Continuous monitoring of enzyme reactions on a microchip: application to catalytic RNA self-cleavage.
Kinetic analysis of RNA enzymes, or ribozymes, typically involves the tedious process of collecting and quenching reaction time points and then fractionating by polyacrylamide gel electrophoresis (PAGE). As a way to automate and simplify this process, continuous analysis of a ribozyme reaction is demonstrated here using completely automated capillary sample introduction onto a microfabricated device with laser-induced fluorescence detection. The method of injection is extremely reproducible thereby standardizing data analysis. A 30-nucleotide ribozyme model, the self-cleaving lead-dependent ribozyme, or "leadzyme", which cleaves into a 24-mer and a 6-mer in the presence of Pb(2+), was end-labeled with fluorescein (FAM) and used to demonstrate the potential of this technique. After manually initiating the cleavage reaction by Pb(2+) addition, reaction samples were automatically injected directly into the parallel separation lanes of the chip via a capillary at predetermined time intervals, thus eliminating the need for additional sample-handling steps. The FAM-labeled leadzyme starting material and products were monitored for 60 min in order to ascertain kinetic information. The effect of lead acetate concentration on cleavage rates was also studied, and the results are in agreement with rates determined by conventional hand-mixing/PAGE analysis. This work demonstrates, through the use of a simple ribozyme model, the potential of this method to provide valuable kinetic information for other, more complex, biologically relevant RNA and protein enzymes.
Paxon
Tracy L
TL
Department of Chemistry and The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Brown
Trevor S
TS
Lin
Hsiao-Yu Nancy
HY
Brancato
Sam J
SJ
Roddy
Elizabeth S
ES
Bevilacqua
Philip C
PC
Ewing
Andrew G
AG
D001322:Autoanalysis; D002384:Catalysis; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D004789:Enzyme Activation; D007700:Kinetics; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry*; D012680:Sensitivity and Specificity
D016337:RNA, Catalytic; C113411:leadzyme; D012313:RNA