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，安全和准确的癌症工具 监视。诊断出患有癌症的患者需要准确评估肿瘤负担 通过他们的治疗过程，对肿瘤监测的需求更加急切 许多惰性的B细胞恶性肿瘤，例如卵泡淋巴瘤和慢性淋巴细胞性白血病。 当前的成像技术，例如超声超声检查，计算机断层扫描和正电子 排放断层扫描昂贵，可靠性有限，并使患者暴露于过多 辐射。我们将通过为已知的生物标志物的台式测定法，以满足这一需求 许多B细胞癌最终都应该导致套件的开发 检测该标记物在循环肿瘤DNA（CTDNA）中的存在。这个生物标志物的水平， 尿嘧啶在正常人细胞的基因组中很低，但在A的DNA中大大升高 大多数B细胞淋巴瘤和白血病。该项目的长期目标是应用此 在B细胞癌患者血浆中检测CTDNA中尿嘧啶作为肿瘤的技术 监视工具。在第一个目标中，我们将提高基于点击化学技术的灵敏度 通过合成和测试与Abasic位点反应的转交荧光标签来量化尿嘧啶 脱氧核糖核酸。这些新颖探针的使用将消除当前测定中的许多步骤，增加 DNA的产量和大幅度降低背景信号。各种交通的化学成分是 可用，我们将使用单击的化学对，该化学对已知/关闭荧光强度比 >> 100。在第二个目标中，我们将设计一种来自smegmatis的新型蛋白质，UDGX，该蛋白 在DNA中的尿嘧啶部位特定和共价链接，作为该稀有碱的探针。该蛋白质 将融合到标志和荧光蛋白上，并用于直接标记含尿嘧啶的蛋白 脱氧核糖核酸。蛋白-DNA复合物的荧光信号可以使用抗flag进一步增强 通过ELISA或Tyramide Chemistry结合了适当的荧光标签的抗体。极限 检测两种技术的检测将使用含有尿嘧啶的合成DNA和 来自尿嘧啶含量高的B细胞淋巴瘤衍生细胞系的基因组DNA。总体目标 项目是为了提高目前尿嘧啶检测方法10至50倍的灵敏度，应 允许我们在大多数B-NHL患者的血浆中检测到尿嘧啶，并最终发展 测定试剂盒将有助于通过常规抽血监测B细胞癌患者。

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