. The most important design parameters for asRNA in Escherichia coli DH10B have been identified by using three target reporter genes in plasmids. A predictive model for asRNA-mediated repression, which relates repression efficiency to the identified parameters by a multiple linear regression analysis, will be developed. To demonstrate the generalizability of this model, three specific aims are proposed. First, the model developed with plasmid reporters in E. coli DH10B will be validated by expanding it to chromosomal gene targets. This task will allow for testing of forward-designed asRNAs in different genetic contexts. Second, the predictive model will be tested in three different bacterial species. This task will test the hypothesis that asRNA design principles are consistent among diverse bacteria. Third, the model will be further validated by building and testing additional asRNAs. A user-friendly online version of the model will also be developed to provide a way to collaboratively advance this RNA-based technology.

RNA主要作为传递遗传信息的信使，而小RNA在基因调控中也起着重要作用。这种调控rna已被发现自然存在于许多生物体中，但它们已被用于相对有限数量的细菌中作为调节基因表达的工具。在这个项目中，研究人员将开发可用于理解和设计各种生物技术重要细菌的通用RNA调节因子。具体来说，反义RNA （asRNA）是细菌中常见的调控RNA之一，其设计原则将为生物学家和生物工程师提供可预测的基因调控RNA工具。此外，该项目通过培养下一代具有广泛的影响力。多名研究生将接受跨学科项目的培训。此外，我们的外展计划将向高中教师介绍最先进的合成生物学RNA工具，并向他们提供将在课堂上使用的教学工具包，扩大学生对科学和工程的参与。该项目的长期目标是确定asrna的定量设计原则，并提供一个可推广的模型，使各种生物中可预测的asrna介导的基因抑制成为可能。asRNA是几种小调控RNA中的一种，它通过结合其靶信使RNA来抑制基因表达。尽管这种结合遵循简单的RNA碱基配对规则发生，但可预测的可调节的asrna介导的抑制仍然具有挑战性。利用质粒中的三个靶报告基因，确定了大肠杆菌DH10B中asRNA最重要的设计参数。将开发一个asrna介导的抑制预测模型，该模型将抑制效率与通过多元线性回归分析确定的参数联系起来。为了证明该模型的普遍性，提出了三个具体目标。首先，用大肠杆菌DH10B的质粒报告者建立的模型将通过扩展到染色体基因靶点来验证。这项任务将允许在不同的遗传背景下测试前瞻性设计的asRNAs。其次，该预测模型将在三种不同的细菌种类中进行测试。这项任务将验证asRNA设计原则在不同细菌中是一致的假设。第三，该模型将通过构建和测试额外的asrna来进一步验证。该模型的一个用户友好的在线版本也将被开发，以提供一种协同推进这种基于rna的技术的方法。

项目成果

Making Security Viral: Shifting Engineering Biology Culture and Publishing

让安全病毒式传播：改变工程生物学文化和出版

• DOI: 10.1021/acssynbio.1c00324
• 发表时间: 2022
• 期刊: ACS Synthetic Biology
• 影响因子: 4.7
• 作者: Mackelprang, Rebecca;Adamala, Katarzyna P.;Aurand, Emily R.;Diggans, James C.;Ellington, Andrew D.;Evans, Samuel Weiss;Fortman, J. L.;Hillson, Nathan J.;Hinman, Albert W.;Isaacs, Farren J.
• 通讯作者: Isaacs, Farren J.

Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System

• DOI: 10.1021/acssynbio.7b00322
• 发表时间: 2018-03-01
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Lee, Young Je;Kim, Soo-Jung;Moon, Tae Seok
• 通讯作者: Moon, Tae Seok

Establishing a Multivariate Model for Predictable Antisense RNA-Mediated Repression

• DOI: 10.1021/acssynbio.8b00227
• 发表时间: 2019-01-01
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Lee, Young Je;Kim, Soo-Jung;Moon, Tae Seok
• 通讯作者: Moon, Tae Seok

Modulating Responses of Toehold Switches by an Inhibitory Hairpin

• DOI: 10.1021/acssynbio.8b00488
• 发表时间: 2019-03-01
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Kim, Soo-Jung;Leong, Matthew;Moon, Tae Seok
• 通讯作者: Moon, Tae Seok

Duplex Structure of Double-Stranded RNA Provides Stability against Hydrolysis Relative to Single-Stranded RNA

• DOI: 10.1021/acs.est.1c01255
• 发表时间: 2021-05-25
• 期刊: ENVIRONMENTAL SCIENCE & TECHNOLOGY
• 影响因子: 11.4
• 作者: Zhang，Ke;Hodge，Joseph;Parker，Kimberly M.
• 通讯作者: Parker，Kimberly M.