Advancing Quantitative DNA Biomarker Detection through Single Molecule Fluorescence Kinetic Fingerprinting

Abstract

DNA (deoxyribonucleic acid), as the primary genetic material in eukaryotic organisms, is one of the major biomarkers for biological processes, pathological processes and drug responses in clinical diagnostics and prognostics. Genetic variations and DNA methylation are two major types of disease-related DNA biomarker. The gold standard for detecting DNA disease-related biomarkers is PCR (polymerase chain reaction) amplification-based approach, consisting over 50% FDA (U.S. Food and Drug Administration)-approved in vitro diagnostic tests. However, it suffers from two significant technical limitations: biased estimation due to unequal amplification and limited specificity. These issues are largely overlooked within the scientific community. In this Dissertation, I developed amplification-free detections for DNA methylation cancer biomarkers and DNA mutation cancer biomarkers by using single-molecule fluorescent kinetic fingerprinting (SMFKF) based on single-molecule recognition through equilibrium Poisson sampling (SiMREPS). SMFKF or SiMREPS-based assays offer high-confidence identification of targeted biomarkers as a fluorescent imager repeatedly probes the same molecule. This dissertation consists of two parts: 1) Chapter 2 and 3, developing quantitative SMFKF biosensor for detecting DNA methylation biomarkers; 2) Chapter 4, developing ultrafast quantitative SMFKF biosensor for detecting DNA mutation biomarkers. Chapter 2 introduced bisulfite Me-SiMREPS (BSM-SiMREPS) that bisulfite-converted methylated 102 nt BCAT1 promoter, a DNA methylation biomarker for colorectal cancer and immobilized it specifically through DNA hybridization with designed probes. Fluorescent DNA imagers then identify and quantify these immobilized molecules by kinetic filtering. This amplification-free approach yielded a sub-femtomolar limit of detection and 99.9999% specificity for pure DNA methylation biomarker. And eventually, BSM-SiMREPS measured a 31% methylation level of BCAT1 promoter in whole blood DNA, exposing the significant underestimation by PCR-based measurement. However, DNA degradation and limited conversion efficiency during bisulfite conversion compromise overall sensitivity and specificity. To address these limitations, Chapter 3 introduced a reversible but specific methylation binder – MBD (methyl-binding domain) and achieved direct, amplification-free methylation detection of 55 nt BCAT1 promoter using MBD-SiMREPS. Effects of methylation patterns and sensor structures on methyl-binding kinetics at single-molecule level were investigated with 36 constructs. We discovered that a “branch” motif on the unmethylated reverse strand in a hemimethylated double-stranded DNA facilitated MBD binding, negating the necessity for methylation on the reverse strand. This unexpected MBD behavior could offer novel insights into gene regulation by MBD superfamily in vivo. In Chapter 4, the potential of fluorogenic imagers was explored. Fluorogenic DNA imagers augment sensitivity of SiMREPS detection by acquiring more fields of view within a similar total acquisition time. We demonstrated fluorogenic SiMREPS (FG-SiMREPS) for detecting three DNA cancer biomarkers: T790M, L858R and HPV. 2-second detections were achieved for all three biomarkers using micromolar fluorogenic imagers. Eventually, we enabled 104-FOV (field of view) scanning detection in just 5 min. However, the inability to distinguish false positives due to significant non-specific interactions was a setback. To address this, further optimizations on surface passivation and imaging conditions are necessary.