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

开发高灵敏度检测 DNA 中 B 细胞肿瘤标志物的新技术

• 批准号: 10217428
• 负责人: ASHOK S BHAGWAT
• 金额: $ 36.55万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-08 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217428
• 关键词: Acute; Aldehydes; B lymphoid malignancy; B-Cell Lymphomas; B-Cell Neoplasm; B-Cell NonHodgkins Lymphoma; B-Lymphocytes; BODIPY; Binding Proteins; Biochemical; Biological Assay; Biological Markers; Biopsy; Blood; Blood Volume; Boron; Cancer Patient; Cell Line; Cells; Chemicals; Chemistry; Chronic Lymphocytic Leukemia; Clinical; Complex; Cyclooctenes; Cytosine; DNA; Detection; Development; Diagnosis; Diagnostic Procedure; Drops; Engineering; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Financial Hardship; Fluorescence; Follicular Lymphoma; Gene Frequency; Genome; Genomic DNA; Goals; Health; Homologous Gene; Human; Human Resources; Image; Imaging Techniques; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Indolent; Knowledge; Label; Laboratories; Lead; Link; Malignant Neoplasms; Methods; Microbiology; Monitor; Mutation; Mycobacterium smegmatis; Non-Hodgkin' s Lymphoma; Patients; Plasma; Positron-Emission Tomography; Problem Solving; Proteins; RNA; Radiation; Residual Neoplasm; Risk; Sampling; Side; Signal Transduction; Signaling Protein; Site; Somatic Mutation; Structure of germinal center of lymph node; Surveillance Methods; Techniques; Technology; Testing; Tissues; Tumor Burden; Tumor Markers; Tumor-Derived; Ultrasonography; Uracil; Work; X-Ray Computed Tomography; activation-induced cytidine deaminase; antibody conjugate; base; cancer cell; cell free DNA; circulating DNA; cost effective; design; detection limit; detection method; detection sensitivity; digital; fluorophore; hybrid protein; improved; innovative technologies; instrument; leukemia; leukemia/lymphoma; liquid biopsy; minimally invasive; multiple myeloma M Protein; new technology; next generation sequencing; non-Hodgkin' s lymphoma patients; novel; overexpression; peripheral blood; prognostic; success; synthetic construct; tool; tumor; tumor DNA; uracil-DNA glycosylase; volunteer

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.

迫切需要开发廉价、安全和准确的癌症治疗工具 监视诊断为癌症的患者需要准确评估肿瘤负荷 在他们的治疗过程中，对肿瘤监测的需求更加迫切， 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，如超声超声波检查，计算机断层扫描和正电子 发射断层扫描是昂贵的，具有有限的可靠性，并且使患者暴露于过量的放射性物质中。 辐射我们将通过为已知的生物标志物创建一个台式测定来帮助满足这一需求， 许多B细胞癌症，最终应该导致开发一种试剂盒，可用于 检测循环肿瘤DNA（ctDNA）中该标记物的存在。这种生物标志物的水平， 尿嘧啶在正常人类细胞的基因组中含量很低，但在正常人细胞的DNA中含量却大大升高。 大多数B细胞淋巴瘤和白血病。该项目的长期目标是将其应用于 技术检测B细胞癌患者血浆中ctDNA中的尿嘧啶细胞作为肿瘤 监视工具。在第一个目标中，我们将增加基于点击化学的技术的灵敏度 通过合成和测试与尿嘧啶中的脱碱基位点反应的开启荧光标记来定量尿嘧啶， DNA.这些新探针的使用将消除当前测定中的许多步骤，增加检测效率， DNA的产量，并大大降低背景信号。各种开启化学物质是 我们将使用已知开/关荧光强度比为 >>100。在第二个目标中，我们将从M. smegalloy，UdgX， 特异性地共价连接在DNA中尿嘧啶的位点上，作为这种罕见碱基的探针。这种蛋白质 将与FLAG标签和荧光蛋白融合，并用于直接标记含尿嘧啶的 DNA.蛋白质-DNA复合物的荧光信号可以使用抗FLAG进一步增强 通过ELISA或酪胺化学，将抗体与适当的荧光标签缀合。极限 将使用含有尿嘧啶的合成DNA确定这两种技术的检测率， 来自具有高尿嘧啶含量的B细胞淋巴瘤衍生细胞系的基因组DNA。总的目标是 该项目的目的是将目前尿嘧啶检测方法的灵敏度提高10至50倍，这应该 使我们能够在大多数B-NHL患者的血浆中检测尿嘧啶，并最终发展为一种新的治疗方法。 这是一种检测试剂盒，可用于通过常规抽血监测B细胞癌症患者。

项目成果

A redox-sensitive iron-sulfur cluster in murine FAM72A controls its ability to degrade the nuclear form of uracil-DNA glycosylase.

• DOI: 10.1016/j.dnarep.2022.103381
• 发表时间: 2022-07
• 期刊: DNA repair
• 影响因子: 3.8
• 作者: Jessica A. Stewart;A. Bhagwat

Human activation-induced deaminase lacks strong replicative strand bias or preference for cytosines in hairpin loops.

• DOI: 10.1093/nar/gkac296
• 发表时间: 2022-05-20
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Sakhtemani, Ramin;Perera, Madusha L. W.;Huebschmann, Daniel;Siebert, Reiner;Lawrence, Michael S.;Bhagwat, Ashok S.
• 通讯作者: Bhagwat, Ashok S.

FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

• DOI: 10.1038/s41586-021-04144-4
• 发表时间: 2021-12
• 期刊: Nature
• 影响因子: 64.8