| The ability to monitor biological processes in the context of living cells with good spatial and temporal resolution offers significant potential for understanding many biological problems. The key to the successful signaling of these processes is the use of molecular probes. Currently, there are limitations for intracellular probes, which include low sensitivity, reduced selectivity, and poor stability. The major goal of this research is to integrate molecular engineering techniques with new signaling materials and approaches to design more sensitive and effective nucleic acid probes.; In an attempt to develop sensitive molecular probes, novel materials were explored for signal amplification and background reduction. Conjugated polymers (CPs) are good candidates for signal amplification because of their excellent light harvesting and superquenching properties. Using a solid phase synthesis method, CP labeled molecular beacons (MBs) were prepared. In addition, superquenchers, a series of macromolecules with exceptional quenching capabilities, were generated through a molecular assembling approach and used for labeling MBs.; To overcome the problem of autofluorescence from biological fluids, a new signaling approach called the excimer light switching signaling technique was developed. An aptamer that selectively binds to platelet derived growth factor (PDGF) was labeled with pyrene molecules on both ends, resulting in a light-switching aptamer. The probe changes its emission from a blue monomer emission to a green excimer emission upon binding to PDGF. Taking advantage of the long fluorescence lifetime of pyrene, time-gated measurements were performed to eliminate biological background signals.; Finally, the stability and false signals of nucleic acid probes were addressed. When used inside cells, normal nucleic acid probes are prone to enzymatic digestion, protein binding, and elicitation of RNase H action, all of which lead to nonspecific signals. The possibility of alleviating these issues by using locked nucleic acid (LNA) bases in the molecular probe design was investigated. With different stem lengths and LNA base ratios, LNA-MBs were designed and their thermodynamic properties, hybridization kinetics, enzymatic resistance, as well as interactions with DNA binding proteins, were studied. In addition to using base modification, we designed a new type of molecular probes called hybrid molecular probe, which does not generate false signal upon digestion by nuclease, binding to SSB. HMP is capable of selectively detecting targets from cellular samples.; The new materials, synthesis methods, and signaling techniques developed in this research have the potential for developing sensitive and effective molecular probes for bioanalysis and intracellular imaging. Future endeavors will include the application of these probes to single living cell gene expression studies. |
