Automated chemical synthesis of RNA

Miriam Catherine Estanislao Schwartz, Purdue University

Abstract

The chemical synthesis of RNA requires an efficient protecting system for the 2$\sp\prime$-hydroxyl on the sugar ring of ribonucleosides. A novel blocking group, o-nitrobenzyloxymethyl, has been studied in our laboratory for this purpose. Protected nucleosides, 5$\sp\prime$-O-dimethoxytrityl-2$\sp\prime$-O-(o-nitrobenzyloxymethyl) derivatives of uridine, 4-N-benzoylcytidine, 6-N-benzoyladenosine, and 2-N-isobutyrylguanosine, have been prepared using o-nitrobenzyl chloromethyl ether, an alkylating agent newly developed for this research project. The monomers can be converted into their corresponding 3$\sp\prime$-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) for use in automated synthesis. Purification of these compounds, and of the various synthetic intermediates, employed silica gel chromatography. Their structures and purity have been confirmed by elemental analysis, mass spectroscopy, thin layer chromatography, and nuclear magnetic resonance spectroscopy ($\sp1$H, $\sp{13}$C, and $\sp{31}$P nuclei). A number of oligoribonucleotides have been constructed using the 2$\sp\prime$-O-(o-nitrobenzyloxymethyl) monomers; RNA oligomers can be made with coupling efficiencies and reaction times comparable to DNA synthesis when standard conditions of the phosphoramidite method are employed. Following ammonia treatment to deprotect the base moieties, selective removal of the o-nitrobenzyloxymethyl is carried out easily by exposure to long-wave ultraviolet light. A variety of biologically-active RNA molecules have been synthesized using the new methodology. These include a hammerhead-motif ribozyme composed of a catalytic oligomer and a natural substrate oligomer with the latter containing a cytidine at the point of breakage. In addition, three substrate analogs were prepared. These contained in place of the cytidine the nucleosides, 2$\sp\prime$-deoxy-2$\sp\prime$-fluorocytidine, 2$\sp\prime$-deoxycytidine, and cytidine with a 2$\sp\prime$-5$\sp\prime$ phosphodiester bond at the cleavable linkage, which were expected to confer cleavage resistance. Upon combining the catalyst and natural substrate, hydrolysis of the labile phosphodiester bond was observed while mixtures containing any of the substrate analogs with the catalyst showed no cleavage activity. These results indicate that the presence of a hydroxyl at the 2$\sp\prime$-position of the cytidine residue is an absolute requirement for cleavage of the hammerhead ribozyme.

Degree

Ph.D.

Advisors

Gilham, Purdue University.

Subject Area

Molecular biology|Biochemistry|Organic chemistry

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