Capillary enzymophoresis of nucleic acid fragments using coupled capillary electrophoresis and capillary enzyme microreactors having surface-immobilized RNA-modifying enzymes.
This report describes the coupling of capillary enzyme reactors to capillary electrophoresis, which is termed capillary enzymophoresis. In the present study, the capillary enzyme reactors were prepared by immobilizing RNA-modifying enzymes, e.g., RNAse T1 and RNAse U2, on the inner walls of 50 microns fused-silica capillaries. These microreactors served to selectively modify the solutes (or substrates) before entering the separation capillary. Capillary enzymophoresis using single or mixed enzyme reactors proved useful in identifying minute amounts of dinucleotides as well as the fingerprinting of tRNAs. The immobilized RNase T1 and RNase U2 displayed their usual enzymic activities toward RNA fragments and in addition exhibited different activity-pH dependency than the soluble enzymes. This was attributed to microenvironmental effects arising from the charged nature of the capillary walls in the close proximity of the immobilized enzymes. The enzyme reactors were reusable for several RNA samples and showed chemical and thermal stability.
Mechref
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Y
Department of Chemistry, Oklahoma State University, Stillwater 74078-0447, USA.
El Rassi
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Z
D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004722:Endoribonucleases / Q000378:metabolism*; D015394:Molecular Structure; D012313:RNA / Q000302:isolation & purification*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism*
D012360:RNA, Transfer, Phe; D012313:RNA; D004722:Endoribonucleases; D006163:Ribonuclease T1; C022544:ribonuclease U2