Oligonucleotides (ON) are commonly used as research reagents to modulate gene expression in cell culture and in animal models. Various chemistries of ON have been synthesized, incorporating modifications to the phosphodiester backbone, sugar or nucleobases portions. Oligonucleotides 2’-O-Methyl-RNA-Phosphorothioates (2’-OMePS) can modulate gene expression through splicing arrest of pre-mRNA and inducing the skipping of specific exons. In these years, we have set up a fully equipped laboratory and we have developed efficient and reliable protocols for the synthesis and purification of 2’-OMePS full length oligonucleotides. All synthesis were performed on a GE ÄKTATM oligopilot plus 10 fully automated synthesizer in the 1 to 50 μmol scale range using a polystyrene-based solid support at different loading, and β-cyanoethyl phosphoroamidite synthons. All purification were performed on a ÄKTA basic UPC HPLC system by reverse phase chromatography. Furthermore, we have studied the synthesis of news 2’-O-bile acids (AB) conjugated nucleosides, because in future we will insert these building blocks in the automated synthesis process to obtain different 2’-Oconjugates oligonucleotides containing one or more units of AB for improvement of cellular uptake efficiency.
Sintesi e Purificazione di Oligonucleotidi e Nucleosidi 2’- funzionalizzati
MARI, Lara
2009
Abstract
Oligonucleotides (ON) are commonly used as research reagents to modulate gene expression in cell culture and in animal models. Various chemistries of ON have been synthesized, incorporating modifications to the phosphodiester backbone, sugar or nucleobases portions. Oligonucleotides 2’-O-Methyl-RNA-Phosphorothioates (2’-OMePS) can modulate gene expression through splicing arrest of pre-mRNA and inducing the skipping of specific exons. In these years, we have set up a fully equipped laboratory and we have developed efficient and reliable protocols for the synthesis and purification of 2’-OMePS full length oligonucleotides. All synthesis were performed on a GE ÄKTATM oligopilot plus 10 fully automated synthesizer in the 1 to 50 μmol scale range using a polystyrene-based solid support at different loading, and β-cyanoethyl phosphoroamidite synthons. All purification were performed on a ÄKTA basic UPC HPLC system by reverse phase chromatography. Furthermore, we have studied the synthesis of news 2’-O-bile acids (AB) conjugated nucleosides, because in future we will insert these building blocks in the automated synthesis process to obtain different 2’-Oconjugates oligonucleotides containing one or more units of AB for improvement of cellular uptake efficiency.File | Dimensione | Formato | |
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