Preparation of group I introns for biochemical studies and crystallization assays by native affinity purification.
The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides -- some containing exons -- were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3' overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules.
Vicens
Quentin
Q
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA. quentin.vicens@colorado.edu
Gooding
Anne R
AR
Duarte
Luis F
LF
Batey
Robert T
RT
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D003014:Clostridium botulinum / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005798:Genes, Bacterial; D007438:Introns; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D014158:Transcription, Genetic
D012329:RNA, Bacterial