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: We synthesized a number of chimeric oligonucleotides that comprised two parts connected with a non-nucleotide linker. These oligonucleotides (Table 1) contained the following parts: M–complementary to the template region (Figure 1A) (hTR position 46–65)–oligonucleotide analogs complementary to hTR within this region were shown to inhibit telomerase activity by interacting with the template (26); J (hTR position 152–168)–complementary to single-stranded chain of the pseudoknot (Figure 1A)–such oligonucleotide was shown to be telomerase inhibitor both in vivo and in vitro (20); N–contains complementary to CR4/CR5 region (Figure 1A)–the hTR position 256–272, region is known to be essential for telomerase assembly (10) and three additional nucleotides at the 5′ end that allow to separate complementary regions in chimeras when N is a part of either MN or JN chimeras. All oligonucleotides were modified with 2′-OMe nucleotides and connected together either via 3′-5′ or 3′-3′ or 5′-5′ ends with or without a 1,3-propane diol linker (c3) (Figure 1B). 3′-ends in all cases were blocked by addition of 6-aminohexanol. If M, N, J parts were used alone, they contained a c3 linker as a modification at the 5′-end. Oligonucleotide G (Figure 1A) (hTR position 42–54), with a sequence of a known telomerase inhibitor that binds to the hTR template (26), modified as described above, was used as a control.
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.