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- aptamer (2)
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The vast majority of RNA splicing in today‘s organisms is achieved by the highly regulated and precise removal of introns from pre-mRNAs via the spliceosome. Here we present a model of how RNA splicing may have occurred in earlier life forms. We have designed a hairpin ribozyme derived spliceozyme that mediates two RNA cleavages and one ligation event at specific positions and thus cuts a segment (intron) out of a parent RNA and ligates the remaining fragments (exons). The cut-out intron then performs a downstream function, acting as a positive regulator of the activity of a bipartite DNAzyme. This simple scenario shows how small RNAs can perform complex RNA processing dynamics, involving the generation of new phenotypes by restructuring segments of given RNA species, as well as delivering small RNAs that may play a functional role in downstream processes.
Boronate esters, formed by the reaction of an oligonucleotide bearing a 5′-boronic acid moiety with the 3′-terminal cis-diol of another oligonucleotide, support the assembly of functional nucleic acid architectures. Reversible formation of boronate esters occurs in templated fashion and has been shown to restore the activity of split DNA and RNA enzymes as well as a split fluorescent light-up aptamer. Apart from their suitability for the design and application of split nucleic acid enzymes and aptamers in the field of biosensing, boronate esters may have played an important role in early life as surrogates of the natural phosphodiester bond. Their formation is reversible and thus fulfills an important requirement for biological self-assembly. Here we discuss the general concept of stimuli-dependent boronate formation and its application in biomolecules with implications for future research.
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.