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The Design and Synthesis of Nucleic Acid Based Imaging Agents
Date of Award
Doctor of Philosophy (PhD)
This dissertation focuses on the improvement of iNOS mRNA imaging in living cells and the development of a novel gene delivery agent based on dendrimer-like DNA (DL-DNA). mRNA imaging can provide important information on the mRNA expression, function, and localization, which is useful for diagnostic and therapeutic purposes. The iNOS mRNA expression has been shown to be greatly upregulated during acute lung injury (ALI) and the resulting high concentration of nitric oxide (NO) is responsible for tissue damage. Therefore, iNOS mRNA serves as a biomarker of ALI and successful targeting of iNOS mRNA is beneficial for the early ALI diagnostics and further therapeutic applications. The antisense imaging of iNOS mRNA in living cells has been explored by previous members in Taylor lab, Dr. Zhenghui Wang and Dr. Yuefei Shen. The aim of the current project is to improve the sensitivity and selectivity of the detection and move one step closer towards viable mRNA imaging in vivo. Three designs were developed for this project, including utilization of a multiple-tyrosine carrying peptide nucleic acid (PNA) to increase the specific activity, use of multiple-tyrosine carrying 2'-O-methyl RNA probes to simplify the preparation process, and development of a radiolabel releasable PNA to reduce the background signal. The last project was intended to develop a naturally degradable DNA nanostructure using highly programmable and controllable assembled dendrimer-like (DL-DNA) as a potential carrier for imaging agents.
The first approach utilized the multiple-tyrosine carrying ("Hi-Y") PNA probes to increase the specific activity so as to increase the radioactivity signal without compromising the selectivity between the antisense and mismatched PNA probes. Prior work by Shen et al. has shown that better selectivity was achieved when a lower dose of PNA probes was used. We therefore intended to increase the signal by increasing the specific activity of the probes. Herein, Hi-Y PNA probe carrying six tyrosines was delivered to the cells by cationic shell-crosslinked knedel-like (cSCK) nanoparticles and used for the iNOS mRNA detection in living cells. We achieved 4.5 times higher specific activity for Hi-Y PNAs than their Low-Y counterparts. Although the cellular uptake experiment showed that antisense Hi-Y PNA was retained about 30% in comparison to the Low-Y PNA at 72-hour time point, the radioactivity in cells may still be higher than that of the Low-Y PNA (~1.4-fold higher), considering the 4.5 times higher specific activity for Hi-Y PNA. This Hi-Y approach may be potentially useful in the in vivo imaging application.
The second approach used Hi-Y 2'-O-methyl RNA probes to replace Hi-Y PNA probes to simplify the nanocomplex preparation process. Similar to PNAs, 2'-O-methyl RNAs also exhibit advantageous properties that make them ideal antisense probes, such as nuclease resistance, higher binding affinity, ability to invade secondary structures, and inability to activate RNase H. Besides, 2'-O-methyl RNAs still possess the same negatively charged phosphodiester backbones like unmodified DNAs and RNAs. Thus, complementary oligonucleotides providing the necessary negative charges to the PNA probes to form nanocomplexes with positively charged cSCK nanoparticles can be omitted by using 2'-O-methyl RNA probes. These Hi-Y 2'-O-methyl RNA probes were successfully prepared by using "copper-free" click reaction to conjugate 2'-O-methyl RNA strands and five-tyrosine bearing peptides. Reverse-phase HPLC, gel electrophoresis, and mass spectrometry were used to characterize the conjugates. However, without the presence of the positively charged cell-penetrating peptide (nonaarginine) as was used with PNA probes, no selectivity between antisense and mismatched 2'-O-methyl RNA probes was observed at most time points. The lack of selectivity indicated the necessity for incorporating a cell-penetrating peptide to efflux the unbound probes.
In the third approach, we used a radiolabel releasable PNA with a cathepsin B sensitive linker (Ala-Leu-Ala-Leu) to reduce the background signal. The background signal was proposed to arise from the entrapment of probes in the lysosomes. Thus, reducing the background signal may result in the improvement of selectivity. Presumably, the radiolabel releasable PNA would be cleaved in the lysosomes and the cleaved radiolabeled tyrosines can be exported outside the cell more easily than the full-length radiolabeled PNA. The cellular uptake experiment showed that the background signal may have been reduced when using the radiolabel releasable PNA probe in comparison to the one without the cathepsin B sensitive linker. Unexpectedly, antisense PNA probe cellular retention was less than the mismatched one. Therefore, the exact intracellular behaviors of these radiolabel releasable PNA probes need to be further examined in the future.
The last part of the dissertation focuses on the self-assembly of DL-DNA nanostructures. Due to the presence of highly negatively charged backbones and high molecular weight, the oligonucleotides generally cannot be transported into the cells efficiently, but certain DNA nanostructures display efficient cellular uptake abilities. A DL-DNA nanostructure was constructed by the self-assembly of well-designed DNA sequences. There were four branches for generation zero and twelve branches for generation one in this nanostructure. These twelve branches on the periphery had two distinct sequences, which enabled the functionalization by aptamers and fluorophore- or radionuclide-labeled PNA probes. The DL-DNA nanostructure was prepared and characterized by polyacrylamide gel electrophoresis (PAGE), agarose gel electrophoresis, and size-exclusion HPLC. The targeting aptamer and functional PNAs were also successfully introduced to the DL-DNA.
Chair and Committee
Kevin Moeller, Liviu Mirica, Timothy Wencewicz, Steven Brody, Yongjian Liu
Li, Zifan, "The Design and Synthesis of Nucleic Acid Based Imaging Agents" (2014). Arts & Sciences Electronic Theses and Dissertations. 350.