Synthesis and properties of triplex-forming oligonucleotides containing 2'-modified nucleoside analogues

Lou, Chenguang (2011) Synthesis and properties of triplex-forming oligonucleotides containing 2'-modified nucleoside analogues. University of Southampton, Chemistry, Doctoral Thesis, 229pp.

Abstract

Triplex-forming oligonucleotides (TFOs) bind to the major groove of the DNA duplex via the Hoogsteen interactions to generate triple helices. Potential applications of triplex technology are in regulation of gene expression, site-directed gene-knockout, mutation correction and as tools in molecular biotechnology. The presence of 2’-modified nucleosides in therapeutic oligonucleotides inhibits enzymatic degradation in vivo. Therefore such sugar modifications have the potential to improve the biological activity of TFOs. We have synthesized the phosphoramidite monomers of six 2’-modified nucleosides from D-ribose via 1-O-methyl-3,5-di-O-benzyl-?-D-ribofuranoside. Three of these are N-linked nucleosides: 2’-O-methoxyethyl-5-propargylamino-uridine (MEPU), 2’-O methoxyethyl-5-methyl-cytidine (MOE-5-MeC) and 2’-O-aminoethylthymidine (AE-T); and three are C-linked nucleosides: 3-methyl-2-amino-pyrdine-2’-O-methyl-ribonucleoside (Me-MAP), 3-methyl-2- amino-pyrdine-2’-O-methoxyethyl-ribonucleoside (MOE-MAP) and 3-methyl-2- amino-pyrdine-2’-O-aminoethyl-ribonucleoside (AE-MAP). These monomers were incorporated into a number of oligonucleotides, on which the biophysical and biochemical studies have been performed. TFOs containing the N-nucleoside (MEPU) showed high duplex affinity and strong nuclease resistance. Studies on two C-nucleosides (Me-MAP and MOE-MAP) revealed that their triplex stability was determined by the sequence context. The incorporation of Me-MAP and MOE-MAP into oligonucleotides renders them much more resistant to the degradation by serum nucleobases compared to their 2’-deoxy derivative (dMAP) and 2’-deoxycytidine (dC). AE-MAP is a promising triplex stabilizer, which not only shows the highest triplex stabilization, but also displays an impressive resistance to enzymatic degradation

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