Guanine-rich oligonucleotides (GROs) are promising therapeutic candidate for cancer treatment and

Guanine-rich oligonucleotides (GROs) are promising therapeutic candidate for cancer treatment and other biomedical application. the lysosome of CL1-0 lung cancer cells after incubation for 2 h. On the contrary the GROs that form nonparallel G4 structures such as human telomeres (HT23) and thrombin binding aptamer (TBA) are rarely detected in the lysosome but found BTZ044 mainly in the mitochondria. Moreover the fluorescence resonant energy transfer studies of fluorophore-labeled GROs show that this parallel G4 structures can BTZ044 be retained in CL1-0 cells whereas the non-parallel G4 structures are likely distorted in CL1-0 cells after cellular uptake. Of interest is that the distorted G4 structure of HT23 from the nonparallel G4 structure can reform to a probable parallel G4 structure induced by a G4 ligand in CL1-0 living cells. These findings are useful to the design and rationale behind the possible targeted drug delivery to specific cellular organelles using GROs. INTRODUCTION A large number of potential guanine-quadruplex-forming sequences are found in the human genome (1-4). The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. It is suggested that this G4 topologies can act as novel therapeutic target (5-8). On the other hand several lines of evidence show that some guanine-rich oligonucleotides (GROs) such as d[(G2T)4TG(TG2)4] (AS1411) (9) d[G3C]4 (“type”:”entrez-protein” attrs :”text”:”T40214″ term_id :”7491594″ term_text :”pirT40214) (10) d[T2AG3]4 (HT24) (11) and d[TG4AG3TG4AG3TG4AAG2] (PU27) (12) could inhibit cancer cell growth and act as anticancer brokers. It appears that GRO can be a target for drug design as well as an anticancer agent. Recently Biffi (13) used G4-specific antibodies linked to a fluorescence tag to quantitatively visualize the G4 structures in cells. Thus the study of the G4 structure in living cells is essential for exploring their possible biological roles in cellular activity and for developing anticancer brokers. Recently several groups have used fluorescence images to demonstrate the cellular uptake of fluorophore-labeled (FL) GROs (12 14 Although the mechanism of the uptake and cellular trafficking of these GROs still remains unclear nevertheless the FL GROs of PU27 (12) and AS1411 (16) can be taken into the living cell without carriers. At present it is not clear whether these GROs can retain their G4 structures in living cells BTZ044 after cellular uptake. In addition some CYFIP1 G-rich sequences can form various G4 structures. Hurley (17) reported that this PU27 in c-gene promoter can form both intramolecular and intermolecular conformations in K+ answer. Dailey (18) reported that AS1411 forms a mixture of monomeric and dimeric G4 structures with several different topologies in K+ answer. Therefore it is important to explore the cellular response to different types of G4 structures and to determine whether their G4 structures can be retained in living cells. In addition it is necessary to examine whether the covalently linked dye to the GROs could perturb their G4 structures. Considering human telomeres compelling evidence suggested the coexistence of at least two different G4 structures of HT24 in K+ answer (19-23). In addition telomere sequences with slight differences can adopt different types of G4 structures such as a hybrid G4 structure of HT23 (24) with three G-quartet layers versus a basket form of HT21-T (25) with two G-quartet layers in K+ answer. Of particular interest is that these telomeric nonparallel G4 structures all convert to the propeller G4 structure on adding 40% v/v polyethylene glycol which provides a molecular crowding effect to mimic the cellular environment (26). Thus the possible conversion from the non-parallel G4 structures of human telomeres to the parallel G4 structure deserves more detailed investigation in living cells. Here we introduce a fluorescence probe 3 6 carbazole diiodide (BMVC) to monitor the cellular response of CL1-0 cancer cells to naked GROs with different G4 structures as well as the localization of these GROs. BMVC was used to verify the presence of G4 structure in the human telomeres of metaphase chromosomes (27 28 Most importantly free BMVC molecules can be taken into the nucleus of CL1-0 lung cancer cells and show hyperfluorescence on conversation with DNA (29). Using BMVC as a fluorescence probe we found that the GROs with parallel G4 structures. BTZ044