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Significant pKa Perturbation of Nucleobases Is an Intrinsic Property of the Sequence Context in DNA and RNA
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
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2004 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 28, 8674-8681 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2004. Vol. 126, no 28, 8674-8681 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96321DOI: 10.1021/ja048484cOAI: oai:DiVA.org:uu-96321DiVA: diva2:170858
Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Targeting RNA by the Antisense Approach and a Close Look at RNA Cleavage Reaction
Open this publication in new window or tab >>Targeting RNA by the Antisense Approach and a Close Look at RNA Cleavage Reaction
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis summarizes the results of studies on two aspects of nucleic acids. Chemically modified antisense oligonucleotides (AONs) have been evaluated with regards to their suitability for mRNA targeting in an antisense approach (Paper I – III). The chemically modified nucleotidic units 2'-O-Me-T, 2'-O-MOE-T, oxetane-T, LNA-T, azetidine-T, aza-ENA-T, carbocyclic-ENA-T and carbocyclic-LNA-T were incorporated into 15-mer AONs and targeted against a 15-mer RNA chosen from the coding region of SV-40 large T antigen. The comparative study showed that a single modified nucleotide in the AON with North-East locked sugar (oxetane-T and azetidine-T) lowered the affinity for the complementary RNA whereas North locked sugars (LNA-T, aza-ENA-T, carbocyclic-ENA-T, and carbocyclic-LNA-T) significantly improved the affinity. A comparative RNase H digestion study showed that modifications of the same type (North-East type or North type) in different sequences gave rise to similar cleavage patterns. Determination of the Michaelis-Menten parameters by kinetic experiments showed that the modified AONs recruit RNase H resulting in enhanced turnover numbers (kcat) although with weaker enzyme-substrate binding (1/Km) compared to the unmodified AON. The modified AONs were also evaluated with regards to resistance towards snake venom phosphodiesterase and human serum to estimate their stability toward exonucleases. The aza-ENA-T and carbocyclic-ENA-T modified AONs showed improved stability compared to all other modified AONs. In general, the modified AONs with North type nucleotides (except LNA-T) were found to be superior to the North-East type as they showed improved target affinity, comparable RNase H recruitment capability and improved exonuclease stability.

The second aspect studied in this thesis is based on physicochemical studies of short RNA molecules utilizing NMR based pH titration and alkaline hydrolysis reactions (Paper IV – V). The NMR based (1H and 31P) pH titration studies revealed the effect of guaninyl ion formation, propagated electrostatically through a single stranded chain in a sequence dependent manner. The non-identical electronic character of the internucleotidic phosphodiesters was further verified by alkaline hydrolysis experiments. The internucleotidic phosphodiesters, which were influenced by guaninyl ion formation, were hydrolyzed at a faster rate than those sequences where such guaninyl ion formation was prevented by replacing G with N1-Me-G.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 355
Keyword
Organic chemistry, mRNA targeting, antisense oligonucleotides, target affinity, RNase H, Michaelis-Menten kinetics, exo-nuclease stability, NMR, pH titration, alkaline hydrolysis, Organisk kemi
National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-8272 (URN)978-91-554-6995-5 (ISBN)
Public defence
2007-11-08, B22, BMC, Box 576, SE-75123, Uppsala, 09:15 (English)
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Available from: 2007-10-18 Created: 2007-10-18 Last updated: 2012-08-03Bibliographically approved

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