DOI
https://doi.org/10.25772/360F-GP50
Author ORCID Identifier
https://orcid.org/0009-0002-8748-9410
Defense Date
2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Dr. Soma Dhakal
Abstract
Reliable and early diagnosis of diseases, although highly desirable, has been a challenging task for some of the diseases including cancers either due to lack of suitable technologies or disease-specific biomarkers. Thanks to the recent progress in the field of molecular diagnostics and sensing, a wide range of biomolecules including circulating DNA and RNA sequences, proteins, enzymes, and other small-molecule metabolites have been identified as markers for diseases. In recent years, microRNA biomarkers have gained significant attention in the field of diagnosis and therapeutics after the realization that circulating sequences (especially microRNAs or miRNAs) in body fluids can serve as indicators for different health conditions such as cancer. Considering that the concentration of miRNAs in body fluids (plasma or serum) is quite low (low pM – fM range), most of the existing methods either require labeling or amplification of miRNAs to reach the desired sensitivity. While hybridization-based techniques have been predominately used for miRNA sensing, they often lack specificity between perfectly matched targets and those with a point mutation. Therefore, there is an increasing demand for simple and amplification-free methods that can detect trace amounts of miRNAs while still being able to distinguish actual targets from mutants. To address this gap in knowledge we have developed and tested a DNA-based fluorescence resonance energy transfer (FRET) sensor that enables ultrasensitive detection of a miRNA biomarker (miRNA-342-3p) associated with triple-negative breast cancer (TNBC) without the need for target/signal amplification strategies. The sensor was developed by adopting the dynamic nature of a four-way junction (also known as Holliday junction, HJ) which is designed in a way that the junction can only form in the presence of a target. The sensor shows a relatively static low-FRET state in the absence of a target, yet continuous FRET transitions between low- and high-FRET states in the presence of the target. Using this approach, we showed that the sensor is highly specific, has a detection limit down to low femtomolar (fM) without having to amplify the target, and has a large dynamic range (3 orders of magnitude) extending to 300,000 fM. We have also demonstrated that the sensor allows detection of miRNA-342-3p in the miRNA-extracts from cancer cell lines and TNBC patient-derived xenografts (PDX). Given the simple-to-design hybridization-based detection, the sensing platform developed here can be used to detect a wide range of miRNAs enabling early diagnosis and screening of other genetic disorders.
Additionally, studies have shown that diagnosis using a single biomarker can limit the accuracy of diagnosis due to possible cross-talk of the biomarker in different diseases. There is emerging evidence that false positives can be avoided or at least minimized by measuring multiple biomarkers instead of just one. However, this requires a technique that can detect multiple biomarkers in one test. For this reason, we have recently expanded the aforementioned sensors for multiplexed detection of miRNAs, which involves the creation of multiple HJ sensors with unique inter-dye distances. Using this approach, we are able to get four distinctly different FRET values and intensity patterns for four different sensors. This work has established that the multiplexed platform is suitable for the detection of DNA mimics of TNBC microRNAs and thus it should enable detection of actual TNBC miRNAs.
Apart from miRNAs, in a pursuit to develop a sensor for the detection of foodborne bacteria - Staphylococcus aureus (S. aureus), we have developed a sensor for S. aureus surface protein called Iron-regulated surface determinant protein A (IsdA). IsdA is known to be a critical protein for the survival and colonization of S. aurues. Given that S. aureus is pathogenic and has been linked to foodborne diseases, its timely detection is critical to prevent diseases caused by this bacterium. Several detection methods such as cell culture, nucleic acid amplification, and other colorimetric and electrochemical methods have been developed for S. aureus, but the detection through the critical protein IsdA is underdeveloped. By combining computational generation of target-guided aptamers and fluorescence resonance energy transfer (FRET)-based single-molecule analysis, we developed a detection method for IsdA. Three different RNA aptamers specific to the IsdA protein were identified and their ability to switch the FRET construct to a high-FRET state in the presence of protein was verified. The method we developed is sensitive down to picomolar levels (×10-12 M, equivalent to ~1.1 femtomoles IsdA) with a dynamic range extending to ~40 nM of IsdA. The FRET-based single-molecule technique that we developed for IsdA can have a broader application in the food industry and aptamer-based sensing field by enabling the quantitative detection of a wide range of pathogen proteins.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
11-22-2023