Expedite Enzymatic Assembly of Glycans via DNA (de)Hybridization-Enabled Catch-and-Release

通过 DNA（去）杂交捕获和释放加速聚糖的酶促组装

• 批准号: 10648697
• 负责人: Lei Li
• 金额: $ 23.4万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10648697
• 关键词: Affinity; Alkynes; Automation; Azides; Biological; Buffers; Carbohydrates; Cells; Chemicals; Chemistry; Complement; Complementary DNA; Complex; Consumption; DNA; Derivation procedure; Disease; Electrostatics; Ensure; Evaluation; Future; Glycoconjugates; High temperature of physical object; Human Milk; Immobilization; Length; Liquid substance; Medical; Methods; Monosaccharides; Nucleic Acids; Oligonucleotides; Oligosaccharides; Organic solvent product; Peptides; Phase; Planets; Plant Resins; Play; Polysaccharides; Procedures; Process; Reaction; Role; Route; Salts; Sepharose; Single-Stranded DNA; Sodium Chloride; Solid; Solvents; Structure; System; Technology; Thigh structure; Time; Vertebral column; Water; Work; base; chemical synthesis; cold temperature; functional group; glycosyltransferase; medical specialties; melting; oligo (dT); rapid technique; reaction rate; success; Affinity; Alkynes; Automation; Azides; Biological; Buffers; Carbohydrates; Cells; Chemicals; Chemistry; Complement; Complementary DNA; Complex; Consumption; DNA; Derivation procedure; Disease; Electrostatics; Ensure; Evaluation; Future; Glycoconjugates; High temperature of physical object; Human Milk; Immobilization; Length; Liquid substance; Medical; Methods; Monosaccharides; Nucleic Acids; Oligonucleotides; Oligosaccharides; Organic solvent product; Peptides; Phase; Planets; Plant Resins; Play; Polysaccharides; Procedures; Process; Reaction; Role; Route; Salts; Sepharose; Single-Stranded DNA; Sodium Chloride; Solid; Solvents; Structure; System; Technology; Thigh structure; Time; Vertebral column; Water; Work; base; chemical synthesis; cold temperature; functional group; glycosyltransferase; medical specialties; melting; oligo (dT); rapid technique; reaction rate; success

Project Summary Every living cell on the planet is covered by a dense layer of glycans. These complicated structures play critical roles in many biological and disease processes. Functional studies and medical applications require well-defined glycan structures. While automated peptide and nucleic acid syntheses have matured and allow non-specialists to access defined standards, the synthesis of glycans and glycoconjugates is still often laborious, time- consuming, and requires specialties. So far, only a few platforms have been introduced to automate chemical syntheses of glycans but suffer from slow reaction rates, low selectivity, efficiency, and yields, especially in preparing complex glycans. A mature and practical automated system to synthesize complex glycans is not available. In the past decade, glycosyltransferases (GTs)-catalyzed reactions have been widely explored to prepare diverse complex glycans. With perfect regio- and stereo-selectivity as well as high conversion rates, they are attractive for automation. The challenge is tedious repeated process of separating intermediates and the final product. This has been well overcome by solid-phase-based automation in peptide and nucleic acid synthesis. But GTs are often much less active when the acceptor substrate is immobilized, causing slow conversions and low yields. Catch-and-release strategies have the potential to solve the problem. In such strategy, acceptors are tagged with a functional group, and “captured” on solid phase through specific interactions between the group and the solid phase. After cleanup, they can be “released” using appropriate solvents to disrupt the interaction. Several “catch-and-release” strategies have been introduced to expedite enzymatic assembly of glycans, but all suffer from one major drawback: chemicals, organic solvents, high concentrations of salts, etc, must be introduced to release glycans from solid phase. Thus, extra steps which often cannot be easily realized on automated platforms have to be involved to clean up each intermediate for the next round reaction and catch-and-release separation. This will greatly complicate and prolong automated synthesis and decrease efficiency. We propose a simple catch-and-release strategy enabled by DNA hybridization (Catch) and dehybridization (Release). In this strategy, the glycan an conveniently “captured” with any GT reaction mixtures, and “released” using pure water. We believe this catch-and-release strategy is the missing puzzle to tackle practical automated glycan synthesis. Iterative enzymatic assembly is already widely employed to prepare complex glycans, and many liquid handling systems are commercially available. Upon the success of current technology, these can be readily integrated to generate a practical and costless automated platform for glycan synthesis (future work).

项目摘要 地球上的每个活细胞都被密集的聚糖覆盖。这些复杂的结构起着至关重要的作用 在许多生物和疾病过程中的作用。功能研究和医疗应用需要明确的 聚糖结构。虽然自动肽和核酸合成已经成熟并允许非专家 为了访问确定的标准，聚糖和糖缀合物的合成通常是实验室，时间 - 消费，需要专业。到目前为止，仅引入了几个平台以自动化化学 聚糖的合成，但反应速率缓慢，选择性低，效率和产量，尤其是在 准备复杂的聚糖。一个成熟且实用的自动化系统，以合成复杂的聚糖不是 可用的。在过去的十年中，已广泛探索了糖基转移酶（GTS）催化反应 准备多样化的复合物聚糖。具有完美的区域和立体视选择性以及高转换率， 它们对自动化很有吸引力。挑战是分离中间体的繁琐重复过程 最终产品。通过基于固相的肽和核酸自动化，这已经很好地克服了这一点 合成。但是，当受体底物固定时，GTS通常会降低活性，从而导致缓慢 转换和低收率。捕获和释放策略有可能解决问题。在这样 策略，受体用功能组标记，并通过特定的特定在固相上“捕获” 组与固相之间的相互作用。清理后，可以使用适当的 已经引入了几种“捕捞和释放”策略以加快 聚糖的酶促组装，但所有人都有一个主要缺点：化学物质，有机溶液，高 必须引入盐的浓度等，以从固相释放聚糖。那，额外的步骤 通常必须在自动化平台上轻松实现，以清理每个中间体的 下一轮反应和捕获和释放的分离。这将极大地复杂并延长自动化 合成和降低效率。我们提出了一个简单的DNA启用捕获和释放策略 杂交（捕获）和脱杂交（释放）。在这种策略中，聚糖可以方便地“捕获” 任何GT反应混合物，并使用纯水“释放”。我们认为，这种捕获和释放策略是 缺少难题来解决实际自动化的聚糖合成。迭代酶组装已经广泛 用于制备复杂的聚糖，并且许多液体处理系统都有商业上可用。在 当前技术的成功，这些技术可以很容易地集成以产生实用且无价的自动化 聚糖合成平台（未来工作）。