Targeting RNA with Drug-Like Small Molecules and Fluorescent Probes for Mapping Cell Fate

Abstract

Part A Ribonucleic acid (RNA) plays a central role in various essential functions such as cellular information transfer and gene regulations in living organisms.1 Recent studies have shown that RNA is involved in various human diseases. The human genome project revealed that 70 % of RNA transcribed from DNA is non-coding RNA and a potential drug target for treating various diseases.2,3 Targeting RNA with small drug-like molecules is a relatively new and rapidly evolving path with the potential to expand the target space in the drug discovery discipline. Recent advanced studies produced a wealth of information about the structure of different RNAs and their unique biology;4,5 however, methodologies to develop small molecules that selectively target these RNAs still need to be discovered in the literature. Thus, this thesis aims to design and develop a small molecules-based probe that selectively binds target RNA and modulates its functional properties such as translations. Barbituric acid is a uracil analog, and it can form stronger and more hydrogen-bonding interactions with adenine. Furthermore, the presence of electron-deficient carbonyl units makes this ring system electron-deficient. Thus one can easily generate a donor-acceptor (DA) dyad by covalently linking it with electron-rich systems. Considering these facts, I designed and synthesized a series of D-A systems comprising barbituric acid (A) and bithiophene (D) moieties. The optical properties and hydrophobicity characteristics of these dyads were systematically fine-tuned by judiciously varying the substituents on D and A moieties. The newly developed dyads bind selectively with RNA and form different coacervates. Real-time analysis of the self-assembly process is monitored by spectroscopic, microscopic, and high-resolution fluorescence imaging (HRFI) techniques. The in vitro assay studies on these nanospheres revealed that the binding of these dyads with RNA inhibits the translation process.6

Part-B Autophagy and apoptosis are essential processes, and maintaining a balance between them is necessary for cell homeostasis.7 Recent studies have shown that these two processes have complex crosstalk and are parallelly involved in deciding the cell fate.8 Thus, capturing these two processes in a single cell is vital to understanding their crosstalk. In the present study, we investigated the cooccurrence of apoptosis and autophagy processes in Cisplatin and Rapamycin-treated cells using a single, dual luminescent probe.9 Detailed photoluminescence (PL) studies revealed that these probes are dual emitters (green and red), and the intensity and the nature [charge transfer (CT) or locally excited (LE)] of the PL band can be tuned by varying the polarity and viscosity of the microenvironment. These probes effectively stained autophagosomes and apoptotic vesicles and exhibited strong luminescence. Thus, these probes enable us to quickly identify apoptotic and autophagic bodies based on their morphologies in a single cell using microscopy. Drosophila was used as a model organism to demonstrate the utility of this probe. Interestingly, these probes selectively and effectively stained autophagic and apoptotic drosophila gut tissues. Thus, all these imaging results collectively demonstrated that these probes could concomitantly picture apoptotic and autophagic cells/tissues differentially in dual-color imaging mode.10 All these intriguing results are discussed in detail in this thesis.