Studies of Hematologic Malignancies by PNA Technology

该项目的目标是建立一种检测血液病恶性肿瘤外体或基因组整合癌病毒dna的可靠方法。考虑到病毒DNA数量有限，需要高特异性和高灵敏度的DNA诊断。为了达到高特异性，将使用合成的DNA模拟物肽核酸（PNA）在双链DNA靶标中序列选择性地形成pd环。滚动圈放大（RCA）是一种强大的污染免疫等温方法，具有很高的灵敏度。RCA固有的大复用能力使不同癌病毒的突变不敏感并行检测成为可能。EBV和HHV-8疱疹病毒和HTLV-1逆转录病毒将是原型癌病毒。它们的基因组具有独特且非常恒定的pd环形成位点（PD-sites）。这些特征使得pd -位点成为以前从未在DNA诊断中使用过的病毒标记物。待开发的测定方法包括三个步骤。首先，寡核苷酸探针通过pd环与dsDNA杂交，随后探针环状化。在病毒特异性PNA开启器的辅助下，这一步主要负责检测的总体特异性。其次，圆形探针在单一温度下的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敏感性。最后，复制的pd位点将被PNA打开剂选择性地暴露，并用分子信标进行荧光检测。最后一步提供了一种方便的检测，另外确保了分析的特异性。选择不同的pd位点作为标记，加上多重RCA和多色信标，将确保对几种特定癌病毒的可靠诊断。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，在超过1 μ g人类DNA的100倍摩尔过量中检测pd位点。这些研究将证明急性血液感染中偶发癌病毒的特征。在第二阶段，该检测方法将用于检测不同病毒载量的细胞系和临床样本中的癌病毒。将利用质粒模型优化选定的癌病毒标记PD-位点的检测，以进一步提高对过量人类DNA背景的敏感性。圆形探针组件的产率和特异性将针对不同的pd环配置进行优化。RCA将使用各种DNA聚合酶和扩增策略进行优化。根据其与pd位点的杂交动力学，将彻底搜索信标的最佳条件和结构。因此，预计每个人类基因组检测不到一种癌病毒，将对来自癌病毒感染细胞系的DNA样本进行测试，最后对临床标本进行测试。该项目的成功将产生一种荧光测定法，用于淋巴瘤/白血病中癌病毒的可靠和高度敏感的等温诊断。它将通过检测像原病毒一样的极少量拷贝的病毒DNA来对恶性样本进行愚弄。

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.