Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates

Abstract

The chemical synthesis of oligoribonucleotides containing vicinal (2'-5')- and (3'-5')-phosphodiester linkages is described. The solid-phase method, based on silyl-phosphoramidite chemistry, was applied to the synthesis of a series of branched RNA [(Xp)nA2' (pN)n3'(pN)n] related to the splicing intermediates derived from Saccharomyces cerevisiae rp51a pre-messenger RNA. The branched oligonucleotides have been thoroughly characterized by nucleoside and branched nucleotide composition analysis. Branched oligoribonucleotides will be useful in the study of messenger RNA splicing and in determining the biological role of RNA 'lariats' and 'forks' in vivo.

Chemical synthesis of an artificially branched hairpin ribozyme variant with RNA cleavage activity

“…19,20 However, the routinely used and easily removable phenoxyacetyl (PAC) groups for N-protection 21 turned out to be too labile under the conditions of hydrazinolysis. Therefore, more stable N-protecting groups were used: benzoyl for A and C, 17 and dimethylformamidine for G. 21,22 The half life of N-benzoyl protected adenosine and cytidine under the conditions of hydrazinolysis was determined to be about 3 and 5 h, respectively, while the dimethylformamidine group was stable for the time of observation. Removal of the levulinyl group by hydrazinolysis takes about 5 min, hence leaving the major part of the N-protected nucleobases intact.…”

Solid-Phase Synthesis of Transition Metal Linked, Branched Oligonucleotides

“…To optimize the yield of 1, our convergent solid-phase strategy requires close proximity of the 5'-OH termini of growing oligonucleotides on the solid support (Scheme 2). [7] Hence, high-density CPG support (56 mmol g À1 ) was used. Bis-phosphoramidite 3 in CD 2 Cl 2 was then added to the solid support under an inert atmosphere, with tetrazole as activator to achieve branching.…”

“…[ Titanaazacyclobutenes and titanaazacyclobutanes are proposed as intermediates in the catalytic cycle, [2a, 4, 5] in analogy to the [Cp 2 Zr(NHR) 2 ]-catalyzed hydroamination of alkynes [6] or the stoichiometric reaction of allenes with zirconium and titanium pyridine imido derivatives. [7] However, the mechanism of the protonation of the titanacycle by the amine is not well understood. Here we report high-level density functional model calculations on the intermediates involved in this step.…”

“…The ruthenium moiety is both redox active and intensely luminescent, and it has been extensively used as a probe of electron transfer processes in proteins. [6] Our convergent solidphase strategy [7] for the preparation of ruthenium-linked parallel oligonucleotides is outlined in Schemes 1 and 2. This strategy starts with the generation of a ruthenium bisphosphoramidite branching complex 3.…”

A general two-step strategy to synthesize lariat RNAs

“…Solid-phase synthesis methods to prepare even the relatively simple 2¢,5¢-branched RNA are tedious (Damha and Ogilvie 1988;Damha and Zabarylo 1989;Damha et al 1992;Ganeshan et al 1995;Reese and Song 1999), and the closed loop of a lariat provides an added measure of complexity (Carriero and Damha 2003). Group II intron ribozymes can synthesize lariats, but numerous sequence requirements for the RNA substrates limit the applicability of this approach.…”