Imidazopyridine/Pyrrole and hydroxybenzimidazole/pyrrole pairs for DNA minor groove recognition

The DNA binding properties of fused heterocycles imidazo[4,5-b]pyridine (Ip) and hydroxybenzimidazole (Hz) paired with pyrrole (Py) in eight-ring hairpin polyamides are reported. The recognition profile of Ip/Py and Hz/Py pairs were compared to the five-membered ring pairs Im/Py and Hp/Py on a DNA restriction fragment at four 6-base pair recognition sites which vary at a single position 5'-TGTNTA-3', where N = G, C, T, A. The Ip/Py pair distinguishes G.C from C.G, T.A, and A.T, and the Hz/Py pair distinguishes T.A from A.T, G.C, and C.G, affording a new set of heterocycle pairs to target the four Watson-Crick base pairs in the minor groove of DNA

Nuclear antisense effects in cyclophilin A pre‐mRNA splicing by oligonucleotides: a comparison of tricyclo‐DNA with LNA

The nuclear antisense properties of a series of tricyclo (tc)‐DNA oligonucleotide 9-15mers, targeted against the 3′ and 5′ splice sites of exon 4 of cyclophilin A (CyPA) pre‐mRNA, were evaluated in HeLa cells and compared with those of corresponding LNA‐oligonucleotides. While the 9mers showed no significant antisense effect, the 11-15mers induced exon 4 skipping and exon 3+4 double skipping to about an equal extent upon lipofectamine mediated transfection in a sequence‐ and dose‐dependent manner, as revealed by a RT-PCR assay. The antisense efficacy of the tc‐oligonucleotides was found to be superior to that of the LNA‐oligonucleotides in all cases by a factor of at least 4-5. A tc‐oligonucleotide 15mer completely abolished CyPA mRNA production at 0.2 µM concentration. The antisense effect was confirmed by western blot analysis which revealed a reduction in CyPA protein to 13% of its normal level. Fluorescence microscopic investigations with a fluorescein labeled tc‐15mer revealed a strong propensity for homogeneous nuclear localization of this backbone type after lipofectamine mediated transfection, while the corresponding lna 15mer showed a less clear cellular distribution pattern. Transfection without lipid carrier showed no significant internalization of both tc‐ and LNA‐ oligonucleotides. The obtained results confirm the power of tc‐DNA for nuclear antisense applications. Moreover, CyPA may become an interesting therapeutic target due to its important role in the early steps of the viral replication of HIV‐

Exploring Hoogsteen and reversed-Hoogsteen duplex and triplex formation with tricyclo-DNA purine sequences

The duplex- and triplex-formation properties of the tricyclo-DNA purine decamer 5'p-gagaaggaaa-3' as a single strand or as part of a hairpin duplex with corresponding parallel and antiparallel pyrimidine DNA and RNA complements, as well as with antiparallel purine DNA and RNA complements, were investigated by UV melting curve analysis, circular dichroism spectroscopy, and gel mobility shift experiments. It was found that tricyclo-DNA forms very stable duplexes with the pyrimidine RNA and DNA complements not only in the Watson-Crick pairing mode, but also in the Hoogsteen one. Below pH 6.0, the tc-DNA/DNA and tc-DNA/RNA Hoogsteen duplexes were found to be more stable than the corresponding Watson-Crick DNA duplexes. Triplexes of the hairpin structure with parallel pyrimidine complements revealed even stronger Hoogsteen pairing relative to the duplexes, presumably due to structural preorganization phenomena. Triplex formation with antiparallel pyrimidine and purine third strands (reversed-Hoogsteen motif) could not be observed and seem to be unstabl

Synthesis of the Sugar Building Block of Bicyclo-RNA

We present the novel synthesis of two sugar units that are central intermediates for the formation of members of the bicyclo-DNA and -RNA family. The synthesis starts from commercially available 1,2: 5,6-di-O-isopropylidene-alpha-D-glucofuranose. The key step involves the elaboration of a carbocyclic ring in a furanoside by rhodium(I)-catalyzed hydroacylation. Via this pathway, one of the sugar units is available in 8 steps and in an overall yield of 27%, while its deoxy derivative is obtained in 11 steps, which is 5 steps fewer than in our previous synthesis of this compound

Antisense properties of tricyclo-DNA

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2′-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10–17 nt in length, show enhanced selectivity and enhanced thermal stability by ∼1°C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37°C. Moreover, tc-DNA–RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human β-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in β-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3′-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2′-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2′-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations

Antisense properties of tricyclo-DNA

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2′-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by ∼1°C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37°C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human β-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in β-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3′-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2′-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2′-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentration