Capillary electrophoresis of RNA oligonucleotides: catalytic activity of a hammerhead ribozyme.
Ribozymes are sequences of catalytic RNA that are being evaluated as possible antisense therapeutics. This paper describes how capillary electrophoresis (CE) could be used to measure the catalytic rate of a synthetic hammerhead ribozyme in cleaving its substrate. This substrate was a synthetic full-RNA 17-mer, whereas the ribozyme was made up of a mixture of 37 2'-OH and 2'-OCH3 RNA nucleotides. After experimental conditions to exclude ribonuclease contamination were successfully met, different CE modes were tried out to separate the ribozyme from its substrate. Only the combination of chemical and thermal denaturation was adequate to disrupt strong secondary structures and to inhibit comigration of the two molecules. Cleavage kinetics were measured by continuous injection from the reaction vial into a polymer-filled capillary, and by determination of the area of the shrinking substrate peak. Compared to the well-established slab gel electrophoresis, CE is at least one order of magnitude faster, may be completely automated, allows easier and more precise quantitation of results, and, due to the small scale and self-contained nature of the apparatus, reduces health risks from dangerous chemicals. Unfortunately, UV detection in a 100-microm internal diameter capillary lacked the sensitivity to perform assays in the nanomolar range, which was necessary for a full Michaelis-Menten analysis.
Saevels
J
J
Laboratory for Pharmaceutical Chemistry and Drug Analysis, K.U. Leuven, Van Evenstraat 4, Leuven, B-3000, Belgium.
Van Schepdael
A
A
Hoogmartens
J
J
D004586:Electrophoresis / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D004795:Enzyme Stability; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009843:Oligoribonucleotides / Q000737:chemistry*; D017433:Protein Structure, Secondary; D016337:RNA, Catalytic / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity
D009843:Oligoribonucleotides; D016337:RNA, Catalytic