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

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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.