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

基本信息

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
项目类别：
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&apos 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&apos s lymphoma patients novel overexpression peripheral blood prognostic success synthetic construct tool tumor tumor DNA uracil-DNA glycosylase volunteer

项目摘要

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、安全且准确的癌症工具监视。诊断患有癌症的患者需要准确评估肿瘤负荷在他们的治疗过程中，对肿瘤监测的需求甚至更加迫切许多惰性 B 细胞恶性肿瘤，如滤泡性淋巴瘤和慢性淋巴细胞白血病。当前的成像技术，例如超声波检查、计算机断层扫描和正电子发射断层扫描价格昂贵，可靠性有限，并且使患者暴露在过多的环境中辐射。我们将通过为已知的生物标志物创建台式测定来帮助满足这一需求许多 B 细胞癌症最终应该会导致开发出一种可用于检测循环肿瘤 DNA (ctDNA) 中是否存在该标记。该生物标志物的水平，尿嘧啶在正常人类细胞的基因组中含量较低，但在人类细胞的 DNA 中含量大大升高大多数 B 细胞淋巴瘤和白血病。该项目的长期目标是应用该技术检测 B 细胞癌患者血浆中 ctDNA 中尿嘧啶的技术监视工具。第一个目标是提高基于点击化学的技术的灵敏度通过合成和测试与脱碱基位点反应的开启荧光标签来量化尿嘧啶脱氧核糖核酸。这些新型探针的使用将消除当前测定中的许多步骤，增加 DNA 产量并显着降低背景信号。多种开启化学物质可用，我们将使用已知开/关荧光强度比的点击化学对 >>100。在第二个目标中，我们将设计一种来自耻垢分枝杆菌的新型蛋白质 UdgX，它特异性地共价连接在 DNA 中的尿嘧啶位点上，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，用于直接标记含有尿嘧啶的脱氧核糖核酸。使用抗 FLAG 可以进一步增强蛋白质-DNA 复合物的荧光信号通过 ELISA 或酪酰胺化学方法将抗体与适当的荧光标签缀合。极限这两种技术的检测将使用含有尿嘧啶和的合成 DNA 来确定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。本次活动的总体目标该项目旨在将现有尿嘧啶检测方法的灵敏度提高 10 至 50 倍，这应该使我们能够检测大多数 B-NHL 患者血浆中的尿嘧啶，并最终开发出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

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
通讯作者：
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.