In vitro selection of a 5′-purine ribonucleotide transferase ribozyme

Here we report in vitro selection of a novel ribozyme that catalyzes the 5 '-nucleotidyl transfer reaction forming the 2 '-5 ' phosphodiester bond. This ribozyme was retrieved as a sole sequence in the pool enriched for the 5 '-triphosphate-dependent activities in incorporating ATP-gamma S. The originally selected ribozyme consisting of 109-nucleotide (nt) was miniaturized to 45-nt M4 ribozyme via a series of mutation studies, and based on this mini-ribozyme a trans-acting system was constructed. One of the most challenging tasks in our study was to determine the chemistry occurring at the 5 '-ppp site. We utilized various analytical methods including MALDI-TOF analysis of the product generated by the trans-acting system and elucidated the chemistry to be 3 '-> 5 ' mononucleotide extension forming the 2 '->-5 ' phosphodiester bond. Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5 '-mono-, di- and triphosphates as substrates. This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5 '-modification of RNAs with unique chemical groups.