Technology to Create Spiegel ERAbodies on Demand: Biostable Universal Antibody Replacements

按需创建Spiegel ER抗体的技术：生物稳定性通用抗体 更换人员 应用分子进化基础 ELISABiondi 摘要 生物医学、诊断和临床领域的研究人员希望根据需要创造(或购买)试剂 与他们正在研究的生物过程中可能涉及的蛋白质和其他靶点结合。安提伯德- 工业工程师长期以来一直担任这一角色。然而，作为生物制品，抗体处于“不可再生性危机”的中心。 在生物医学研究方面，即使在合适的情况下，也需要几个月和数千美元的制造。这有 推动创造抗体替代品的努力，既有蛋白质(例如DARPins)，也有RNA(例如适配子)。第一 很难操纵，而第二种稳定性低，亲和力令人失望。 我们假设，一个非自然的平台具有“扩展的RNA字母表”(ERA)和额外的功能 具有额外约束潜力的团体将满足这一长期未得到满足的需求。而扩展的DNA字母表 现在都是先进的，第一个创新是时代还没有成为任何初步的目标 数据。我们假设纳米分子结合将被常规地实现，因为ER抗体将能够访问 为了(A)更高的信息密度，这将导致(B)通过使用RNA支架和通过 具有支持折叠的功能，(C)更大的结构多样性，为ERA提供更多模式 紧密结合，以及(D)更多的折叠基序，使ERAbs具有更紧密的结构。他们是 也假设具有经典适配子的所有优势，包括作为 后续几轮演进、使用信令实体的可修改性、低成本、快速周转和直接 化学合成。 这个R21项目将证明这个新技术平台的价值，它将允许研究人员订购或 直接在几周内为他们自己选择的目标创建活页夹。我们假设会有进一步的创新 通过将尚未探索的ERA技术与镜像对称的经典概念相结合。要创建 ERA在生物系统中是稳定的，我们将以镜像(“Spiegel”)的形式制造它们。 目标1.一个单一的R21演示项目将显示，ERAbs可以由积木制成 由钯化学产生。尽管该技术对于应用程序是不可知的(它可以创建 任何目标的粘合剂)，这一演示将通过使用超大医学靶向蛋白质来产生影响 意义，PD-L1(程序性死亡配体1)。我们将检验L-ERA试剂的假设，很像 标准的L核糖核酸对生命系统中的核糖核酸酶是稳定的。此工作流程步骤将耗时18个月。 我们将扩展ERA测序的方法，并对酶合成的保真度进行基准测试。在 工作流程，这将在前6个月内完成，作为音译模式的各种颠倒 转录酶将被用来定义适用于各种6字母和8字母ERA系统的测序程序。 目标指标是结合亲和力(纳摩尔)、结合特异性(100：1区分)和体外 血清稳定性(2小时内0.1%的核糖核酸酶降解)。

Technology to Create Spiegel ERAbodies on Demand: Biostable Universal Antibody Replacements Foundation for Applied Molecular Evolution Elisa Biondi ABSTRACT Researchers in biomedical, diagnostic, and clinical areas want to create (or buy), on demand, reagents that bind to proteins and other targets that may be involved in a biological process that they are studying. Antibod- ies have long served this role. However, as biologics, antibodies are at the center of an "irreproducibility crisis" in biomedical research, and, even when suitable, take months and thousands of dollars to make. This has driven efforts to create antibody replacements, both protein (e.g. Darpins) and RNA (e.g. aptamers). The first are difficult to manipulate, while the second have low stability and disappointing affinity. We hypothesize that an unnatural platform with an "Expanded RNA Alphabet" (ERA) and extra functional groups with extra binding potential will meet this long-standing unmet need. While expanded DNA alphabets are now advanced, a first innovation is that ERAs have not yet been the target of any preliminary data. We hypothesize that nanomolar binding will be routinely achieved because ERAbodies will have access to (a) higher information density that will lead to (b) better defined folds, both by using an RNA scaffold and by having functionality that supports folding, (c) greater structural diversity that gives ERAbodies more modes for tight binding, and (d) more folding motifs that allow ERAbodies to have more compact structures. They are also hypothesized to have all of the advantages of classical aptamers, including value as the starting points for subsequent rounds of evolution, modifiability using signaling entities, low cost, fast turnaround, and direct chemical synthesis. This R21 project will prove the value of this new technology platform, which will allow researchers to order or directly create in weeks, binders for targets that they themselves select. We hypothesize a further innovation by merging yet unexplored ERA technology with the classical concept of mirror symmetry. To create ERAbodies that are stable in biological systems, we will make these in mirror image ("Spiegel") form. Aim 1. A single R21 demonstration project will show that ERAbodies can be made with building blocks created by palladium chemistry. Although this technology is agnostic with respect to applications (it can create binders for any target), this demonstration will have impact by targeting a protein with outsized medical significance, PD-L1 (Programmed Death Ligand 1). We will test the hypothesis that L-ERA reagents, much like standard L-RNA, are stable against RNases in living systems. This workflow step will consume 18 months. We will expand methods to sequence ERA and benchmark the fidelity of enzymatic synthesis. In the workflow, this will be completed in the first 6 months, as patterns of transliteration by various reverse transcriptases will be used to define a sequencing procedure applicable to various 6- and 8-letter ERA systems. The target metrics are binding affinity (nanomolar), binding specificity (100:1 discrimination), and in vitro sera stability (<0.1% RNase degradation over 2 hours).