Direct Identification and Counting of MicroRNAs in Single Cells by Transient Binding and Kinetic Fingerprinting

Abstract

Cancer is the second leading cause of mortality worldwide. Within the U.S. alone, it is estimated that 1.9 million people will be diagnosed this year and over 610,000 people are expected to perish. Despite advances in diagnostic and treatment strategies, a significant number of patients still develop late-stage cancer, where treatment options are inadequate. This suggests early detection is critical to reducing cancer morbidity and mortality, motivating interest in liquid biopsies, an alternative to invasive procedures. Body fluids like blood and urine are analyzed for biomarkers to detect cancer and regularly monitor disease. Of various biomarkers, circulating tumor cells (CTCs) provide substantial information for better understanding tumor biology and the metastatic cascade. With recent technological developments, molecular characterization of CTCs at the single-cell resolution is now possible, opening new windows into metastasis and laying groundwork for development of single cell technologies to diagnose and monitor disease. MiRNAs are non-coding RNAs with pervasive gene regulatory function in higher eukaryotes. Although short in length, miRNAs regulate essentially all cellular pathways relevant to human health. Over the past few decades, it has become clear that due to their abundance and relative stability in body fluids, the levels of miRNAs in liquid biopsies can be compared between healthy individuals and cancer patients, making them suitable biomarkers of disease. Several technologies have been investigated and established for nucleic acid profiling. Current gold standards include the Polymerase Chain Reaction (PCR) and Next Generation Sequencing (NGS). These techniques have high sensitivity and throughput, respectively, however they come with practical shortcomings. For instance, detection via PCR often introduces enzymatic amplification bias leading to false negatives. To overcome these pitfalls, our group developed a novel, innovative technology capable of direct single-molecule identification and counting of various analytes. Our approach, termed Single-Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS), measures the transient binding of a detection probe to a target immobilized on a glass surface. The equilibrium binding of the fluorescent detection probe to the target is detected in a single molecule microscope and is distinctive in its kinetic signature, or fingerprint, a feature used to achieve ultra-high specificity. The ultimate vision of this dissertation is to develop a technology platform for the rapid, robust single molecule analysis of a panel of microRNA (miRNA) biomarkers in single human cancer cells utilizing SiMREPS. After an introduction in Chapter 1, Chapter 2 focuses on the development of an in situ based detection platform, expanding the capabilities of SiMREPS. Here, a FRET detection scheme is developed to complement the new detection environment. In this chapter, the optimization of the technology workflow is described with a detailed strategy for FRET probe development to obtain desirable kinetic behavior and applied to miRNAs in single cells in situ. Chapter 3 focuses on the development of an assay utilizing microfluidics for isolation and lysis of single cells that will undergo SiMREPS analysis. Details of the optimization of the acquisition time, slide preparation, and incorporation of a system to improve sensitivity are discussed. A final Chapter 4 concludes the thesis and discusses future directions. Together, this thesis presents a proof-of-concept for using either in situ or microfluidic approaches to detect miRNAs in single cells with the potential to better understand the mechanisms behind miRNA variability in single cells for application in cancer diagnosis, prognosis, and monitoring.