Nucleic Acid Innovations to Manage Pathogen Sequence Divergence

管理病原体序列分歧的核酸创新

• 批准号: 9375498
• 负责人: STEVEN A BENNER
• 金额: $ 18.5万
• 依托单位: FOUNDATION FOR APPLIED MOLECULAR EVOLUTN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9375498
• 关键词: Accident and Emergency department; Arboviruses; Architecture; Authorization documentation; Benchmarking; Binding; Biological; Biological Assay; Bite; Centers for Disease Control and Prevention (U.S.); Chemicals; Chemistry; Clinical; Culicidae; DNA; DNA Structure; Data; Defect; Dengue; Detection; Development; Diagnosis; Diagnostic; Ebola virus; Emergency Situation; Enzymes; Evolution; Exanthema; FDA approved; Foundations; Frequencies; Hydrogen Bonding; Informatics; Information Systems; Investigation; Laboratories; Learning; Left; Length; Ligation; Measures; Medical; Methods; Molecular; Molecular Biology; Molecular Evolution; Molecular Structure; Morphologic artifacts; Mutation; Nested PCR; Nucleic Acids; Nucleotides; Outcome; Patients; Performance; Physicians' Offices; Polymerase; Population; Pregnant Women; Preparation; Purines; Pyrimidine; Pyrimidines; RNA; RNA Sequences; RNA Viruses; RNA-Directed DNA Polymerase; Reagent; Running; Sampling; Specificity; Structure; System; Technology; Temperature; Testing; Uncertainty; Urine; Variant; Viral; Viral Genome; Virus; Work; Yang; Zika Virus; base; chikungunya; cold temperature; design; experience; flexibility; genetic information; individual patient; innovation; molecular recognition; novel; nucleobase; pathogen; point of care; prevent; recombinase; respiratory virus; sugar; synthetic biology; tool; viral RNA

Nucleic Acid Innovations to Manage Pathogen Sequence Divergence Foundation for Applied Molecular Evolution Steven A. Benner Zunyi Yang ABSTRACT One of the most important outcomes from synthetic biology has been the recognition that many limitations of diagnostics tools that target the DNA and RNA (collectively xNA) arise from defects in the xNA molecular framework. These defects can be fixed by changing the structure of DNA used in these assays. The Benner lab has created over a dozen reagent, enzyme, and architecture innovations based on this recognition. These are now allowing assays to move from the clinical lab to emergency rooms to physicians offices. In this progression, one problem arising from the molecular biology of the RNA viruses has remained recalci- trant. The sequences of RNA viral genomes diverge rapidly. Thus, we do not know the exact sequence of the xNA molecules that we are trying to detect in any patient. With increasing frequency, this allows RNA viruses to escape primers and probes needed to detect their xNA. This, in turn, renders simple tests unable to detect a virus with certainty. When these are intended to diagnose individual patients, this all but prevents FDA approval under anything but emergency use authorizations. Further, this uncertainty in the target xNA makes medically informative variation difficult to detect amid the medically uninformative variation. This work will introduce NextGen “biversal” nucleic acid innovations to manage this problem. We begin with by noting that viral sequence divergence occurs under strong adaptive constraints with error biases intrinsic in relevant polymerases. Thus, transitions (purines replace purines, pyrimidines replace pyrimidines) are more common than transversions (purines replace pyrimidines, or vice versa). This means that we can manage viral genome divergence with pyrimidine biversals (Y) that pair to both A and G, and purine biversals (R) that pair to both C and T. Biversals have advantages over “universal bases”, which create primers so promiscuous that they get lost by binding to background xNA, abundant in real biological samples. These facts set up these aims. Aim 1. Add NextGen biversals to isothermal amplification architectures, which allow diagnostics kits to move closer to points-of-sampling and points-of-care. We will benchmark their performance relative to the performance of amplifications with all-standard primers, and quantiate specificity footprints. Aim 2. Characterize the details of how polymerases handle NextGen biversals, specifically, what standard nucleotides are placed opposite these biversals by polymerases used in low-temperature amplifications. Aim 3. Integrate NextGen biversals into architectures for SNP detection that exploit ligation and cleavage. Rich preliminary data allow this project to move directly to a "development" stage. The emergence of Zika, Ebola, chikungunya, dengue, and other RNA viral pathogens, and the soon-to-emerge Mayoro virus, points to the immediate significance of this project. Especially innovative in this project is its integrated investigation of alternative molecular structures, the enzymes called upon to handle them, and the architectures where they will be used. These will allow FDA approvable diagnostics products to move towards points of sampling.

管理病原体序列差异的核酸创新 应用分子进化基础 史蒂文·A·本纳 杨遵义 摘要 合成生物学最重要的成果之一是认识到许多限制 以DNA和RNA(统称XNA)为靶点的诊断工具的产生源于XNA分子中的缺陷 框架。这些缺陷可以通过改变这些检测中使用的DNA的结构来修复。本纳实验室 基于这一认识，已经创造了十几种试剂、酶和结构创新。这些是 现在允许化验从临床实验室转移到急诊室到医生办公室。 在这一进展中，RNA病毒的分子生物学产生的一个问题仍然存在。 川特。核糖核酸病毒基因组序列迅速分化。因此，我们不知道 我们试图在任何患者身上检测到的XNA分子。随着频率的增加，这使得RNA病毒 以逃避检测其XNA所需的引发物和探针。这进而导致简单的测试无法检测到 肯定会感染病毒。当这些计划用于诊断个别患者时，这几乎阻止了FDA 除紧急使用授权外，任何情况下的批准。此外，目标XNA中的这种不确定性使 医学上信息丰富的变异很难在医学上没有信息的变异中被发现。 这项工作将引入下一代“生物”核酸创新来管理这一问题。我们先从 通过注意到病毒序列分歧发生在具有内在误差偏向的强适应性约束下 相关聚合酶。因此，转变(嘌呤取代嘌呤，嘧啶取代嘧啶)更多 比颠倒更常见(嘌呤取代嘧啶，或相反)。这意味着我们可以管理病毒 与A和G配对的嘧啶生物体(Y)和配对的嘌呤生物体(R)的基因组差异 对于C和T.来说，通用碱基都比“全能碱基”更有优势，因为“全能碱基”会产生如此混杂的引物，以至于 它们通过与丰富的真实生物样本中的背景XNA结合而丢失。这些事实确立了这些目标。 目标1.将NextGen Biversals添加到等温放大体系结构中，从而允许诊断试剂盒移动 更接近采样点和护理点。我们将以他们的表现为基准 使用全标准引物的扩增性能，并量化特异性足迹。 目标2.描述聚合酶如何处理下一代生物的细节，具体是什么标准 核苷酸被低温扩增中使用的聚合酶放在与这些生物相反的位置。 目的3.将NextGen Biversals整合到利用连接和切割的SNP检测体系中。 丰富的初步数据使该项目能够直接进入“开发”阶段。寨卡病毒的出现， 埃博拉、基孔肯雅热、登革热和其他RNA病毒病原体，以及即将出现的马约罗病毒，指出 这个项目的现实意义。这个项目的创新之处在于它对 可供选择的分子结构，需要处理它们的酶，以及它们将 被利用。这些将允许FDA批准的诊断产品进入采样点。