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. 2002 Jul 1;30(13):2751-7.
doi: 10.1093/nar/gkf412.

Antisense properties of tricyclo-DNA

Dorte Renneberg  1 , Emilie Bouliong, Ulrich Reber, Daniel Schümperli, Christian J Leumann
Affiliations

Antisense properties of tricyclo-DNA

Dorte Renneberg et al. Nucleic Acids Res. .

Abstract

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 approximately 1 degrees C/modification in binding to RNA _targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree 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 beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-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.

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Figures

Figure 1
Figure 1
Representation of the chemical structure of tc-DNA as compared with DNA.
Figure 2
Figure 2
UV-melting curves (260 nm) of the duplexes of 2′-O-Me-PS-AS (diamonds) and tcd-AS (triangles) with the corresponding RNA complement r(pUCUUUCAGGGCAAUAAUG) in 10 mM NaH2PO4, 150 mM NaCl, pH 7.0 (2 µM duplex concentration).
Figure 3
Figure 3
Polyacrylamide gel (20%) showing the time course of the FCS mediated degradation of oligonucleotides d-1 and tcd-1 (A) and tcd-AS (B) at the indicated time intervals. Bands were visualized by staining with ‘stains all’.
Figure 4
Figure 4
Autoradiogram of a 20% denaturing polyacrylamide gel showing the RNase H mediated cleavage of r(AACUGUCACG) by d-1 (lanes 1–4), tcd-1 (lanes 5–8) and r-1 (lanes 9–12) at 1, 30, 120 and 300 min each.
Figure 5
Figure 5
Correction of β-globin mRNA splicing after lipofection of tc-DNA oligonucleotide. Total RNA from HeLa cells expressing the human β-globin gene with either the IVS2-654 or IVS2-705 mutation, which result in the inclusion of an aberrant exon between the normal exons 2 and 3, was subjected to RT–PCR with primers in exons 2 and 3. Concentrations (µM) of the transfected oligonucleotides are indicated above the lanes. pos, RT–PCR products from cells expressing both the aberrant IVS2-654 and correct β-globin mRNA; neg (lanes 2 in A, B and D), RT–PCR with RNA from a mouse cell line not expressing β-globin; mock (lane 2 in C, lane 3 in A and B), RT–PCR with RNA from cells transfected with empty liposomes; hd, heteroduplexes between aberrant and correct RT–PCR products. Note that the aberrantly spliced product is longer in HeLa-705 than in HeLa-654 cells.
Figure 6
Figure 6
Correction of β-globin mRNA splicing after co-incubation (without liposomes) of tc-DNA oligonucleotide. Total RNA from HeLa-654 and HeLa-705 cells was subjected to RT-PCR with primers in exons 2 and 3. Concentrations (µM) of the transfected oligonucleotides are indicated above the lanes. pos, neg, mock and hd, as in Figure 5.
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