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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?
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2017 (English)In: Molecular Biosystems, ISSN 1742-206X, E-ISSN 1742-2051, Vol. 13, no 5, 921-938 p.Article in journal (Refereed) Published
Abstract [en]

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 similar to 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 (pK(a) 9.3, similar to 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 50-Pu-p-Py-30 stacking promotes preferential 5'-GpN-3' and 5'-ApN-3' cleavage, providing similar to 90% of the total products, compared to similar to 50% in that of the native one, because of the cLNA/ LNA substituent effect on the neighboring 50-Pu-p-Py-30 sites, providing both local steric flexibility and additional hydration. This facilitates both the water and water/Mg-2+ 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.

Place, publisher, year, edition, pages
2017. Vol. 13, no 5, 921-938 p.
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-323445DOI: 10.1039/c6mb00762gISI: 000400659600011PubMedID: 28352859OAI: oai:DiVA.org:uu-323445DiVA: diva2:1119711
Funder
Swedish Research Council
Available from: 2017-07-04 Created: 2017-07-04 Last updated: 2017-07-04Bibliographically approved

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Plashkevych, OleksandrLi, QingChattopadhyaya, Jyoti
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