A 2'-O-methyl-RNA oligonucleotide containing a single free 2'-OH group flanking a branching phosphotriester linkage was prepared as a model for phosphate-branched RNA by using an orthogonally protected dimeric phosphoramidite building block in solid-phase synthesis. The strategy allows the synthesis of phosphate-branched oligonucleotides, the three branches of which may be of any desired sequence. Hydrolytic reactions of the phosphotriester linkages in such oligonucleotides were studied at physiological pH in the presence (and absence) of various complementary oligonucleotides. The fully hybridized oligonucleotide model is an order of magnitude more stable than its single-stranded counterpart, which, in turn, is an order of magnitude more stable than its trinucleoside phosphotriester core lacking any oligonucleotide arms. Furthermore, kinked structures obtained by hybridizing the phosphate-branched oligonucleotide with partially complementary oligonucleotides are three to five times more stable than fully double-stranded ones and only approximately three times less stable than the so-called RNA X structure, which has been postulated to incorporate an RNA phosphotriester linkage. The results indicate that when the intrinsically unstable RNA phosphotriester linkage is embedded in an oligonucleotide of appropriate tertiary structure, its half-life can be at least several hours.
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