Improved HIV Assays by Combining Four Innovations in Nucleic Acid Chemistry

描述(申请人提供)：2010年，Benner团队宣布开发了四项与在复杂生物样本中检测人类免疫缺陷病毒(HIV)的工具相关的创新：(A)一种人工扩展的遗传信息系统(Aegis)，支持“六核苷酸聚合酶链式反应”，允许独立扩增少量HIV RNA，而不受环境中其他DNA的干扰。(B)一种自避免分子识别系统(SAMRS)，在DNA探测、启动和多重PCR扩增中支持基本上无限制的多重。(C)将标准DNA转化为含有宙斯盾的DNA的程序，支持下游正交捕获，使DNA靶向分析具有灵活性和适应性，可能允许将新靶点添加到多路分析试剂盒中，而不需要对已靶向试剂盒的部分进行返工。(D)可逆终止子，作为三磷酸盐，被假设为允许检测和相对定量变异的艾滋病毒序列。我们假设，通过结合这些创新，我们可以改进艾滋病毒诊断工具，扩大它们的能力，以在更复杂的生物环境中检测更少的病毒粒子，具有更大的动态范围和更大的亚型特异性，以及更多的多路复用。此外，这些技术应该提供灵活性；这应该是可能的 要快速添加功能以检测新的变种、同时感染的病原体，甚至识别 在诊断艾滋病毒感染的过程中，艾滋病毒基因组中特定位置以前未知的变异。为了验证这一假设，我们将进行一系列分阶段的分析开发，将这些创新逐一加入到艾滋病毒靶标序列检测中日益具有挑战性的问题中，从在相对简单的环境中对单个HIV靶标进行单链检测开始，随着我们降低靶标分子的数量、增加多路复用水平并使环境变得更加复杂而增加创新。在每个阶段，我们将驱动系统发生故障，并记录系统发生故障的参数(敏感度、复杂性、多路复用级)。这些定义了一个“参数空间”，它为进度提供了一个衡量标准。该项目还将以可交付套件的形式提供引子、探针和检测捕获珠，以供有兴趣进行基准测试或使用它们的艾滋病毒研究人员使用。尽管Benner实验室的技术支持分支DNA(BDNA)3.0工具，该工具目前广泛用于测量HIV病毒载量，但这是Benner实验室首次为艾滋病研究寻求资金。因此，这项工作的另一个目标将是允许Benner实验室的创新更广泛地用于解决NIAID的许多与艾滋病毒相关的问题。这将有助于NIAID实现国家艾滋病毒/艾滋病战略确立的目标，即到2015年将美国对艾滋病毒状况的知晓率从79%提高到90%。 公共卫生相关性：准确的艾滋病毒诊断检测继续构成挑战，在110万艾滋病毒感染者中，近20%的人没有意识到自己的感染。去年，本纳实验室在DNA/RNA化学方面开发了四项创新，这些创新被认为能够提高灵敏度，增强低成本多路复用，并在复杂的生物混合物中检测新出现的艾滋病毒变种。这项提案寻求资金来检验这些假设，并提供实验室准备好的混合物，以支持对这些混合物中艾滋病毒及其变种的分析。

DESCRIPTION (provided by applicant): In 2010, the Benner group announced the development of four innovations relevant to tools to detect human immunodeficiency virus (HIV) in complex biological samples: (a) An artificially expanded genetic information systems (AEGIS) that supports "six nucleotide PCR", allowing independent amplification of small amounts of HIV RNA without interference from other DNA in the environment. (b) A self-avoiding molecular recognition system (SAMRS) that supports essentially unlimited multiplexing in DNA probing, priming, and multiplexed PCR amplification. (c) Procedures that convert standard DNA into AEGIS-containing DNA, supporting downstream orthogonal capture that allows DNA-targeted assays to be flexible and adaptive, possibly allowing new targets to be added to a multiplexed assay kit without demanding a reworking of the parts of that kit already targeted. (d) Reversible terminators that, as triphosphates, are hypothesized to allow detection and relative quantitation of variant HIV sequences. We hypothesize that by combining these innovations, we can improve HIV diagnostics tools, expanding their power to detect fewer virions in more complex biological environments with greater dynamic range and greater subtype specificity, together greater multiplexing. Further, these technologies should deliver flexibility; it should be possible to rapidly add capabilities to detect new variants, co-incident infectious agents, or even identify previously unknown variants at specific sites in the HIV genome in the course of diagnosing HIV infections. To test this hypothesis, we will perform a staged series of assay development, adding each of these innovations in series to increasingly challenging problems in the detection of HIV target sequences, starting with singleplexed detection of single HIV targets in relatively simple environments, adding innovations as we lower the amount of target molecules, increase the level of multiplexing, and make the environment more complex. At each stage, we will drive the system to fail, and note the parameters (sensitivity, complexity, multiplexing level) at which the system fails. These define a "parameter space" which provides a metric for progress. This project will also make available as deliverables kits of primers, probes, and detection capture beads, to be provided HIV researchers interested in benchmarking or using them. Although technology from the Benner laboratory stands behind the branched DNA (bDNA) 3.0 tool now widely used to measure HIV viral load, this is the first time that the Benner laboratory has sought funding for AIDS research. Thus, a further goal of this work will be to allow innovations from the Benner laboratory to be more widely used to solve the many HIV-related problems at the NIAID. This will help the NIAID help meet the goal established by the National HIV/AIDS Strategy of increasing the awareness of HIV status from 79% to 90% by 2015 in the US. PUBLIC HEALTH RELEVANCE: Accurate HIV diagnostic testing continues to pose challenges, and nearly 20% of the 1.1 million individuals infected with HIV are unaware of their infection. Last year, the Benner laboratory developed four innovations in DNA/RNA chemistry that are hypothesized to be able to improve sensitivity, enhance low cost multiplexing, and detect emerging variants of HIV in complex biological mixtures. This proposal seeks funding to test those hypotheses, and to deliver laboratory-ready mixtures to support analysis of HIV and its variants in those mixtures