title: Antisense oligonuclotides with oxetane-constrained cytidine enhance heteroduplex stability, and elicit satisfactory RNase H response as well as showing improved resistance to both exo and endonucleases.
abstract: Antisense oligonucleotides (AONs) with single and double oxetane C modifications [1',2'-oxetane constrained cytidine, 1-(1',3'-O-anhydro-beta-D-psicofuranosyl)cytosine] have been evaluated, in comparison with the corresponding T-modified AONs, for their antisense potentials by targeting to a 15mer complementary RNA. Although the C modified mixmer AONs show approximately 3 degrees C drop per modification in melting temperature (Tm) of their hybrid AON-RNA duplexes, they are found to be good substrates for RNase H, in comparison with the native AON-RNA duplex. An AON with double C modifications along with 3'-DPPZ (dipyridophenazine) conjugation shows the Tm of the hybrid duplexes as high as that of the native, and the RNase H activity as good as its unconjugated counterpart. A detailed Michaelis-Menten kinetic analysis of RNase H cleavage showed that the single and double C modified AON-RNA duplexes as well as double C modifications along with 3'-DPPZ have catalytic activities (kcat) close to the native. However, the R Nase H binding affinity (1/Km) showed a slight decrease with increase in the number of modifications, which results in less effective enzyme activity (kcat/Km) for C modified AON-RNA duplexes. All oxetane modified AON-RNA hybrids showed a correlation of Tm with the 1/Km, Vmax, or Vmax/Km. The C modified AONs (with 3'-DPPZ), as in the T counterpart, showed an enhanced tolerance towards the endonuclease and exonuclease degradation compared to the native (the oxetane-sugar and the DPPZ based AONs are non-toxic to K562 cell growth, ref. 18). Thus a balance has been found between exo and endonuclease stability vis-a-vis thermostability of the heteroduplex and the R Nase H recruitment capability and cleavage with the oxetane-constrained cytidine incorporated AONs as potential antisense candidates with a fully phosphate backbone for further biological assessment.
authors.affiliation: Department of Bioorganic Chemistry, Biomedical Center, University of Uppsala, Box 581, S-751 23 Uppsala, Sweden.
authors.forename: Pushpangadan I
authors.initials: PI
authors.lastname: Pradeepkumar
authors.forename: Nariman V
authors.initials: NV
authors.lastname: Amirkhanov
authors.forename: Jyoti
authors.initials: J
authors.lastname: Chattopadhyaya
mesh_terms: D001483:Base Sequence; D003562:Cytidine / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000494:pharmacology*; D004988:Ethers, Cyclic / Q000737:chemistry*; D005092:Exonucleases / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D016914:Ribonuclease H / Q000737:chemistry*; D013696:Temperature
chemical_list: D004988:Ethers, Cyclic; D009711:Nucleotides; D016376:Oligonucleotides, Antisense; D003562:Cytidine; D012313:RNA; D004720:Endonucleases; D005092:Exonucleases; C022650:spleen exonuclease; D003850:Deoxyribonuclease I; D016914:Ribonuclease H; C005287:oxetane