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-gammaS. 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.
Kang
Taek Jin
TJ
Research Center for Advanced Science and Technology, The University of Tokyo, 153-8904 Tokyo, Japan.
Suga
Hiroaki
H
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D019020:Directed Molecular Evolution; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011685:Purine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry*; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D011685:Purine Nucleotides; D016337:RNA, Catalytic; D012265:Ribonucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate