title: Antisense properties of tricyclo-DNA.
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.
authors.affiliation: Departement für Chemie und Biochemie der Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
authors.forename: Dorte
authors.initials: D
authors.lastname: Renneberg
authors.forename: Emilie
authors.initials: E
authors.lastname: Bouliong
authors.forename: Ulrich
authors.initials: U
authors.lastname: Reber
authors.forename: Daniel
authors.initials: D
authors.lastname: Schümperli
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Leumann
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002417:Cattle; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D016373:DNA, Antisense / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D005312:Fetal Blood / Q000502:physiology; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D018075:RNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
chemical_list: D016373:DNA, Antisense; D009841:Oligonucleotides; D018075:RNA, Complementary; D012333:RNA, Messenger; D005914:Globins; D004247:DNA; D016914:Ribonuclease H