RNA aptamers are structured motifs that bind to specific molecules. A growing number of RNAs bearing aptamer elements, whose functions are modulated by direct binding of metabolites, have been found in living cells. Recent studies have suggested that more small RNAs binding to metabolites likely exist and may be involved in diverse cellular processes. However, conventional methods are not necessarily suitable for the discovery of such RNA aptamer elements in small RNAs with lengths ranging from 50 to 200 nucleotides, due to the far more abundant tRNAs in this size range. Here, we describe a new in vitro selection method to uncover naturally occurring small RNAs capable of binding to a ligand of interest, referred to as small RNA transcriptomic SELEX (smaRt-SELEX). By means of this method, we identified a motif in human precursor microRNA 125a (hsa-pre-miR-125a) that interacts with folic acid. Mutation studies revealed that the terminal loop region of hsa-pre-miR-125a is important for this binding interaction. This method has potential for the discovery of new RNA aptamer elements or catalytic motifs in biological small RNA fractions.

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By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids.

The means of in vitro selection has yielded a number of artificial ribozymes with functions that have not been discovered as yet in modern biological systems. Like naturally occurring ribozymes, most artificial ribozymes also use metal ions for the support of catalysis. Here we choose two such ribozymes, flexizyme and ribox, that exhibit specific activities of tRNA aminoacylation and redox chemistry, respectively, and comprehensively summarize the roles of metal ions in conjunction with their structure and function.

Here we report engineering efforts on an artificially evolved redox ribozyme, known as ribox02, and its more compact pseudoknot construct generated, combox02. The rigorous mutational works in the various regions suggested that the catalytically essential domain composed of three joining regions flanked by three stems. The architecture of combox02 can be an excellent framework for constructing libraries by randomizing sequences in this region for in vitro selection of novel ribozymes capable of catalyzing various chemical conversions of 5′-substrates.