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…
Contents
Introduction
Solid-phase oligonucleotide synthesis
5´ Hydroxyl protection
Aglycone protection
Phosphate protection
2´ Hydroxyl protection
Oligosynthesis cycle and deprotection conditions
Purification of oligonucleotides
Functionalized solid support as a purification aid
Oligonucleotide purification using RPC
Present work
Paper I. A method for RPC oligonucleotide purification
Paper II. Solid-phase RNA synthesis using 2´-O-tert-butyldithiomethyl (2´-O-DTM) protecting group
Paper III and IV. Synthesis of 3´-O-alkyldithiomethyl analogues and their
application in oligonucleotide synthesis
Summary in Swedish
Acknowledgements
References
Author: Semenyuk, Andrey
Source: Uppsala University Library
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