Chimeric bifunctional oligonucleotides as a novel tool to invade telomerase assembly.
Bottom Line: Telomerase RNA and protein reverse transcriptase subunits are essential for the appearance of active telomerase in vitro.The approach is based on the application of chimeric bifunctional oligonucleotides that contain two oligonucleotide parts complementary to the functional domains of telomerase RNA connected with non-nucleotide linkers in different orientations (5'-3', 5'-5' or 3'-3').Such chimeras inhibited telomerase in vitro in the nM range, but were effective in vivo in sub-nM concentrations, predominantly due to their effect on telomerase assembly and dimerization.
Affiliation: Department of Chemistry and A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119992, Russian Federation Skolkovo Institute of Science and Technology, Novaya Street, 100, Skolkovo, Odintsovsky District, Moscow Region, 143025, Russian Federation.Show MeSH
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Mentions: To test whether chimeras interfere telomerase assembly fractionation gradient was done on a discontinuous sucrose. Extracts obtained from cells transfected with a corresponding chimeric oligonucleotide at a concentration that exceeds IC50 were subjected to sucrose gradient centrifugation to separate assembled telomerase complexes from free hTR. Fractions were collected and the amounts of telomerase RNA and telomerase activity were measured. The data are shown in Figure 3. In HEK293 cells observed peak for hTR amount correlates with the peak of telomerase activity (Figure 3A) thus proving that this peak corresponds to assembled telomerase. The comparison of hTR distribution for hTR in HEK293 cells and cells with M*c3M chimera (the most effective telomerase inhibitor in vivo) shown in Figure 3B reveals a significant drop in the hTR (22% remained) in assembled telomerase peak. The data for c3J and other efficient chimeras Nc3J, M*c3N, c3MN are shown in Supplementary Figure S8 and summarized in Figure 3C. All most active chimeras significantly affected telomerase assembly. The strongest effect was observed in the case of Nc3J, where almost no assembled telomerase complex was detected, and significant loss of assembled complex was detected for all other chimeras. It should be mentioned that c3G only slightly interfered telomerase complex formations (Figure 3C).
Affiliation: Department of Chemistry and A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119992, Russian Federation Skolkovo Institute of Science and Technology, Novaya Street, 100, Skolkovo, Odintsovsky District, Moscow Region, 143025, Russian Federation.