Applications of functional nucleic acids in imaging, sensing and drug delivery and investigations of DNAzyme-metal interactions

Functional nucleic acids (FNA), including nucleic acids catalysts (ribozymes and DNAzymes) and ligands (aptamers), have been discovered in nature or isolated in a laboratory through a process called in vitro selection. They are nucleic acids with functions similar to protein enzymes or antibodies. They have been developed into sensors with high sensitivity and selectivity; it is realized by converting the reaction catalyzed by a DNAzyme/ribozyme or the binding event of an aptamer to a fluorescent, colorimetric or electrochemical signal. While a number of studies have been reported for in vitro sensing using DNAzymes or aptamers, there are few reports on in vivo sensing or imaging. MRI is a non-invasive imaging technique; smart MRI contrast agents were synthesized for molecular imaging purposes. However, their rational design remains a challenge due to the difficulty to predict molecular interactions. Chapter 2 focuses on rational design of smart T1-weighted MRI contrast agents with high specificity based on DNAzymes and aptamers. It was realized by changing the molecular weight of the gadolinium conjugated DNA strand with the analytes, which lead to analyte-specific water proton relaxation responses and contrast changes on an MRI image. The designs are general; the high selectivity of FNA was retained. Most FNA-based fluorescent sensors require covalent labeling of fluorophore/quencher to FNAs, which incurrs extra expenses and could interfere the function of FNAs. Chapter 3 describes a new sensor design avoiding the covalent labeling of fluorophore and quencher. The fluorescence of malachite green (MG) was regulated by the presence of adenosine. Conjugate of aptamers of MG and adenosine and a bridge strand were annealed in a solution containing MG. The MG aptamer did not bind MG because of its hybridization to the bridge strand, resulting in low fluorescence signal of MG. The hybridization was weakened in the presence of adenosine, leading to the binding of MG to its aptamer and a fluorescence increase. The sensor has comparable detection limit (20 micromolar) and specificity to its labeled derivatives. Enzymatic activity of most DNAzymes requires metal cations. The research on the metal-DNAzyme interaction is of interest and challenge to scientists because of the lack of structural information. Chapters 4 presents the research on the characterization of the interaction between a Cu2+-dependent DNAzyme and Cu2+. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopy were used to probe the binding of Cu2+ to the DNAzyme; circular dichroism was used to probe the conformational change of the DNAzyme induced by Cu2+. It was proposed that the conformational change by the Cu2+ binding is important for the activity of the DNAzyme. Chapter 5 reports the dependence of the activity of 8-17 DNAzyme on the presence of both Pb2+ and other metal cations including Zn2+, Cd2+ and Mg2+. It was discovered that presence of those metal cations can be cooperative or inhibitive to 8-17 activity. It is hypothesized that the 8-17 DNAzyme had multiple binding sites for metal cations based on the results. Cisplatin is effective killing tumor cells, but with significant side effects, which can be minimized by its targeted delivery. Chapter 6 focuses on the effort to functionalize liposomes encapsulating cisplatin by an aptamer that selectively bind nucleolin, an overexpressed protein by breast cancer cells. The study proved the selective cytotoxicity to breast cancer cells of the aptamer-functionalized liposome.

Using a voxel-based krogh cylinder array to simulate microvascular contrast enhancement

The goal of this study is to better simulate microscopic and voxel-based dynamic contrast enhancement in magnetic resonance imaging. Specifically, errors imposed by the traditional two-compartment model are reduced by introducing a novel Krogh cylinder network. The two-compartment model was developed for macroscopic pharmacokinetic analysis of dynamic contrast enhancement and generalizing it to voxel dimensions, due to the significant decrease in scale, imposes physiologically unrealistic assumptions. In the project, a system of microscopic exchange between plasma and extravascular-extracellular space is built while numerically simulating the local contrast agent flow between and inside image elements. To do this, tissue parameter maps were created, contrast agent was introduced to the tissue via a flow lattice, and various data sets were simulated. The effects of sources, tissue heterogeneity, and the contribution of individual tissue parameters to an image are modeled. Further, the study attempts to demonstrate the effects of a priori flow maps on image contrast, indicating that flow data is as important as permeability data when analyzing tumor contrast enhancement. In addition, the simulations indicate that it may be possible to obtain tumor-type diagnostic information by acquiring both flow and permeability data.