Structurally engineered biocompatible molecular probes for live cell-imaging and localization of native DNA (PhD)

Abstract

Because of their vitality toward genetic inheritance, governing and functioning of cellular activities, nucleic acids have earned the prime focus out of other biomolecules. Therefore, deep insight into their structural dynamics and its influence on the cellular functioning could be of great importance in the physiological and pathological investigations. Hence, it became crucial to visualize their dynamics in the cellular milieu, and the same directed immense attention of the researcher for the development of useful techniques for their real time monitoring at cellular level. In this continuation, fluorescence microscopy using emissive molecular probes has grown as a non-invasive optical tool for in-vitro/in-vivo cellular imaging to understand the role of various biomolecules toward physiological changes and their influences at the cellular level. The promising molecular markers with enhanced optical properties, cytocompatibility, photobleaching resistance and target specificity constitute the backbone of fluorescence microscopy. Although, the dedicated efforts led to the appreciable development of the molecular probes and their successful employment for visualization of nucleic acids, the common shortcomings such as poor photostability, cell-impermeant nature and photoinduced toxicity restrict their real applicability in the biological fields. Moreover, the incorporation of aforementioned requisite parameters into a single molecular probe has been rare and a challenging task. In this concern, we became interested to crack these challenges and to bring the appreciable contributions toward the advanced biochemical research. The molecular probes with tailored donor-acceptor conjugated (D-π-A and D-π-A-π-D pull-push systems) molecular architectures have been devised and synthesized. Their optical behavior was studied both in solution as well as cellular milieu to explore their candidature as promising DNA markers. The thesis entitled “Structurally engineered biocompatible molecular probes for live cell-imaging and localization of native DNA” begins with the development of indole based “turn-on” green fluorescent molecular probes PA1-PA5 with rationally designed D-ᴨ-A framework. Thorough study of their optical behavior in solution and cell-based assays successfully demonstrated that how a single atom modification (from carbon to selenium through oxygen and sulfur) in the acceptor unit can govern their biocompatibility and affinity toward DNA. The thesis also emphasizes on the importance of rationally incorporated chemical-functionality and structurally devised crescent geometry toward the selective DNA binding of a molecular probe as compared to other biomolecules. In this context, probes P1-P5 were developed and further their structure-interaction relationship investigation established the importance of rational chemical functionality towards DNA binding. Further, the development of RD2 through the synthesis of a well-planned chemical library RD1-RD8 and a careful structure-interaction relationship study led us to report that probe RD2 having more curved geometry than others exhibited stronger binding affinity towards DNA. Additionally, RD2 established its standing as a highly photostable DNA marker for chromosomal staining and micronuclei detection. Molecular simulation studies employing density functional theory (DFT) contributed toward the understanding of the electronic delocalization in pull-push system and the worth of crescent geometry of the probe towards DNA specificity. Additionally, Autodoc 4.0 and circular dichroism (CD) helped to establish the binding mode and nature of interactions between DNA-probe ensembles. At last, the study includes the development of a structurally engineered orange emissive probe RF1 through the synthesis of a series of probes RF1-RF10. Thorough photophysical study clearly established that the improved optical properties (high quantum efficiency, brightness and especially long range excitation/emission) can be tuned by a rational design of highly conjugated pull-push system (D-ᴨ-A). Additionally, the steric influence of the substituents towards the DNA binding affinity of the cationic moiety was also explored. Furthermore, a careful cellular --investigation established the promising candidature of RF1 as the first DNA marker used for the real-time monitoring of the reproductive and proliferative potential of the live cells beyond nine days without involving any tedious transfection. Hence, the thesis presents an extended combinatorial approach which includes synthesis of rationally designed molecular probes as DNA markers, photophysical investigations in steady as well as excited state, understanding of intramolecular charge transfer (ICT) through DFT studies followed by time-resolved fluorescence measurements, establishment of binding mechanism employing molecular modeling supported by CD experiment and evaluation of the biological utility of the synthesized probes for live cell imaging and various biomedical applications.