High-Performance Liquid Chromatography for DNA Structure Purification and Characterization

用于 DNA 结构纯化和表征的高效液相色谱

• 批准号: RTI-2022-00685
• 负责人: Sleiman, Hanadi
• 金额: $ 8.19万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=742982
• 关键词: Performance; Liquid; Chromatography; DNA; Structure

We urgently request a high-performance liquid chromatography (HPLC) instrument to replace our current HPLC system, which is 9 years old and has recently broken down. This has significantly slowed down the research of 20 HQP in my research group, as all the DNA conjugates that we synthesize require HPLC purification and characterization. The requested instrument will support our research program which focuses on developing DNA structures for biological applications. Specifically, this proposal introduces a new, highly scalable method to develop stimuli-responsive DNA hydrogels for tissue regeneration, antimicrobial and biosensing applications. We recently discovered that a small, inexpensive and non-toxic molecule, cyanuric acid, can induce DNA to assemble into a triple helix that grows into long nanofibers. We propose to chemically polymerize adenosine to form poly(adenine) DNA at scale, followed by the addition of cyanuric acid derivatives and crosslinking, to produce biocompatible hydrogels in a single pot with environmentally benign reagents and solvents. We will develop these hydrogels to promote cellular growth, and to respond to multiple biological stimuli through changes in mechanical and drug release properties. In developing the first small molecule-promoted DNA hydrogel, we expand the application space of biocompatible materials: our hydrogels are scalable, mechanically robust, stimuli-responsive, and recyclable, enabling a new, green methodology for the development of DNA hydrogel materials for widespread medical use. Our second project involves the development of sequence-controlled polymers as carriers of nucleic acid therapeutics. We aim to overcome important barriers that prevent their translation to the clinic, such as poor cellular uptake, and entrapment in organelles called endosomes. DNA strands conjugated to sequence-controlled polymers assemble in aqueous media to form monodisperse spherical structures, with a hydrophobic core and a dense nucleic acid corona (spherical nucleic acids, SNAs). We propose to develop these structures are highly effective delivery vehicles, where their nucleic acid exterior silences the expression of disease-causing proteins even in naked form, and can efficiently escape endosomal entrapment. We will also perform high-throughput cell selection studies, using a new class of DNA-encoded polymers developed in our lab. The objective is to select polymers that mediate the escape of nucleic acid therapies from endosomes, and that allow them to operate in the cell cytosol. Overall, the requested HPLC will ensure that productivity in our laboratory is not slowed down, and it will accelerate the discovery of new DNA biomaterials for tissue regeneration and for gene therapy. It will help train HQPs in nucleic acid research, an area that is critically important to ensure Canada's preparedness to any future pandemic.

我们迫切需要一台高效液相色谱仪，以取代我们目前使用了9年、最近出现故障的高效液相色谱系统。这大大减缓了我的研究小组对20个HQP的研究，因为我们合成的所有DNA缀合物都需要HPLC纯化和表征。所要求的仪器将支持我们的研究计划，重点是开发生物应用的DNA结构。 具体而言，该提案引入了一种新的，高度可扩展的方法来开发用于组织再生，抗菌和生物传感应用的刺激响应性DNA水凝胶。我们最近发现，一种小的，廉价的，无毒的分子，氰尿酸，可以诱导DNA组装成一个三螺旋，生长成长的纳米纤维。我们建议化学修饰腺苷以形成大规模的聚（腺嘌呤）DNA，然后加入氰尿酸衍生物和交联，在一个锅中用环境友好的试剂和溶剂生产生物相容性水凝胶。我们将开发这些水凝胶来促进细胞生长，并通过机械和药物释放特性的变化来响应多种生物刺激。在开发第一个小分子促进的DNA水凝胶时，我们扩展了生物相容性材料的应用空间：我们的水凝胶具有可扩展性、机械稳定性、刺激响应性和可回收性，为开发广泛医疗用途的DNA水凝胶材料提供了一种新的绿色方法。 我们的第二个项目涉及开发序列控制聚合物作为核酸治疗剂的载体。我们的目标是克服阻止其翻译到临床的重要障碍，如细胞吸收不良，以及在称为内体的细胞器中的捕获。与序列控制聚合物缀合的DNA链在水性介质中组装以形成单分散球形结构，具有疏水核心和致密核酸冠（球形核酸，SNA）。我们建议开发这些结构是非常有效的运载工具，其中它们的核酸外部沉默致病蛋白的表达，即使在裸露的形式，并可以有效地逃脱内体截留。我们还将使用我们实验室开发的一类新的DNA编码聚合物进行高通量细胞选择研究。目的是选择介导核酸疗法从内体逃逸并允许它们在细胞胞质溶胶中操作的聚合物。总的来说，所要求的HPLC将确保我们实验室的生产率不会降低，并将加速发现用于组织再生和基因治疗的新DNA生物材料。它将帮助培训核酸研究方面的HQP，这一领域对于确保加拿大对未来任何大流行病的准备至关重要。