Studies of Hematologic Malignancies by PNA Technology

利用 PNA 技术研究血液恶性肿瘤

• 批准号: 6664334
• 负责人: MAXIM D FRANK-KAMENETSKII
• 金额: $ 28.14万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6664334
• 关键词: Epstein Barr virus; blood /lymphatic neoplasm; communicable disease diagnosis; diagnosis design /evaluation; gel mobility shift assay; human T cell lymphotropic virus type 1; human herpesvirus 8; human immunodeficiency virus 1; human tissue; neoplasm /cancer diagnosis; nucleic acid probes; oligonucleotides; peptide nucleic acids; plasmids; provirus; ultraviolet spectrometry; virus DNA; virus related neoplasm /cancer

The project goal is a robust assay for detecting episomal or genome- integrated oncoviral DNAs in hematologic malignancies. Considering a limited amount of viral DNA, both high specificity and high sensitivity of DNA diagnostics are required. To reach high specificity, a synthetic DNA mimic, peptide nucleic acid (PNA), will be used for sequence- selective formation of PD-loops in double-stranded (ds) DNA targets. High sensitivity will be provided by rolling-circle amplification (RCA), a powerful contamination-immune isothermal method. Large multiplexing capacity intrinsic in RCA enables mutation-insensitive parallel detection of different oncoviruses. EBV and HHV-8 herpesviruses and HTLV-1 retrovirus will be prototype oncoviruses. Their genomes have PD-loop forming sites (PD-sites) that are unique and very constant. These features make PD-sites promising viral markers, which have never been used before in DNA diagnostics. The assay to be developed includes three steps. First, an oligonucleotide probe hybridizes to dsDNA through PD-loops with subsequent probe circularization. This step, assisted by viral-specific PNA openers, is mostly responsible for the overall specificity of the assay. Secondly, the RCA hyperamplification of circular probes at a single temperature yields the dsDNA product in which the original PD-site repeats more than million times. This step will provide requisite sensitivity. Finally, thus multiply copied PD-sites will be selectively exposed by PNA openers and fluorescently detected with molecular beacons. The last step provides a convenient detection and additionally secures the specificity of the assay. Choice of various PD-sites as markers, together with multiplex RCA and multicolor beacons, will ensure reliable diagnosis of several specific oncoviruses. In Phase I, proof-of-principle experiments will be performed on a model system comparable to Phase II samples. The goal is to detect, after initial optimization, the PD-site in a hundred-fold molar excess over 1 mu g of human DNA. These studies will prove that episomal oncoviruses can be characterized in acute blood infections. In Phase II, the assay will be developed for detecting oncoviruses in cell lines and clinical samples with different viral loads. Detection of selected oncoviral marker PD- sites will be optimized using plasmid models to further increase the sensitivity on the excessive human DNA background. The yield and the specificity of the circular probe assembly will be optimized for different configurations of PD-loops. RCA will be optimized using various DNA polymerases and amplification strategies. Optimal conditions and constructions of beacons in terms of their hybridization kinetics with PD-sites will be thoroughly searched. As a result, detection of less than one oncovirus per human genome is projected, which will be tested on DNA samples from oncoviral-infected cell lines and, finally, on clinical specimens. The success in the project will yield a fluorescent assay for a reliable and highly sensitive isothermal diagnosis of oncoviruses in lymphomas/leukemias. It will allow fool roof characterization of malignant samples by detecting viral DNA in a very low number of copies like in case of provirus.

该项目的目标是建立一种稳健的检测方法，用于检测血液系统恶性肿瘤中的上体或基因组整合的肿瘤病毒DNA。考虑到病毒DNA的数量有限，要求DNA诊断既有高特异性又有高灵敏度。为了达到高特异性，一种合成的DNA模拟物--肽核酸(PNA)将用于双链(DS)DNA靶标中PD-环的序列选择性形成。滚环扩增(RCA)将提供高灵敏度，这是一种强大的免污染等温法。RCA固有的大的多路传输能力使得对不同的肿瘤病毒的突变不敏感的并行检测成为可能。EBV、HHV-8疱疹病毒和HTLV-1逆转录病毒将是肿瘤病毒的原型。它们的基因组具有独特且非常恒定的PD环形成位点(PD位点)。这些特征使PD-Site有希望成为以前从未在DNA诊断中使用过的病毒标记。待开发的分析方法包括三个步骤。首先，寡核苷酸探针通过PD-环与dsDNA杂交，随后的探针环化。这一步骤在病毒特异性PNA开放剂的辅助下，主要负责检测的总体特异性。其次，在同一温度下对环状探针进行RCA扩增，可得到原始Pd位点重复百万次以上的dsDNA产物。这一步骤将提供必要的敏感性。最后，这样复制的多个Pd位点将被PNA开放剂选择性地曝光，并用分子信标进行荧光检测。最后一步提供了一种方便的检测方法，另外还确保了检测的特异性。选择不同的PD位点作为标记，再加上多重RCA和多色信标，将确保对几种特定的肿瘤病毒进行可靠的诊断。在第一阶段，将在与第二阶段样品相当的模型系统上进行原则证明实验。目标是在初步优化后，在超过1微克人类DNA的100倍摩尔中检测Pd位点。这些研究将证明，附体肿瘤病毒可以在急性血液感染中表现出特征。在第二阶段，该方法将用于检测不同病毒载量的细胞系和临床样本中的肿瘤病毒。对选定的肿瘤病毒标志物PD位点的检测将使用质粒模型进行优化，以进一步提高对过量人类DNA背景的敏感性。圆形探头组件的成品率和专属性将针对不同配置的PD环进行优化。RCA将使用各种DNA聚合酶和扩增策略进行优化。根据信标与Pd位点的杂交动力学，将彻底寻找最佳条件和信标结构。因此，预计每个人类基因组将检测到不到一种肿瘤病毒，这将在肿瘤病毒感染细胞系的DNA样本上进行测试，最后在临床样本上进行测试。该项目的成功将产生一种用于淋巴瘤/白血病中肿瘤病毒的可靠和高度敏感的等温诊断的荧光分析方法。它将允许通过检测拷贝数非常少的病毒DNA来描述恶性样本的愚蠢屋顶，就像前病毒的情况一样。