Development of functional and responsive nucleic acid systems for applications in biotechnology

开发用于生物技术应用的功能性和响应性核酸系统

• 批准号: RGPIN-2020-05043
• 负责人: OFlaherty, Derek
• 金额: $ 1.75万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748031
• 关键词: Development; functional; responsive; nucleic; acid

My research program aims at developing biotechnology using nucleic acid polymers. By introducing well-defined chemical modifications into DNA/RNA scaffolds, we will develop probes and tools to investigate biochemical processes ranging from redox reactions to genome repair pathways. This research area will advance our understanding of nucleic acids in ways well-beyond their widely known role in modern biology as information carriers. The proposed research aims to find chemical triggers to activate or silence functional nucleic acid materials. Much effort has been devoted to identifying nucleic acid sequences (such as DNA/RNA) with function such as catalysis (ribozymes) and molecular recognition (aptamers). Ribozymes catalyze various chemical transformations, which can be harnessed to control gene expression. On the other hand, aptamers are short single-stranded nucleic acid sequences that can selectively bind to a specific target (including proteins, peptides, and small molecules such as toxins). The proposed strategy involves modulating nucleic acid function via an external stimulus. Switching function on (or off) will be possible by embedding site-specific chemical modifications in the nucleic acid sequence, that intrinsically respond to specific stimuli. An oxidative desulfurization strategy will serve as an initial platform to this research program. Thiolated nucleotide units within RNA strands, such as 2-thiouridine or 2-thiocytidine (2sU / 2sC), are susceptible to oxidative desulfurization reactions with various oxidants. Interestingly, the desulfurized products have a dramatic shift in the base-pairing preferences; for instance, 2sU pairs more strongly with A compared to U, whereas U is more promiscuous in forming a pair with G (in addition to A). Desulfurization chemistry can thus be used to fine tune structure and functionality of RNA-based technology, such as ribozymes (DNAzymes), riboswitches and aptasensors. Given that the presence of 2sU (or 2sC) can disrupt critical base pairs necessary for RNA activity, a novel redox switching mechanism will be explored to either silence or activate functional RNA sequences. Thio modifications within RNA has precedence in the literature, making this avenue promising for in vivo applications. The approach outlined here will pave the way to utilizing redox-sensitive nucleic acid probes in a unique fashion. In addition to desulfurization, this program will be expanded to other types of biocompatible triggers. For instance, the introduction of certain alkyl groups within nucleic acid-based technologies can be corrected by cellular repair mechanisms. In doing so, a similar activation/silencing mechanism can be summoned. The proposed research will introduce an additional gate to modulate the specific function of defined nucleic acid sequences, and enhance the capabilities of current biotechnology.

我的研究项目旨在利用核酸聚合物开发生物技术。通过将定义明确的化学修饰引入DNA/RNA支架，我们将开发探针和工具来研究从氧化还原反应到基因组修复途径的生化过程。这一研究领域将推动我们对核酸的理解，远远超出其在现代生物学中作为信息载体的广为人知的作用。本研究旨在寻找激活或沉默功能性核酸物质的化学触发器。对具有催化（核酶）和分子识别（适体）等功能的核酸序列（如DNA/RNA）的鉴定已经投入了大量的努力。核酶催化各种化学转化，可以用来控制基因表达。另一方面，适体是短的单链核酸序列，可以选择性地与特定靶标（包括蛋白质，肽和小分子如毒素）结合。提出的策略包括通过外部刺激调节核酸功能。通过在核酸序列中嵌入特定位点的化学修饰，可以打开（或关闭）功能，这些修饰本质上对特定刺激作出反应。氧化脱硫策略将作为该研究计划的初始平台。RNA链内的硫代核苷酸单元，如2-硫脲或2-硫代胞苷（2sU / 2sC），容易与各种氧化剂发生氧化脱硫反应。有趣的是，脱硫产物的碱基配对偏好发生了巨大的变化；例如，与U相比，2sU与A的配对更强，而U与G（除了A）的配对更混杂。因此，脱硫化学可以用于微调基于rna的技术的结构和功能，例如核酶（DNAzymes），核开关和适配体传感器。鉴于2sU（或2sC）的存在会破坏RNA活性所必需的关键碱基对，研究人员将探索一种新的氧化还原开关机制，以沉默或激活功能性RNA序列。RNA内的硫代修饰在文献中具有优先权，使这一途径在体内应用前景广阔。本文概述的方法将为以独特的方式利用氧化还原敏感的核酸探针铺平道路。除脱硫外，该计划将扩展到其他类型的生物相容性触发器。例如，在以核酸为基础的技术中引入某些烷基可以通过细胞修复机制加以纠正。在此过程中，可以调用类似的激活/沉默机制。提出的研究将引入一个额外的门来调节已定义的核酸序列的特定功能，提高当前生物技术的能力。