Functionalized DNA as glycan recognition molecules

项目摘要 在自然界中，所有细胞和大多数生物大分子都被糖修饰。这些糖的变化， 其被称为“聚糖”，调节和介导多种细胞过程。毫不奇怪， 异常聚糖结构与多种人类疾病有关。聚糖结构的研究 和功能-被称为糖组学领域-是一个新兴的领域，这将大大提高我们的 了解人类健康和疾病。糖组学面临的一个重大挑战是了解糖的结构。 在人类糖组内的巨大的聚糖决定簇库的功能， 估计有数千人这一挑战因缺乏容易获得的聚糖而变得更加复杂。 识别分子（GRM）的这些决定因素。正如蛋白质的高亲和性试剂一直是一种 对蛋白质组学的发展有布恩，获得GRM有望使研究人员能够测试 关于特定聚糖结构和功能的假设。人类糖组的GRM储存库是 极其有限。此外，许多GRM，特别是凝集素，已被证明与多聚糖结合 凝集素的结构和商业制剂已经证明在结合方面存在很大的不一致性， 亲和力这些问题刺激了开发替代选择性和高亲和力GRM的研究 超越传统GRM的性能。尽管他们的承诺，许多这些替代品， 包括抗体和核酸适体的技术存在几个缺点。在这 我们的建议旨在通过开发基于以下内容的新一类GRM来满足对强大GRM的迫切需求： 沿着稳定的ssDNA支架沿着显示化学功能性的异多价。利用我们最近 设计了一种T4 DNA连接酶介导的官能团沿着的序列特异性掺入方法 ssDNA支架，我们将开发一个体外进化系统，以确定GRM从大型库， 功能化的ssDNA（>10个成员）。在aim 1中，我们将限定一个高保真密码子用于体外 GRM的发展。在目标2中，我们将应用该平台发现功能化的ssDNA GRM， 识别不同的双触角聚糖结构。然后，我们将证明这种方法使 通过针对不同聚糖的阳性和阴性体外进化的迭代循环来控制聚糖特异性 目标的

PROJECT SUMMARY In nature, all cells and most biological macromolecules are decorated with sugars. These sugar modifications, which are referred to as “glycans”, modulate and mediate a wide array of cellular processes. Not surprisingly, aberrant glycan structures have been implicated in a variety of human diseases. The study of glycan structure and function – known as the field of glycomics – is a burgeoning field, which promises to greatly enhance our understanding of human health and disease. A significant challenge facing glycomics is to understand the function of the enormous repertoire of glycan determinants within the human glycome, which has been estimated to be many thousands. This challenge is compounded by the deficit of readily accessible glycan recognition molecules (GRMs) for these determinants. Just as high affinity reagents for proteins have been a boon to the development of proteomics, access to GRMs are anticipated to enable researchers to test hypotheses regarding specific glycan structure and function. The repository of GRMs for the human glycome is extremely limited. Furthermore, many GRMs, especially lectins, have been shown to bind to multiple glycan structures, and commercial preparations of lectins have demonstrated great inconsistencies in binding affinities. These issues have stimulated research in developing alternative selective and high-affinity GRMs that exceed the performance of traditional GRMs. Despite their promise, many of these alternative technologies, including antibodies and nucleic acid aptamers, suffer from several shortcomings. In this proposal we aim to address the critical need for robust GRMs by developing a new class of GRMs based on the heteromultivalent display of chemical functionality along a stable ssDNA scaffold. Using our recently devised method for T4 DNA ligase-mediated sequence-specific incorporation of functional groups along ssDNA scaffolds, we will develop an in vitro evolution system to identify GRMs from large libraries of functionalized ssDNA (>10¹² members). In aim 1 we will qualify a high-fidelity codon to be used during in vitro evolution of GRMs. In aim 2, we will apply this platform to the discovery of functionalized ssDNA GRMs that recognize different biantennary glycan structures. We will then demonstrate that this approach enables the control of glycan specificity by iterated rounds of positive and negative in vitro evolution against different glycan targets.