| Summary: | 博士 === 國立陽明大學 === 生醫光電研究所 === 102 === The importance of guanine-quadruplex (G-quadruplex) is not only in protecting the ends of chromosomes for human telomeres, but also in regulating gene expression for several gene promoters. However, wether G-quadruplex structures exist in living cells is still under debate. In the first half of the work, we characterize a fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G-quadruplexes from duplexes. o-BMVC has large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G-quadruplexes and duplex DNA, making it a good candidate for probing G-quadruplex DNA structures. The fluorescence decay time of o-BMVC upon interaction with G-quadruplex structures of telomeric G-rich sequences is ~2.6 ns while with duplex structure of calf thymus is ~1.2 ns. By analyzing its fluorescence decay time and histograms, we were able to detect one G-quadruplex out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy (FLIM), we demonstrated an innovative methodology for visualizing the localization of G-quadruplexes as well as mapping the localization of different G-quadruplexes in living cells.
Guanine-rich oligonucleotides (GROs) are promising therapeutic candidate for cancer treatment and other biomedical application. In the second half of the work, we have introduced a G-quadruplex ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) and its derivatives, to monitor the cellular uptake of naked GROs and map their intracellular localizations in living cells by using confocal microscopy and FLIM. The GROs that form parallel G-quadruplex structures, such as PU22, T40214, and AS1411, are detected mainly in the lysosome of CL1-0 lung cancer cells after incubation for 2 h. On the contrary, the GROs that form nonparallel G-quadruplex structures, such as human telomeres (HT23) and thrombin binding aptamer (TBA), are rarely detected in the lysosome, but found mainly in the mitochondria. Moreover, the FRET studies of fluorophore-labeled GROs in confocal microscopy and the fluorescence decay in FLIM show that the parallel G-quadruplex structures can be retained in CL1-0 cells, while the nonparallel G-quadruplex structures are likely distorted in CL1-0 cells after cellular uptake. Of interest is that the distorted G-quadruplex structure of HT23 from the nonparallel G-quadruplex structure can reform to a probable parallel G-quadruplex structure induced by a BMVC derivative in CL1-0 living cells. These findings are valuable to the design and rationale behind the possible targeted drug delivery to specific cellular organelles using GROs.
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