PubMed · 8429915
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H Kambara, S Takahashi. 1993-02-11. Multiple-sheathflow capillary array DNA analyser.. https://doi.org/10.1038/361565a0
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Enzymes are capable of a highly specific interaction with a variety of substances including their respective substrates. This review summarizes how such interactions may be used in analytical (bio-)chemistry, e.g., for the elucidation of the binding mechanism, the determination of the binding strength, the carting of the binding site, or the screening of possible substrate/inhibitor molecules. Possible assay formats such as analytical affinity chromatography, affinity capillary electrophoresis (ACE), conventional affinity gel electrophoresis (AEP), and related techniques are discussed together with examples of recent applications. In addition a brief section on enzyme-substrate reactions as tools in analytical chemistry is included, since these are perhaps even more important to analytical (bio-)chemistry. The development and application of bioanalytical systems and especially biosensors in various fields including medicine, biotechnology, agriculture, defense and foodstuffs are considered.
We are studying dye-imidazole conjugates ("IMI dyes") as reagents for labeling phosphomonoesters such as nucleotides. Previously we have employed a BODIPY dye in our IMI reagents, and analyzed the labeled products by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) involving an argon ion laser. (The BODIPY fluorophore is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). Here we broaden the technology by preparing a DBD-IMI dye [DBD = 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole], and using a helium-cadmium laser. While DBD-IMI (IMI3) is about 50x more stable photolytically than a BODIPY-IMI dye (IMI2, a conjugate of a BODIPY dye with histamine, was tested), the detection limit for IMI2 (5.10(-11) M; S/N = 5, CE-LIF with an argon ion laser) is tenfold better than that for IMI3 (5.10(-10) M, S/N = 5, helium-cadmium laser). IMI3 conjugates of the four major DNA nucleotides were prepared and detected by CE-LIF.
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.