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Aspects of Antisense and Antigene Chemistry of Oligonucleotides Tethered to Intercalators
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Synthetic and physicochemical studies on appropriately functionalized ODN-conjugates have been performed to evaluate their abilities to act as antisense agents against RNA or as intramolecular DNA cross-linking agents. Intercalating aromatic systems [phenazine (Pnz), dipyridophenazine (DPPZ)] and metallointercalators such as Ru2+(phen)2(DPPZ) and Ru2+(tpy)(DPPZ)L [where L = chemically or photochemically labile ligand, phen = phenanthroline, tpy = terpyridine], which are covalently tethered to the oligo-deoxynucleotides (ODNs), have been chosen for this purpose. The ODN-conjugates were typically prepared by automated solid phase synthesis using phosphoramidite building blocks, or on solid supports, both functionalized with the chromophore groups. The photosensitive metal complex, Ru2+(tpy)(DPPZ)(CH3CN), has been incorporated by post-synthetic coupling to the amino-linker modified ODNs via an amide bond. The intercalating ability of the tethered chromophores gave enhanced stability of the duplexes and triplexes formed with ODN-conjugates and their complementary targets: DNA, RNA, or double-stranded DNA. The conjugation of DPPZ chromophore to ODN (at 3', 5' or at the middle) led us to incorporate Ru2+(phen)2(DPPZ) through the DPPZ ligand, for the first time. The corresponding (Ru2+-ODN)•DNA duplexes showed dramatic stabilization (ΔTm = 19.4 – 22.0ºC). The CD and DNase I footprinting experiments suggest that the stabilization is owing to metallointercalation by threading of the Ru2+(phen)2 moiety through the ODN•DNA duplex core, thus "stapling" the two helical strands from the minor to major groove. On the other hand, Ru2+(tpy)(DPPZ)(CH3CN)-ODN conjugates represent a new class of oligonucleotides containing the photoactivatible Ru2+ complexes, which can successfully crosslink to the complementary strand. The mechanism of cross-linking upon photoirradiation of [Ru2+(tpy)(DPPZ)(CH3CN)-ODN]•DNA involves in situ conversion to the reactive [Ru2+(tpy)(DPPZ)(H2O)-ODN]•DNA which are subsequently cross-linked through the G residue of the complementary DNA strand. All starting materials and products have been purified by HPLC and/or by PAGE and subsequently characterized by MALDI-TOF as well as ESI mass spectroscopy. Terminal conjugation of the planar Pnz and DPPZ groups through the flexible linkers were also shown to improve thermal stability of the ODN•RNA hybrid duplexes without alteration of the initial AB-type global helical structure as revealed from CD experiments. As a result, RNase H mediated cleavage of the RNA strand in the intercalator-tethered ODN•RNA duplexes was more efficient compared to the natural counterpart. The RNase H cleavage pattern was also found to be dependent on the chemical nature of the chromophore. It appeared that introduction of a tether at the 3'-end of the ODN can be most easily tolerated by the enzyme regardless of the nature of the appending chromophore. The tethered DPPZ group has also been shown to chelate Cu2+ and Fe3+, like phenanthroline group, followed by the formation of redox-active metal complex which cleaves the complementary DNA strand in a sequence-specific manner. This shows that the choice of appropriate ligand is useful to (i) attain improved intercalation giving Tm enhancement, and (ii) sequence-specifically inactivate target RNA or DNA molecules using multiple modes of chemistry (RNase H mediated cleavage, free-radical, oxidative pathways or photocross-linkage).

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2002. , p. 67
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 735
Keywords [en]
Bioorganic chemistry, oligonucleotides, intercalators, ruthenium complexes, RNase H cleavage, photocross-linkage
Keywords [sv]
Bioorganisk kemi
National Category
Organic Chemistry
Research subject
Biorganic Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-2220ISBN: 91-554-5365-1 (print)OAI: oai:DiVA.org:uu-2220DiVA, id: diva2:161831
Public defence
2002-10-05, lecture hall B 21 (corridor A2, floor 1) at the Biomedical Center, Uppsala University, Uppsala, 10:00
Opponent
Available from: 2002-08-21 Created: 2002-08-21Bibliographically approved
List of papers
1. Synthesis of 1'-Phenazine-Tethered Psicofuranosyl Oligonucleotides: The Thermal Stability and Fluorescence Properties of Their Duplexes and Triplexes
Open this publication in new window or tab >>Synthesis of 1'-Phenazine-Tethered Psicofuranosyl Oligonucleotides: The Thermal Stability and Fluorescence Properties of Their Duplexes and Triplexes
1998 In: TetrahedronArticle in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89935 (URN)
Available from: 2002-08-21 Created: 2002-08-21Bibliographically approved
2. Dipyrido[3,2-a:3',2'-c]phenazine-Tethered OligoDNA: Synthesis and Thermal Stability of Their DNA•DNA and DNA•RNA Duplexes and DNA•DNA•DNA Triplexes.
Open this publication in new window or tab >>Dipyrido[3,2-a:3',2'-c]phenazine-Tethered OligoDNA: Synthesis and Thermal Stability of Their DNA•DNA and DNA•RNA Duplexes and DNA•DNA•DNA Triplexes.
1999 In: Helvetica Chimica ActaArticle in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89936 (URN)
Available from: 2002-08-21 Created: 2002-08-21Bibliographically approved
3. 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.
2001 In: TetrahedronArticle in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89937 (URN)
Available from: 2002-08-21 Created: 2002-08-21Bibliographically approved
4. Synthesis of [Ru(phen)2(dppz)]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)2(dppz)]2+-Tethered Oligo-DNA and Studies on the Metallointercalation Mode into the DNA Duplex.
2001 In: American Chemical SocietyArticle in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89938 (URN)
Available from: 2002-08-21 Created: 2002-08-21Bibliographically approved
5. First Synthesis and Reactivity of the DNA-[Ru(tpy)(dppz)(CH3CN)]2+ Conjugates
Open this publication in new window or tab >>First Synthesis and Reactivity of the DNA-[Ru(tpy)(dppz)(CH3CN)]2+ Conjugates
2002 In: Journal of the American Chemical SocietyArticle in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-89939 (URN)
Available from: 2002-08-21 Created: 2002-08-21Bibliographically approved

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