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
基本信息
- 批准号:10648697
- 负责人:
- 金额:$ 23.4万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AffinityAlkynesAutomationAzidesBiologicalBuffersCarbohydratesCellsChemicalsChemistryComplementComplementary DNAComplexConsumptionDNADerivation procedureDiseaseElectrostaticsEnsureEvaluationFutureGlycoconjugatesHigh temperature of physical objectHuman MilkImmobilizationLengthLiquid substanceMedicalMethodsMonosaccharidesNucleic AcidsOligonucleotidesOligosaccharidesOrganic solvent productPeptidesPhasePlanetsPlant ResinsPlayPolysaccharidesProceduresProcessReactionRoleRouteSaltsSepharoseSingle-Stranded DNASodium ChlorideSolidSolventsStructureSystemTechnologyThigh structureTimeVertebral columnWaterWorkbasechemical synthesiscold temperaturefunctional groupglycosyltransferasemedical specialtiesmeltingoligo (dT)rapid techniquereaction ratesuccess
项目摘要
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).
项目概要
地球上的每个活细胞都被一层致密的聚糖覆盖,这些复杂的结构至关重要。
在许多生物和疾病过程中的作用需要明确定义。
聚糖结构。虽然自动化肽和核酸合成已经成熟并且允许非专业人士进行。
为了达到规定的标准,聚糖和糖复合物的合成通常仍然很费力、耗时
消耗,并且需要专业知识,到目前为止,只有少数平台被引入自动化化学。
聚糖的合成,但存在反应速率慢、选择性、效率和产率低的问题,特别是在
制备复杂聚糖还没有成熟实用的自动化系统来合成复杂聚糖。
在过去的十年中,糖基转移酶(GT)催化的反应已被广泛探索。
制备多种复杂聚糖,具有完美的区域和立体选择性以及高转化率,
它们对自动化很有吸引力,挑战是分离中间体和中间体的繁琐的重复过程。
最终产品中基于固相的肽和核酸自动化技术已经很好地克服了这一问题。
但当受体底物固定时,GT 的活性通常要低得多,导致合成缓慢。
转化率和低产量策略有可能解决这个问题。
策略中,受体被标记为官能团,并通过特定的方法“捕获”在固相上
清理后,可以使用适当的方法“释放”基团和固相之间的相互作用。
已经引入了几种破坏相互作用的溶剂。
聚糖的酶促组装,但都存在一个主要缺点:化学品、有机溶剂、高浓度
必须引入一定浓度的盐等以从固相中释放聚糖,因此需要额外的步骤。
通常在自动化平台上不容易实现,必须参与清理每个中间体
下一轮反应和捕获和释放分离这将大大复杂化并延长自动化时间。
我们提出了一种由 DNA 实现的简单捕获和释放策略。
杂交(捕获)和去杂交(释放)在此策略中,可以方便地“捕获”聚糖。
任何 GT 混合物反应,并使用纯水“释放”,我们相信这种捕获和释放策略是最有效的。
缺少解决实际自动化聚糖合成的难题 迭代酶促组装已被广泛使用。
用于制备复杂的聚糖,并且许多液体处理系统是可商购的。
当前技术的成功,这些可以很容易地集成以生成实用且无成本的自动化
聚糖合成平台(未来的工作)。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('Lei Li', 18)}}的其他基金
Center for the Investigation of Factor VIII Inhibitors and Glycosylation
因子 VIII 抑制剂和糖基化研究中心
- 批准号:
10406318 - 财政年份:2018
- 资助金额:
$ 23.4万 - 项目类别:
Center for the Investigation of Factor VIII Inhibitors and Glycosylation
因子 VIII 抑制剂和糖基化研究中心
- 批准号:
10227911 - 财政年份:2018
- 资助金额:
$ 23.4万 - 项目类别:
Facile Synthesis of O-Glycans and O-Glycopeptides
O-聚糖和 O-糖肽的简便合成
- 批准号:
8985647 - 财政年份:2015
- 资助金额:
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Battling AIDS via Mechanistic Understanding of the tRNA Phe modification enzyme T
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8229462 - 财政年份:2012
- 资助金额:
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