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 underrepresented in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-γS, and thus it could have been undiscovered previously using similar selection strategies. 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.
저자
Taek Jin KANG [ Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. ]
Hiroaki SUGA [ Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan. ]
한국생물공학회 [The Korean Society for Biotechnology and Bioengineering]
설립연도
1984
분야
공학>생물공학
소개
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