Open AccessKinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions.
Author(s) -
David M. Long,
Olke C. Uhlenbeck
Publication year - 1994
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.91.15.6977
Subject(s) - hammerhead ribozyme , intramolecular force , stem loop , ribozyme , hairpin ribozyme , rna , cleavage (geology) , cleave , intermolecular force , chemistry , nucleotide , stereochemistry , biology , molecule , biochemistry , gene , dna , paleontology , organic chemistry , fracture (geology)
A method is described to obtain intramolecular cleavage rates for the hammerhead ribozyme during in vitro transcription. By avoiding RNA purification and renaturation, the potential for formation of inactive conformations of the RNA is minimized. By showing that an intramolecular hammerhead and a closely related intermolecular hammerhead cleave at the same rate under a given set of conditions, we confirm that both reactions probably have the same rate-limiting step. An in vitro selection strategy was used to isolate active hammerheads from a library of molecules where six randomized nucleotides replaced stem-loop II. The sequence and number of nucleotides which replace stem-loop II have large effects on hammerhead cleavage activity. The relative activities of three sequences selected from the intramolecular library are the same when the sequences are transferred into an intermolecular hammerhead background.