MFB: Partnerships to Transform Emerging Industries - RNA Tools/Biotechnology: Stabilizing Hairpin Inserts in RNA Virus Induced Gene Silencing Vectors

在这个分子生物技术基金会（MFB）项目中，马里兰大学的Anne Simon博士和Silvec Biologics， Inc.的Feng Gao博士将开发有助于稳定病毒RNA基因组的方法，这些方法可用于治疗植物疾病。这些良性病毒将rna引入植物，关闭入侵作物的病原体基因，从而通过一种称为病毒诱导基因沉默（VIGS）的过程增强植物抵抗病原体的能力。该项目将使用计算方法，包括人工智能方法，设计发夹形状的RNA结构，当插入病毒基因组时，将增加病毒RNA基因组的固有稳定性，或者当遇到负责复制基因组的酶时，发夹的稳定性。因此，VIGS rna将保持完整，并能够沉默入侵植物宿主的病原体基因。这种类型的RNA疗法对农业经济具有重大影响，可直接应用于治疗柑橘绿色病，例如柑橘绿色病，这是一种严重影响国内外柑橘类水果生产的疾病。此外，研究生和博士后学员将获得在学术/私营公司合作下工作的经验。病毒诱导的基因沉默（VIGS）是利用正链RNA病毒作为载体，利用植物的天然抗病毒防御（RNA沉默）。Drs。Simon和Gao已经确定，每个病毒发夹的热力学特征有助于整个病毒基因组的稳定性，自然发夹结构反映了长期热力学进化的终点。因此，插入病毒RNA基因组的发夹保持内源性发夹的热力学性质应该是稳定的。研究人员将扩展我们对稳定发夹设计所需参数的理解，重点关注插入柑橘黄静脉相关病毒（CYVaV）时不稳定的少数发夹的特征。他们将调查是否类似的参数决定了插入不相关的烟草病毒和柑橘病毒时的发夹稳定性。如果CYVaV发夹在这些病毒中不稳定，我们将评估模仿其自身发夹的发夹是否稳定。他们还将验证病毒编码的RNA依赖RNA聚合酶（RdRp）由于这些聚合酶固有的保真度不足而导致插入物不稳定的假设。此外，他们还将确定未优化的发夹是否会给生产效率带来新的RdRp障碍。合理的设计将用于提高CYVaV RdRp的保真度，以确定这种增强的性能是否会导致先前不稳定的发夹的稳定。改变对RdRp保真度/连续性的影响将通过纳米孔测序来确定，以评估是否有更少的5 '端截断rna和缺失产物，如缺陷rna，以及不稳定的发夹插入物是否现在稳定。最后，为了帮助其他希望在病毒中稳定插入的研究人员，他们将开发稳定发夹设计的计算方法，这将需要实验结果和算法设计软件之间的反复相互作用。该项目得到了数学和物理科学理事会化学部、生物科学理事会分子和细胞生物科学部以及植物基因组研究计划的支持。该奖项反映了美国国家科学基金会的法定使命，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。

In this Molecular Foundation for Biotechnology (MFB) project, Dr. Anne Simon from the University of Maryland and Dr. Feng Gao from Silvec Biologics, Inc. will develop methods to help stabilize RNA genomes in viruses that can be used as therapies to treat plant diseases. These benign viruses introduce RNAs into plants to turn off the genes of pathogens that invade crops, thereby enhancing the ability of the plant to fight off pathogens through a process called virus-induced gene silencing (VIGS). This project will use computational approaches, including artificial intelligence methods, to design hairpin shaped RNA structures that, when inserted into the virus genome, will increase the inherent stability of the viral RNA genome or the hairpin’s stability when encountering the enzyme that is responsible for replicating the genome. The VIGS RNAs will, thus, remain intact and capable of silencing the genes of pathogens that invade the plant hosts. This type of RNA therapy has significant impact on the agricultural economy, with direct application to treating, for example, citrus greening, a disease that has significant affected both domestic and foreign production of citrus fruits. In addition, graduate students and postdoctoral trainees will gain experience in working under an academic/private company collaboration.Virus-induced gene silencing (VIGS) makes use of plus-strand RNA viruses as vectors to exploit natural antiviral defenses (RNA silencing) in plants. Drs. Simon and Gao have determined that thermodynamic features of every viral hairpin contribute to stability of the entire viral genome, and natural hairpin structures reflect the end point of long-term thermodynamic evolution. Thus, hairpins inserted into viral RNA genomes that maintain the thermodynamic properties of endogenous hairpins should be stable. The investigators will extend our understanding of parameters required for stable hairpin design, focusing on features found in the few hairpins that were unstable when inserted into the citrus yellow vein associated-virus (CYVaV). They will investigate whether similar parameters dictate hairpin stability when inserted into unrelated tobacco rattle and citrus tristeza plant viruses. If CYVaV hairpins are not stable in these viruses, we will assess if hairpins that mimic their own hairpins are stable. They will also test the hypothesis that the virus-encoded RNA-dependent RNA polymerase (RdRp) is responsible for insert instability due to the inherent lack of fidelity of these polymerases. Additionally, they will determine if non-optimized hairpins introduce new RdRp obstructions to processivity. Rational design will be used to increase the fidelity of the CYVaV RdRp to ascertain if this enhanced property leads to stabilization of previously unstable hairpins. Effect of alterations on the fidelity/processivity of the RdRp will be determined using Nanopore sequencing to assess if there are fewer 5’ terminal truncated RNAs and deletion products like defective RNAs, and if unstable hairpin inserts are now stabilized. Finally, to assist other researchers who desire stable inserts in their viruses, they will develop computational methodologies for stable hairpin design, which will require repeated interplay between experimental results and algorithm design software.This project is supported by the Division of Chemistry in the Directorate for Mathematical and Physical Sciences, and by the Division of Molecular and Cellular Biosciences and the Plant Genome Research Program in the Directorate for Biological Sciences.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.