Boronic Acid Assisted Self-Assembly of Functional RNAs

Abstract

Boronate esters formed by reaction of an oligonucleotide carrying a 5′-boronic acid moiety with the 3′-terminal cis-diol of another have been shown previously to assist assembly of fragmented DNAzymes. Here we demonstrate that boronate esters replacing the natural phosphodiester linkage at selected sites of two functional RNAs, the hairpin ribozyme and the Mango aptamer, allow assembly of functional structures. The hairpin ribozyme, a small naturally occurring RNA that supports the reversible cleavage of appropriate RNA substrates, is very sensitive to fragmentation. Splitting the ribozyme at four different sites led to a significant decrease or even loss of cleavage and ligation activity. Ribozymes assembled from fragments capable of boronate ester formation showed restoration of cleavage activity in some but not all cases, dependent on the split site. Ligation proved to be more challenging, no supportive effect of the boronate ester was observed. Split variants of the Mango aptamer also showed a dramatic loss of functionality, which however, was restored when 5′-boronic acid modified fragments were used for assembly. These studies show for the first time that boronate esters as internucleoside linkages can act as surrogates of natural phosphodiesters in functional RNA molecules.