Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides

Abstract

The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing North-East conformationally constrained 1′,2′-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2′,4′-ethylene-bridged (aza-ENA-T) nucleoside, and 2′-alkoxy modified nucleosides (2′-O-Me-T and 2′-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (∆T m ) 2.5-4°C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4-5.5°C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of ∼5-6°C per modification. The 2′-O-Me-T and 2′-O-MOE-T modifications, on the other hand, showed an increased of T m by 0.5°C per modification in their AON/ RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The K m and V max values obtained from the RNA concentrationdependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (K cat ) of RNase H were found to be greater (∼1.4-2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/K m ) showed a decrease (∼1.7-8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (∼1.1-3.2-fold) enzyme activity (K cat /K m ) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2′-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2′-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3′-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3′-exonuclease preferentially hydrolyzed the 3′-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3′-exonuclease cleaved at the...

New methylene-bridged hexopyranosyl nucleoside modified oligonucleotides (BHNA): synthesis and biochemical studies

“…This is very similar to what we have observed earlier with other North-type conformationally constrained systems such as oxetane 15 azitidine 16,17 or aza-ENA 12 based AONs/RNA duplexes. This can be attributed to the local conformational change from DNA•RNA type to the RNA•RNA type conformation, which is not a substrate to RNase H. This local conformational change for a stretch of 5-6 nucleotides is however not detectable either by CD spectra or by the molecular model.…”

“…) or the carbocylic chain (carba-LNA 13 and carba-ENA 13,14 ), or by a covalent linkage between C1′ and C2′ (such as oxetane 15 or azetidine 16,17 types of constraints) result in to a conformationally-constrained North-type sugar. Oligonucleotides with these types of covalently constrained nucleosides can also potentially preorganize the single-stranded oligonucleotide in to a helical form, which, in turn, show much improved affinity toward complementary RNA (3-8ºC/modification, with the exception of C1′ and C2′ constrained systems) as well as to DNA 18 ;…”

“…13,17 (3) The AONs produced by the replacement of the pentofuranosyl sugar with the hexopyranosyl sugar 19 give thermodynamically stable duplexes with the target DNA or RNA. It is because, first, the sixmembered ring system adopt a rigid chair conformation, requiring a less negative entropy change during the duplex formation; and second, the interstrand phosphate distance in the six-membered pyranosyl-modified system is larger than in the natural nucleic acid duplexes giving a relatively less interstrand charge repulsion compared to the native counterpart.…”

Intramolecular Free-Radical Cyclization Reactions on Pentose Sugars for the Synthesis of Carba-LNA and Carba-ENA and the Application of Their Modified Oligonucleotides as Potential RNA Targeted Therapeutics

“…48 In the radical precursors 29, an allyl is attached to the 5′-O through an ether bond. After treatment with Bu 3 SnH and AIBN, intramolecular cyclization of the obtained hept-6-enyl radical (30) occurred exclusively in the 6-exo mode, formatting the 3′,4′-β-fused six-membered heterocyclic ring (31). The alkenyl function can also be introduced to the radical precursor through a bond other than an ether.…”

Molecular Structure of the Core-Modified siRNA Duplexes Containing Diastereomeric Pair of [C6′(R)-OH]- versus [C6′(S)-OH]-carba-LNAs Suggests a Model for RNAi Action

“…In this regard, LNA and its carba-modifications ( Figure 1, panel B) have emerged as promising alternatives to native siRNAs. [12][13][14][15][16][17][18] Our recent study has demonstrated in vivo (measured by relative IC 50 ) [1] intriguing remarkable sensitivity of the silencing efficiency of siRNA against the target HIV-1 TAR RNA to the type of diastereomeric configuration of the C6 center (S-versus R-) of the conformationally locked [6 -(R/S)-O-(p-Toluoyl)-7 (S)-methyl]-carba-LNA ( Figure 1) nucleotides incorporated into the core region of siRNA at T 13 position. Thus, the introduction of C6 (S)-O-(p-Toluoyl)-7 (S)-methyl-carba-LNA nucleotide into T 13 position of siRNA resulted in dramatically reduced silencing activity [IC 50 of 79.8 ± 17 nM], [1] which is nearly 24 times less potent in silencing the RNA of HIV-1 TAR compared to that of the R-diastereoomer C6 (R)-O-(p-Toluoyl)-7 (S)-methyl-carba-LNA [IC 50 of 3.32 ± 0.17 nM] [1] modified siRNA.…”