Studies of Hematologic Malignancies by PNA Technology

利用 PNA 技术研究血液恶性肿瘤

• 批准号: 6682804
• 负责人: MAXIM D FRANK-KAMENETSKII
• 金额: $ 28.97万
• 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/6682804
• 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检测血液系统恶性肿瘤中游离型或基因组整合型肿瘤病毒DNA的稳健检测方法。考虑到病毒DNA的量有限，需要DNA诊断的高特异性和高灵敏度。为了达到高特异性，合成的DNA模拟物肽核酸（PNA）将用于在双链（ds）DNA靶标中序列选择性形成PD环。滚环扩增（RCA），一种强大的污染免疫等温方法，将提供高灵敏度。RCA固有的大的多重能力使得能够对不同的肿瘤病毒进行突变不敏感的平行检测。EBV和HHV-8疱疹病毒和HTLV-1逆转录病毒将是原型肿瘤病毒。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）。这些特征使得PD位点成为有前途的病毒标记物，这些标记物以前从未用于DNA诊断。待开发的测定法包括三个步骤。首先，寡核苷酸探针通过PD环与dsDNA杂交，随后进行探针环化。在病毒特异性PNA开放剂的辅助下，该步骤主要负责测定的总体特异性。其次，在单一温度下环状探针的RCA超扩增产生dsDNA产物，其中原始PD位点重复超过百万次。这一步骤将提供必要的灵敏度。最后，因此多重复制的PD位点将被PNA开放剂选择性地暴露，并用分子信标进行荧光检测。最后一步提供了方便的检测，并额外确保了测定的特异性。选择不同的PD位点作为标记物，结合多重RCA和多重信标，将确保几种特异性肿瘤病毒的可靠诊断。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理验证实验。目标是在初始优化后检测超过1 μ g人DNA的百倍摩尔过量的PD位点。这些研究将证明，附加型肿瘤病毒可以在急性血液感染的特点。在II期，将开发用于检测细胞系和不同病毒载量的临床样本中的肿瘤病毒的检测方法。将使用质粒模型优化选定的肿瘤病毒标志物PD位点的检测，以进一步提高对过量人DNA背景的灵敏度。将针对PD环的不同构型优化环形探针组装的产率和特异性。RCA将使用各种DNA聚合酶和扩增策略进行优化。最佳条件和建设的信标在其杂交动力学与PD网站将彻底搜索。因此，预计每个人类基因组检测不到一种肿瘤病毒，这将在肿瘤病毒感染细胞系的DNA样本上进行测试，最后在临床标本上进行测试。该项目的成功将产生一种荧光检测方法，用于淋巴瘤/白血病中肿瘤病毒的可靠和高度灵敏的等温诊断。它将允许通过检测非常低拷贝数的病毒DNA来对恶性样本进行傻瓜屋顶表征，如在前病毒的情况下。