title: How RNase HI (Escherichia coli) promoted site-selective hydrolysis works on RNA in duplex with carba-LNA and LNA substituted antisense strands in an antisense strategy context?
abstract: A detailed kinetic study of 36 single modified AON-RNA heteroduplexes shows that substitution of a single native nucleotide in the antisense strand (AON) by locked nucleic acid (LNA) or by diastereomerically pure carba-LNA results in site-dependent modulation of RNase H promoted cleavage of complementary mRNA strands by 2 to 5 fold at 5'-GpN-3' cleavage sites, giving up to 70% of the RNA cleavage products. The experiments have been performed using RNase HI of Escherichia coli. The 2nd best cleavage site, being the 5'-ApN-3' sites, cleaves up to 23%, depending upon the substitution site in 36 isosequential complementary AONs. A comparison of the modified AON promoted RNA cleavage rates with that of the native AON shows that sequence-specificity is considerably enhanced as a result of modification. Clearly, relatively weaker 5'-purine (Pu)-pyrimidine (Py)-3' stacking in the complementary RNA strand is preferred (giving ∼90% of total cleavage products), which plays an important role in RNase H promoted RNA cleavage. A plausible mechanism of RNase H mediated cleavage of the RNA has been proposed to be two-fold, dictated by the balancing effect of the aromatic character of the purine aglycone: first, the locally formed 9-guanylate ion (pKa9.3, ∼18-20% N1 ionized at pH 8) alters the adjoining sugar-phosphate backbone around the scissile phosphate, transforming its sugar N/S conformational equilibrium, to preferential S-type, causing preferential cleavage at 5'-GpN-3' sites around the center of 20 mer complementary mRNA. Second, the weaker nearest-neighbor strength of 5'-Pu-p-Py-3' stacking promotes preferential 5'-GpN-3' and 5'-ApN-3' cleavage, providing ∼90% of the total products, compared to ∼50% in that of the native one, because of the cLNA/LNA substituent effect on the neighboring 5'-Pu-p-Py-3' sites, providing both local steric flexibility and additional hydration. This facilitates both the water and water/Mg2+ion availability at the cleavage site causing sequence-specific hydrolysis of the phosphodiester bond of scissile phosphate. The enhancement of the total rate of cleavage of the complementary mRNA strand by up to 25%, presented in this work, provides opportunities to engineer a single modification site in appropriately substituted AONs to design an effective antisense strategy based on the nucleolytic stability of the AON strand versus RNase H capability to cleave the complementary RNA strand.
authors.affiliation: Chemical Biology Program, Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 581, SE-751 23 Uppsala, Sweden. jyoti@boc.uu.se oleksandr@boc.uu.se.
authors.forename: Oleksandr
authors.initials: O
authors.lastname: Plashkevych
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.forename: Jyoti
authors.initials: J
authors.lastname: Chattopadhyaya
mesh_terms: D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid; D012313:RNA; D016914:Ribonuclease H; C078856:ribonuclease HI