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Synthesis and Structure of Novel Conformationally Constrained 1',2'-Azetidine-Fused Bicyclic Pyrimidine Nucleosides: Their Incorporation into Oligo-DNAs and Thermal Stability of the Heteroduplexes
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
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2006 (English)In: Journal of Organic Chemistry, ISSN 0022-3263, Vol. 71, 299-314 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2006. Vol. 71, 299-314 p.
URN: urn:nbn:se:uu:diva-97297OAI: oai:DiVA.org:uu-97297DiVA: diva2:172169
Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2015-05-19Bibliographically approved
In thesis
1. Conformationally Constrained Nucleosides, Nucleotides and Oligonucleotides: Design, Synthesis and Properties
Open this publication in new window or tab >>Conformationally Constrained Nucleosides, Nucleotides and Oligonucleotides: Design, Synthesis and Properties
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is based on six original research publications describing synthesis, structure and physicochemical and biochemical analysis of chemically modified oligonucleotides (ONs) in terms of their potential diagnostic and therapeutic applications. Synthesis of two types of bicyclic conformationally constrained nucleosides, North-East locked 1',2'-azetidine and North locked 2',4'-aza-ENA, is described. Study of the molecular structures and dynamics of bicyclic nucleosides showed that depending upon the type of fused system they fall into two distinct categories with their respective internal dynamics and type of sugar conformation. The physicochemical properties of the nucleobases in the conformationally constrained nucleosides found to be depended on the site and ring-size of the fused system.

The incorporation of azetidine modified nucleotide units into 15mer ONs lowered the affinity toward the complementary RNA. However, they performed better than previously reported isosequential 1',2'-oxetane modified analogues. Whereas aza-ENA-T modification incorporated into ONs significantly enhanced affinity to the complementary RNA. To evaluate the antisense potential of azetidine-T and aza-ENA-T modified ONs, they were subjected to RNase H promoted cleavage as well as tested towards nucleolytic degradation. Kinetic experiments showed that modified ONs recruit RNase H, however with lower enzyme efficiency due to decreased enzyme-substrate binding affinity, but with enhanced turnover number. Both, azetidine-T and aza-ENA-T modified ONs demonstrated improved 3'-exonuclease stability in the presence of snake venom phosphodiesterase and human serum compared to the unmodified sequence.

Oligodeoxynucleotides (ODNs) containing pyrene-functionalized azetidine-T (Aze-pyr X) and aza-ENA-T (Aza-ENA-pyr Y) modifications showed different fluorescence properties. The X modified ODNs hybridized to the complementary DNA and RNA showed variable increase in the fluorescence intensity depending upon the nearest-neighbor at the 3'-end to X modification (dA > dG > dT > dC) with high fluorescence quantum yield. However, the Y modified ODNs showed a sensible enhancement of the fluorescence intensity only with complementary DNA. Also, the X modified ODN showed decrease (~37-fold) in the fluorescence intensity upon duplex formation with RNA containing a G nucleobase mismatch opposite to the modification site, whereas a ~3-fold increase was observed for the Y modified probe.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 71 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 440
Bioorganic chemistry, antisense oligonucleotides, conformationally constrained nucleosides, azetidine, aza-ENA, target affinity, RNase H, exonuclease stability, pyrene-functionalized nucleotides, fluorescence, mismatch discrimination, Bioorganisk kemi
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-8887 (URN)978-91-554-7219-1 (ISBN)
Public defence
2008-06-05, C10:301, BMC, Husargatan 3, Uppsala, 13:00 (English)
Available from: 2008-05-14 Created: 2008-05-14 Last updated: 2010-03-03Bibliographically approved

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