Exploration of Linking Chemistry in the Design of Aptamer-Molecularly Imprinted Polymer Hybrids (aptaMIPs)

分子印迹包括在聚合物中制造一个结合口袋，该口袋在化学上和形状上都是特定于目标化合物的。与抗体和酶等生物分子识别元件相比，这些“智能塑料”提供了健壮性。他们也有在极端环境条件下工作的能力。然而，它们有时可能缺乏必要的特异性/亲和力。适配子是一小段DNA/RNA，具有靶向蛋白质和小分子的能力，并以高度的特异性和亲和力与它们结合。它们无毒，是有吸引力的抗体替代品。它们主要用于研究，因为它们对酶和化学降解很敏感，尽管这种情况正在慢慢改变，它们正在变得具有商业意义。到2020年，全球适配子市场预计将达到24亿美元，高于2015年的11亿美元。一项为期12个月的概念验证研究由EPSRC支持，由PI(分子印迹专家)领导，通过将适配子掺入分子印迹聚合物(MIP)来制造新型杂化材料。简单地说，适体结构被修改，使其能够直接结合到聚合物中，因此它将保持其形状，同时不受环境条件的影响。新的、高亲和力和稳定的材料被创造出来。这些“aptaMIP”表现出卓越的分子识别能力，并且在稳定性和特异性靶标识别方面比MIP和适配子都有显著的改进，有效地保持了这两类材料的最佳性能。这项提议寻求通过对用于创造这些新材料的核心化学的两年研究来探索aptaMIP的潜力。我们将以初步研究的结果为基础，创造有用、有效、具有高商业潜力的材料。这项建议的研究将集中在：(I)确定正确的连接物化学；(Ii)为所有四个碱基开发可聚合的修饰；(Iii)确定需要多少连接物；(Iv)确定这些连接物的最佳位置。对这四点的深入研究将使我们能够充分了解适配子如何将自己结合到聚合物中的关键化学，并通过这一点，使我们能够理解什么是好的aptaMIP以及为什么。此外，还将分析所使用的合成策略，以确保创建这些杂交物是简单和有效的。已选择两个目标来研究这些化学成分。它们在大小和应用上有所不同：一种蛋白质和一种生物活性药物，但这两种靶点都具有巨大的商业潜力。通过这些模型系统，我们旨在展示aptaMIP材料的有效性和潜力。在PI旁边，两个项目合作伙伴组成了研究团队：Watts小组是先导研究的合作者，总部设在马萨诸塞大学RNA治疗研究所(世界领先的新型适配子合成学校)。他们将通过获得最先进的合成设备，结合低聚物合成和应用方面的技术诀窍来支持这项提议。适配子集团是一家总部位于约克的商业适配子开发公司。他们的专业知识将通过提供已知的低聚物序列使该项目受益，这些序列将作为我们研究和获得专业仪器的基础。该项目的影响将得到他们对适配子领域和商业前景的详细了解的支持。整个团队的经验将使这项涵盖聚合物、核酸、蛋白质和分析化学领域的跨学科提案获得成功。我们将把aptaMIP从现有的概念验证阶段开发出来，并将它们及其合成工艺开发成人工分子识别领域的竞争对手，准备在可以利用其功能的系统中应用。

Molecular imprinting involves making a binding pocket in a polymer which is chemically and shape specific for the target compound. These "smart plastics" offer robustness compared to biological molecular recognition elements such as antibodies and enzymes. They also have the ability to work in extreme environmental conditions. However, they can sometimes lack the necessary specificity/affinity. Aptamers are small pieces of DNA/RNA that have the ability to target proteins and small molecules and bind to them with high specificity and affinity. They are not toxic and are attractive alternatives to antibodies. They have been used primarily in research due to their susceptibility to enzymatic and chemical degradation, though this is slowly changing and they are becoming commercially relevant. The global aptamers market is projected to reach $2.4 billion by 2020, up from $1.1 billion in 2015.A 12-month proof-of-concept study, supported by the EPSRC and led by the PI (a molecular imprinting specialist), created novel hybrid materials made by incorporating aptamers into molecularly imprinted polymers (MIPs). In simple terms, the aptamer structure is modified to allow it to be directly incorporated into a polymer, so it will hold its shape while being protected from environmental conditions. Novel, high affinity and stable materials were created.These "aptaMIPs" demonstrated exceptional molecular recognition and offer significant improvements on both MIPs and aptamers in terms of stability, and specific target recognition, effectively maintaining the best properties of both classes of materials. This proposal seeks to explore the potential of aptaMIPs through a two year study into the core chemistry used to create these novel materials. We will build on the results of the pilot study and create useful, effective materials with high commercial potential.The research in this proposal will focus on: (i) Identifying the right linker chemistry;(ii) Developing polymerisable modifications for all four bases;(iii) Identifying how many linkers are needed;(iv) Identifying the best position for these linkers. An in-depth study on these four points will enable a full understanding of the key chemistry of how the aptamer incorporates itself into the polymer and, through this, allow us to understand what makes a good aptaMIP and why. Alongside these the synthetic strategies used will be analysed to ensure the creation of these hybrids is simple and effective.Two targets have been selected to study these chemistries. These differ in size and application: a protein and a bioactive drug, but both targets have significant commercial potential. Through these model systems we aim to demonstrate the validity and potential of aptaMIP materials.Alongside the PI, two project partners form the research team:The Watts group were collaborators on the pilot study and are based at the University of Massachusetts RNA Therapeutics Institute (a world leading school in novel aptamer synthesis). They will support the proposal through access to state-of-the-art synthesis equipment, combined with know-how in oligomer synthesis and application.Aptamer Group are a commercial aptamer development company based in York. Their expertise will benefit the project by providing the known oligomer sequences which will act as the basis for our studies and access to specialised instrumentation. The impact of the project will be supported by their detailed knowledge of the aptamer field and commercial outlook.The experience of the whole team will allow this interdisciplinary proposal, covering the fields of polymer, nucleic acid, protein and analytical chemistries to succeed. We will take aptaMIPs from the existing proof-of-concept stage and develop them, and their synthetic process, into viable competitors in artificial molecular recognition, ready for application in systems where their functionality can be exploited.