Abstract
Oligoribonucleotides containing a photocaged 2′-amino-5′-S-phophorothiolate linkage have potential applications as therapeutic agents and biological probes to investigate the RNA structure and function. We envisioned that oligoribonucleotides containing a 2′-amino-5′-S-phosphorothiolate linkage could provide an approach to identify the general base within catalytic RNAs by chemogenetic suppression. To enable preliminary tests of this idea, we developed synthetic approaches to a dinucleotide, trinucleotide, and oligoribonucleotide containing a photocaged 2′-amino-5′-S-phosphorothiolate linkage. We incorporated the photocaged 2′-amino-5′-S-phosphorothiolate linkage into an oligoribonucleotide substrate for the hepatitis delta virus (HDV) ribozyme and investigated the pH dependence of its cleavage following UV irradiation both in the presence and absence of the ribozyme. The substrate exhibited a pH-rate profile characteristic of the modified linkage but reacted slower when bound to the ribozyme. Cleavage inhibition by the HDV ribozyme could reflect a non-productive ground-state interaction with the modified substrate’s nucleophilic 2′-NH2 or a poor fit of the modified transition state at the ribozyme’s active site.
Highlights
The synthesis of modified nucleosides, nucleotides, and oligonucleotides has been extensively investigated and motivated, in part, by creation of potential therapeutic agents[1−6] and biological probes for the investigation of the relationship between the RNA structure and function.[7]
The reaction mixture was stirred at rt overnight
The reaction mixture was stirred at rt for 16 h
Summary
The synthesis of modified nucleosides, nucleotides, and oligonucleotides has been extensively investigated and motivated, in part, by creation of potential therapeutic agents (antisense, antiviral, and anticancer agents)[1−6] and biological probes for the investigation of the relationship between the RNA structure and function.[7]. Owing to the weak nucleophilicity of the amino group toward the adjacent phosphodiester bond, 2′-amino substitution renders the ribose phosphate backbone inert to cleavage via internal transphosphorylation.[11] Analogously, substitution of the 5′-bridging oxygen atom of the phosphodiester linkage with a sulfur atom (Figure 1B) alters hydrogen bonding, metal-ion coordination properties, and leaving group ability. In contrast to the 2′-amino group, a 5′-sulfur renders the phosphodiester backbone much more susceptible to transphosphorylation, owing to the greater leaving ability of sulfur relative to oxygen. This hyperactivation of the leaving group underpins a chemogenetic strategy to identify groups that activate the 5′oxygen leaving group within the active site of a biological catalyst.[12−14]
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