In vitro evolution of new ribozymes with polynucleotide kinase activity.
We have isolated a large number of polynucleotide kinase ribozymes from a pool of RNA molecules consisting of an ATP-binding domain flanked by regions of random sequence. Different classes of kinases catalyse the transfer of the gamma-thiophosphate of ATP-gamma S to the 5'-hydroxyl or to internal 2'-hydroxyls. An engineered version of one class is able to catalyse the transfer of thiophosphate from ATP-gamma S to the 5'-hydroxyl of an exogenous oligoribonucleotide substrate with multiple turnover, thus acting as a true enzyme.
Lorsch
J R
JR
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Szostak
J W
JW
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D005075:Biological Evolution; D002384:Catalysis; D003001:Cloning, Molecular; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000145:classification / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D012641:Selection, Genetic; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013873:Thionucleotides / Q000378:metabolism
D016337:RNA, Catalytic; D013873:Thionucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D012313:RNA; D000255:Adenosine Triphosphate; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D015719:Single-Strand Specific DNA and RNA Endonucleases