Assays for Specific DNA, microRNA, and Proteins Using DNA Nanostructures

Abstract

Detection of biomolecules that are over- or under-expressed in diseases is an important field for early diagnosis, monitoring treatment, and improving patient prognosis. The main goal of my thesis is to utilize DNA nanotechnology combined with nanomaterials to provide sensitive and specific detection of the desired target molecules that exist in low abundance. I have developed three analytical assays for the detection of DNA, microRNA, proteins, and for the characterization of functionalized nanomaterials. I have developed a binding induced three-way junction (3WJ) with fluorescence resonance energy transfer (FRET) detection using a quantum dot (QD) as both a FRET donor and scaffolding for the DNA nanostructure. I designed probes to bind different portions of the nucleic acid sequence and optimized this assay for the detection of a DNA target. When both probes bind, a short complementary sequence allows the QD FRET donor and a dye acceptor to be in close proximity; detection occurs through the generation of a fluorescent signal. This developed assay was also applied to the detection of single base mismatches in the target DNA sequence with excellent discrimination between the mismatch and the wild-type sequence. With further optimization, this developed technique was also applied to the detection of a microRNA sequence with a specific and sensitive concentration-dependent response. Building on the QD-FRET detection technique, I have designed a binding-induced DNA assembly (BINDA) assay for the detection of a protein target. This technique utilized two aptamers, which bind to specific epitopes on target molecules. When combined with the formation of a BINDA nanostructure, the QD donor again acted as both the FRET donor and scaffolding for other components of BINDA. This technique was able to detect trace levels of the desired protein target with minimal background signal. I also have developed a toehold-mediated strand displacement assay for the characterization of surface coverage on DNA functionalized gold nanoparticles (AuNPs). Through these strand displacement reactions, multifunctionalized AuNPs can be characterized without needing different fluorescent labeling on each strand. This work was compared to conventional characterization methods with similar results. I also found that the use of a TAMRA fluorescent label reduced surface coverage of DNA onto AuNPs. All of the developed assays are sensitive and specific for the desired targets, do not require any separation steps, have simple procedures, are rapid, and require only a fluorescence reader. These aspects are very important for developing point-of-care diagnostic assays to improve patient care, diagnosis, and prognosis.