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Synthesis of RNA Using 2'-O-DTM Protection
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
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2006 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 38, 12356-12357 p.Article in journal (Refereed) Published
Abstract [en]

tert-Butyldithiomethyl (DTM), a novel hydroxyl protecting group, cleavable under reductive conditions, was developed and applied for the protection of 2′-OH during solid-phase RNA synthesis. This function is compatible with all standard protecting groups used in oligonucleotide synthesis, and allows for fast and high-yield synthesis of RNA. Oligonucleotides containing the 2′-O-DTM groups can be easily deprotected under the mildest possible aqueous and homogeneous conditions. The preserved 5′-O-DMTr function can be used for high-throughput cartridge RNA purification.

Place, publisher, year, edition, pages
2006. Vol. 128, no 38, 12356-12357 p.
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-94935DOI: 10.1021/ja0636587ISI: 000240612700003PubMedID: 16984152OAI: oai:DiVA.org:uu-94935DiVA: diva2:168961
Available from: 2006-10-02 Created: 2006-10-02 Last updated: 2011-06-10Bibliographically approved
In thesis
1. Novel Methods for Synthesis of High Quality Oligonucleotides
Open this publication in new window or tab >>Novel Methods for Synthesis of High Quality Oligonucleotides
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The first part of the work describes a procedure of oligonucleotide purification using a reversed-phase cartridge. The developed method employs a very efficient yet mild oligonucleotide detritylation on the cartridge support allowing fast purification of oligonucleotides regardless of their 5´-modification. Thiol- and amino-modified oligonuc-leotides were detritylated and purified with the same high efficiency as non-modified oligonucleotides. The method enables fast, parallel and automated purification of many oligonucleotide probes that was not possible before. In combination with the method of removal of tritylated failure fragments oligonucleotides were produced with purity superior to that of oligonucleotides purified using RP HPLC.

In the second part of the present study a method of solid-phase RNA synthesis using 2´-tert-butyldithiomethyl (2´-O-DTM) is discussed. The stability of the DTM group during oligonucleotide assembly and deprotection in ammonia, together with its ability for rapid deprotection under mild conditions, allowed the synthesis of RNA with the quality similar to that of synthetic DNA oligonucleotides. The advantage of the 2´-O-DTM group is that it is completely orthogonal to all protecting groups used for the traditional solid-phase DNA synthesis. Therefore, the synthesis can be performed using a standard DNA synthesis procedure – no changes are needed for the product assembly. RNA oligonucleotides synthesized with retained 5´-terminal trityl group can be subjected to a cartridge-based purification using the procedure described in the first part of the study. The phosphoramidite synthesis was optimized for a large scale preparation and gives versatility for introduction of other alkyldithiomethyl groups according to the preference to their certain properties.

The third part of the thesis describes the synthesis of a dithiomethyl linker and its utility for reversible conjugation of oligonucleotides. A dithiomethyl group, cleavable under mild conditions, was introduced onto 3´-OH of tritylated nucleosides via 3´-O-methylthiomethyl derivatives. The influence of different alkyl substituents on the disulfide bond stability was investigated, and stable analogues were employed in oligosyntheses. Two applications were developed using the present linker: 1) purification of oligonucleotides linked to the solid support; and 2) cartridge-based purification of tritylated oligonucleotides having an additional hydrophobic group on their 3´- terminus.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 62 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 179
Organic chemistry, oligonucleotide, solid-phase synthesis, RNA synthesis, phosphoramidite, abasic sites, depurination, 2'-O-DTM, protecting group, cartridge, conjugate, dithiomethyl linker, base-stable linker, oligonucleotide purification, nucleoside, Organisk kemi
urn:nbn:se:uu:diva-7172 (URN)91-554-6672-9 (ISBN)
Public defence
2006-10-23, Rudbecksalen, Rudbeck Laboratory, Dag Hammarskjölds väg, 20, Uppsala, 09:15
Available from: 2006-10-02 Created: 2006-10-02Bibliographically approved

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