Quantitation of in vitro activity of synthetic trans-acting ribozymes using HPLC.
The cleavage activity of synthetic ribozymes needs to be characterized by reliable and rapid methods. A chromatographic method to simultaneously quantitate the amounts of substrate, cleavage fragments and ribozyme is described. The method allows the rapid normalization of analytical data because the sum of the 260-nm peak areas of remaining substrate and obtained fragments is essentially equal to the initial substrate peak area. Moreover, the simultaneous determination of the ribozyme content improves the accuracy of the evaluation of kinetic parameters compared with conventional densitometric methods. Finally, the characterization of two different hammerhead motifs indicated that the method is suitable for a rapid screening of synthetic ribozyme activity.
Citti
L
L
Istituto di Mutagenesi e Differenziamento CNR, Pisa, Italy.
Boldrini
L
L
Nevischi
S
S
Mariani
L
L
Rainaldi
G
G
D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007700:Kinetics; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D013379:Substrate Specificity
D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D012333:RNA, Messenger; D019853:O(6)-Methylguanine-DNA Methyltransferase