Development of new technologies for high-sensitivity detection of a B-cell tumor marker in DNA

迫切需要开发廉价、安全和准确的癌症治疗工具 监视系统。被诊断为癌症的患者需要准确地评估肿瘤负担 在他们的治疗过程中，对肿瘤监测的需求对 许多惰性B细胞恶性肿瘤，如滤泡性淋巴瘤和慢性淋巴细胞白血病。 目前的成像技术，如超声波、计算机断层扫描和正电子 发射断层扫描昂贵，可靠性有限，并使患者暴露在过量的辐射中 辐射。我们将通过创建一种针对已知生物标记物的台式检测来帮助满足这一需求 许多B细胞癌症，最终应该会导致一种试剂盒的开发，这种试剂盒可以用来 检测循环肿瘤DNA(CtDNA)中该标志物的存在。这个生物标记物的水平， 尿嘧啶在正常人类细胞的基因组中含量很低，但在人的DNA中却显著升高 大多数B细胞淋巴瘤和白血病。该项目的长期目标是将这一点应用于 B细胞癌患者血浆ctDNA尿嘧啶检测技术的研究 监控工具。在第一个目标中，我们将提高基于点击化学的技术的灵敏度 通过合成和测试与碱性位点反应的开启的荧光标记来量化尿嘧啶 DNA这些新型探针的使用将省去目前检测中的许多步骤，增加 DNA的产量和大大降低了背景信号。各种令人兴奋的化学物质包括 可用，我们将使用已知开/关荧光强度比的点击化学对 &gt；&gt；100。在第二个目标中，我们将从耻垢分枝杆菌中设计出一种新的蛋白质UdgX，它 DNA中尿嘧啶的特异性和共价性连接，作为这一稀有碱基的探针。这种蛋白质 将与标志标签和荧光蛋白融合，并用于直接标记含有尿嘧啶的 DNA利用抗FLAG可以进一步增强蛋白质-DNA复合体的荧光信号 抗体通过酶联免疫吸附试验或酪胺化学结合到适当的荧光标记上。极限 检测这两种技术的可能性将使用含有尿嘧啶和 尿嘧啶含量高的B细胞淋巴瘤来源细胞系的基因组DNA。这个项目的总体目标是 项目是将目前尿嘧啶检测方法的灵敏度提高10%到50%，这应该会 使我们能够在大多数B-NHL患者的血浆中检测尿嘧啶并最终发展成一种 检测试剂盒，将有助于通过常规抽血监测B细胞癌患者。

There is a critical need for the development of cheap, safe, and accurate tools for cancer surveillance. Patients diagnosed with cancer require an accurate assessment of tumor burden through the course of their treatment, and the need for tumor surveillance is even more acute for many indolent B-cell malignancies such as follicular lymphoma and chronic lymphocytic leukemia. Current imaging techniques such as ultrasound sonography, computed tomography and positron emission tomography are expensive, have limited reliability and expose patients to excessive radiation. We will help satisfy this need by creating a bench-top assay for a known biomarker for many B-cell cancers that should eventually lead to the development of a kit that may be used to detect the presence of this marker in circulating tumor DNA (ctDNA). The levels of this biomarker, uracil, are low in the genomes of normal human cells, but are greatly elevated in the DNA of a majority of B-cell lymphomas and leukemias. The long-term goal of this project is to apply this technology to the detection of uracils in ctDNA in blood plasma of B-cell cancer patients as a tumor surveillance tool. In the first aim, we will increase the sensitivity of a click chemistry-based technology to quantify uracils by synthesizing and testing turn-on fluorescent tags that react with abasic sites in DNA. The use of these novel probes will eliminate many steps in the current assays, increase the yield of DNA and drastically lower the background signal. A variety of turn-on chemistries are available and we will use a click chemistry pair that has known on/off fluorescence intensity ratio of >>100. In the second aim, we will engineer a novel protein from M. smegmatis, UdgX, which specifically and covalently links at sites of uracils in DNA, as a probe for this rare base. This protein will be fused to a FLAG tag and a fluorescent protein, and used to directly label uracil-containing DNA. The fluorescence signal of the protein-DNA complex may be enhanced further using anti-FLAG antibodies conjugated to appropriate fluorescent tags, through ELISA or tyramide chemistry. The limit of detection of both the techniques will be determined using synthetic DNA containing uracils and genomic DNA from B-cell lymphoma-derived cell lines with high uracil content. The overall goal of this project is to increase the sensitivity of current uracil detection methods 10- to 50-fold, which should allow us to detect uracils in the blood plasma of a majority of B-NHL patients and eventual develop an assay kit that will be useful to monitor B-cell cancer patients through a routine blood draw.