Research

Detection of pathogens and cancer biomarkers utilizing custom designed DNA-binding proteins and investigation of the engineered protein-DNA interactions

Figure 1. A schematic diagram of a ZFP array on the COC chip

1. Detection of pathogens

The sensitive detection of DNA is of great interest and demand for an increasing number of biomedical studies and diagnostic applications. The development of simple, rapid, and robust technologies for detection of pathogens is becoming particularly critical, considering the increasing rate of mortality and risk of bacteremia. The use of DNA-binding proteins provides a useful scaffold for ‘direct’ reading of the sequence information, avoiding the need for denaturation and subsequent renaturation required by the DNA hybridization method. Thus, DNA-binding domains such as zinc fingers (ZFs) and novel transcriptional activator-like effectors (TALEs) are engineered for recognition of specific sequences present in multiple pathogens. We also probe DNA recognition and binding behaviors of engineered TALE proteins using a combined biochemical, biophysical, and computational (bioinformatics) approach to provide a new insight into protein-DNA interactions. We envision a protein array capable of multiplexed detection of various pathogens. Our effort to incorporate a molecular diagnostic on a microfluidic technology could possibly facilitate the development of a point-of-care device.


Figure 2. Project overview and specific aims
2. Detection of cancer biomarkers

We also plan to explore the potential use of our system for the detection of specific sites of DNA methylation linked to cancer. Current methylated CpG detection methods are PCR-based and involve the bisulfate modification of DNA, followed by DNA sequencing or methylation-specific PCR. These methods require extensive bisulfate reaction times (4-18 hrs) along with false positive problems caused by incomplete bisulfate reactions. The availability of a simple molecular method to directly determine CpG methylation in a promoter-specific manner would provide a powerful method for the detection of a specific cancer. Combined with the methyl-CpG binding domains, our system will be further developed to detect promoter hypermethylation of tumor suppressor genes in urine DNA sample from bladder cancer patients. Detection of aberrant methylation of candidate genes with our system can provide a non-invasive method for bladder cancer detection by obviating the need for a procedure to obtain biopsy material. It can also serve as early diagnosis and prognosis for bladder cancer.