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Chemically Modified Oligonucleotides: Synthesis, Physicochemical and Biochemical Properties of their Duplexes with DNA and RNA
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is based on 9 papers dealing with the synthesis, physicochemical and biochemical properties of two types of chemically modified oligonucleotides which have the potential to down-regulate gene expression: (i) The first set is comprised of antisense oligonucleotides (AONs) conjugated with different chromophores of varying size, charge and π-electron density. Conjugation of the chromophores at the 3'- or 5'-end enhanced the target RNA binding affinity and RNase H recruitment capabilities compared to the native counterpart without changing the global helical conformation of their AON/RNA hybrid duplexes. The 3'-dipyridophenazine (DPPZ) has emerged as the most promising non-toxic chromophore in this series. (ii) The second set encompasses a new class of AONs containing North-East conformationally constrained 1',2'-oxetane-nucleosides. The introduction of oxetane-T and -C units imparts lowering of the Tm by ~ 6º and ~ 3 ºC/modification, respectively, of the AON/RNA hybrids, whereas the incorporation of the corresponding oxetane-A and-G units into AONs did not alter the thermostability in comparison with that of the native hybrid duplex. The oxetane-modified AONs have been found to possess enhanced serum stability compared to that of the native, whereas oxetane-T and -C containing AONs were more endonuclease-resistant than oxetane-A and-G modified AONs. All oxetane-modified mixmer AON/ RNA hybrid duplexes were, however, found to be excellent substrates for RNase H cleavage, which has been analyzed by Michaelis-Menten kinetics. The oxetane-modified mixmer AONs have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells, which was analyzed by QRT-PCR and Western Blot. Based on the amount of AON uptake after delivery, determined by slot blot, it was apparent that the oxetane-modified AONs are 5-6 times more effective antisense agents than the corresponding isosequential phosphorothioate analogues. The electrochemical assay based on sensitive nucleic acid mediated charge transport (CT) has revealed that the presence of oxetane-T unit causes more stacking perturbations in a DNA/DNA duplex than in a DNA/RNA duplex.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2004. , p. 56
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 973
Keywords [en]
Bioorganic chemistry, Antisense oligonucleotides, Dipyridophenazine, Oxetane, RNase H, Gene down-regulation, DNase 1, Charge transport
Keywords [sv]
Bioorganisk kemi
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-4247ISBN: 91-554-5957-9 (print)OAI: oai:DiVA.org:uu-4247DiVA, id: diva2:164536
Public defence
2004-05-21, Floor 3, C10: 301, Biomedical Centre (BMC), Uppsala, 13:30
Opponent
Supervisors
Available from: 2004-04-26 Created: 2004-04-26Bibliographically approved
List of papers
1. The 3'-modified antisense oligos promote faster hydrolysis of the target RNA by RNase H than the natural counterpart
Open this publication in new window or tab >>The 3'-modified antisense oligos promote faster hydrolysis of the target RNA by RNase H than the natural counterpart
Show others...
2001 In: Tetrahedron, Vol. 57, p. 593-606Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91705 (URN)
Available from: 2004-04-26 Created: 2004-04-26Bibliographically approved
2. Transmission of the conformational information in the antisense/RNA hybrid duplex influences the pattern of the RNase H cleavage reaction
Open this publication in new window or tab >>Transmission of the conformational information in the antisense/RNA hybrid duplex influences the pattern of the RNase H cleavage reaction
2000 In: Tetrahedron Letters, Vol. 41, p. 8601-8607Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91706 (URN)
Available from: 2004-04-26 Created: 2004-04-26Bibliographically approved
3. Conformation-specific cleavage of antisense oligonucleotide-RNA duplexes by RNase H
Open this publication in new window or tab >>Conformation-specific cleavage of antisense oligonucleotide-RNA duplexes by RNase H
2001 In: Journal of Chemical Society, Perkin Transactions 2, p. 402-408Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91707 (URN)
Available from: 2004-04-26 Created: 2004-04-26Bibliographically approved
4. Oxetane modified antisense oligonucleotides promote RNase H cleavage of the complementary RNA strand in the hybrid duplex as efficiently as the native, and offer improved endonuclease resistance
Open this publication in new window or tab >>Oxetane modified antisense oligonucleotides promote RNase H cleavage of the complementary RNA strand in the hybrid duplex as efficiently as the native, and offer improved endonuclease resistance
2001 In: Journal of Chemical Society, Perkin Transactions 2, p. 2074-2083Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91708 (URN)
Available from: 2004-04-26 Created: 2004-04-26Bibliographically approved
5. Antisense oligonucleotides with oxetane-constrained cytidine enhance heteroduplex stability, elicit satisfactory RNase H response as well as show improved resistance to both exo and endonucleases
Open this publication in new window or tab >>Antisense oligonucleotides with oxetane-constrained cytidine enhance heteroduplex stability, elicit satisfactory RNase H response as well as show improved resistance to both exo and endonucleases
2003 In: Organic and Biomolecular Chemistry, Vol. 1, p. 81-92Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91709 (URN)
Available from: 2004-04-26 Created: 2004-04-26Bibliographically approved
6. Synthesis, physicochemical and biochemical studies of 1',2'-oxetane constrained adenosine and guanosine modified oligonucleotides, and their comparison with those of the corresponding cytidine and thymidine analogs
Open this publication in new window or tab >>Synthesis, physicochemical and biochemical studies of 1',2'-oxetane constrained adenosine and guanosine modified oligonucleotides, and their comparison with those of the corresponding cytidine and thymidine analogs
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In: Journal of American Chemical SocietyArticle in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-91710 (URN)
Available from: 2004-04-26 Created: 2004-04-26 Last updated: 2015-05-19Bibliographically approved
7. Rationally targeted, conformationally-constrained, oxetane modified oligonucleotides are highly efficient gene silencing molecules
Open this publication in new window or tab >>Rationally targeted, conformationally-constrained, oxetane modified oligonucleotides are highly efficient gene silencing molecules
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In: Proceedings of National Academy of Sciences, USAArticle in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-91711 (URN)
Available from: 2004-04-26 Created: 2004-04-26Bibliographically approved
8. Synthesis of [Ru(phen)2dppz]2+-tethered oligo DNA and studies on the metallointercalation mode into the DNA duplex
Open this publication in new window or tab >>Synthesis of [Ru(phen)2dppz]2+-tethered oligo DNA and studies on the metallointercalation mode into the DNA duplex
2001 In: Journal of American Chemical Society, Vol. 123, p. 3551-3562Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91712 (URN)
Available from: 2004-04-26 Created: 2004-04-26Bibliographically approved
9. An electrochemical probe of DNA stacking in an antisense oligonucleotide containing C3'-endo locked sugar
Open this publication in new window or tab >>An electrochemical probe of DNA stacking in an antisense oligonucleotide containing C3'-endo locked sugar
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2002 In: Angewandte Chemie International Edition, Vol. 41, p. 3402-3405Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91713 (URN)
Available from: 2004-04-26 Created: 2004-04-26Bibliographically approved

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