In the Laboratory Concepts in Biochemistry
Monitoring Hammerhead Ribozyme-Catalyzed Cleavage with a Fluorescein-Labeled Substrate: Effects of Magnesium Ions and Antibiotic Inhibitors
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A Biochemistry Laboratory: Part 2 Christine S. Chow,* Smita Somne, and Beatriz Llano-Sotelo Department of Chemistry, Wayne State University, Detroit, MI 48202; *
[email protected] This experiment is highly recommended for a biological chemistry laboratory involving junior and senior undergraduate chemistry majors. It combines current techniques used in biochemistry and molecular biology with modern concepts in RNA research. In the undergraduate curriculum, students will already have learned about general nucleic acid structure and function. Nucleic acids can be visualized directly by electron microscopy or other sophisticated techniques, which require advanced training and expertise. Alternatively, nucleic acids can be monitored by radioactive labeling techniques, but these methods are not practical in undergraduate laboratories. Ethidium bromide staining can also be used to visualize small amounts of RNA or DNA, but careful handling and disposal of ethidium bromide are necessary because it is a potent carcinogen. Thus, gaining hands-on experience working with and visualizing picomole amounts of RNA or DNA requires alternative methods, such as fluorescent tagging. The experiment as outlined in Figure 1 involves the study of hammerhead ribozyme activity. The ribozyme is an RNAbased catalyst with current applications in both biotechnology and medicine. Cleavage of a substrate RNA in the presence of magnesium ions (1) and the antibiotic inhibitor neomycin (2) is examined. Several papers are recommended for background reading on ribozyme function (3–6 ). The cleavage reaction is carried out with picomole amounts of RNA and visualized by fluorescent labeling at the 5′ end of the substrate RNA. Although several other highly sensitive methods for visualization of RNA are available (7 ), we have found that the fluorescent tag is generally applicable
and simple to use in the undergraduate laboratory. The substrate RNA is cleaved in half as can be determined by denaturing polyacrylamide gel electrophoresis and quick visualization of the fluorescence with UV light. In this manner, the cleavage reaction can be carried out in a period of 90 minutes and monitored without the use of radioactive labeling. The setting up and running of the polyacrylamide gel requires an additional 2 to 2.5 h. The entire experiment can be completed in one laboratory period of ca. 4 h. The students gain experience with polyacrylamide gel electrophoresis and observe fluorescence of the substrate RNA. In addition, the importance of magnesium ions in ribozyme activity and the effects of a common antibiotic (neomycin) on RNA reactivity are demonstrated. Experimental Procedure
A mini-gel apparatus (such as the Mini-PROTEAN II from Bio-Rad, Hercules, CA) for each pair of students, power supplies, a UV light box (transilluminator), heat block (or water bath), and photodocumentation camera are required. The ribozyme and fluorescein-labeled substrate can be obtained from Commonwealth Biotechnologies, Inc. (Richmond, VA), or synthesized as described in the companion paper (8). Other components required are 0.4 M MgCl 2, 0.2 M neomycin sulfate (Sigma Chemical Co., St. Louis, MO), and 1 M Tris?HCl, pH 7.3. For gel preparation, a 40% 19:1 acrylamide– bisacrylamide solution, 10% (w/v) ammonium persulfate in H2O, and TEMED (N,N,N′,N′-tetramethylethylenediamine) are necessary (components can be obtained from Fisher Scientific). The electrophoresis buffer required is 10× TBE (900 mM Tris, 90 mM 9 1 boric acid, 25 mM EDTA, pH 8.3). The 3' fluorescein fluorescein GGGAACGUC-3' loading buffer consists of 16 M urea, 30% 38 C . G 1 + C. G Mg2+ glycerol, and 1× TBE. The loading dye con5' GUCGUCGC 3' C. G Substrate 10 17 U. A (17 nucleotides) tains the same components as loading buffer + 37 °C Ribozyme U. A G. C (38 nucleotides) plus 0.25% each of xylene cyanol and free ribozyme C. G cleavage site bromophenol blue. These chemicals are A. U A C G U C G U C G C17 3' A available in typical biochemistry departA G . . . . . . . . A G G C C . . . . C A G U A G C G 5' ments or can be purchased (Sigma). uncleaved substrate 2+ A C Mg 1 G C C G GA + The hammerhead ribozyme (30 pmol, G U A GU 37 °C ribozyme 1.5 µ M) and the fluorescein-labeled subneomycin substrate-bound ribozyme strate (100 pmol, 5 µM) are annealed in 18 µ L of 50 mM Tris?HCl, pH 7.3. Nine Figure 1. The hammerhead ribozyme (38 nucleotides) is shown in a base-pairing scheme samples are prepared in 600- µ L 2+ with the fluorescently labeled RNA substrate (17 nucleotides). In the presence of Mg microcentrifuge tubes, mixed thoroughly, and heating at 37 °C, the substrate will be cleaved specifically by the ribozyme into and placed in a boiling water bath for 2 one labeled product (with fluorescein) and one unlabeled product (upper scheme). In min. The tubes are removed from the the presence of neomycin, the reaction will be inhibited (lower scheme). JChemEd.chem.wisc.edu • Vol. 76 No. 5 May 1999 • Journal of Chemical Education
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In the Laboratory
water bath and slowly cooled to room temperature (over ca. 15 min). This step will renature the ribozyme and the substrate. MgCl2 (1 µL of 0.4 M) is added to tubes 2–9. Onemicroliter portions of neomycin solutions of decreasing concentrations are added to tubes 6–9 (5, 0.5, 0.05, 0.005 mM final concentration, respectively) and the reaction mixtures are incubated at 37 °C. The first tube (control) contains no magnesium. The incubations for tubes 2–5 are stopped at 15–30-min intervals. Loading buffer (5 µ L) is added to the reaction mixtures at 15, 30, 60, and 90 min for tubes 2– 5, respectively, and they are immediately placed on ice. Reactions in tubes 1 and 6–9 are stopped at 90 min in a similar manner. During the incubation period a 15% denaturing (7 M urea) polyacrylamide (19:1 acrylamide–bisacrylamide) (1× TBE) gel is prepared. The glassware, gel plates, gel box, casting stand, spacers, and combs are washed extensively with distilled deionized water and rinsed with ethanol to remove any possible contaminants. Before the samples are loaded onto the gel, they are boiled in a water bath for two minutes and immediately placed on ice. Samples 1–9 are loaded in consecutive lanes. Loading dye (10 µL) is loaded in the 10th lane to monitor the movement of the oligonucleotides through the gel. The gel is run at 100 V in 1× TBE for ca. 2 h until the lower dye band (bromophenol blue) is ca. 3/4 of the way to the bottom of the gel. The plates are disassembled and the gel is placed on a UV light-box (transilluminator) to visualize the fluorescent substrate and cleavage product(s). The results are recorded in the form of a Polaroid photograph. Discussion This experiment has been performed many times and has given excellent reproducible results. Representative results are shown in Figure 2. The RNA substrate and cleavage products are separated by polyacrylamide gel electrophoresis and visualized on a standard transilluminator box. Cleavage of the substrate takes place only in the presence of magnesium. The intensity of the band corresponding to the cleavage product (nine nucleotides containing fluorescein) increases with time and at 90 minutes the majority of the substrate is cleaved (Fig. 2, lanes 2–5). In addition, neomycin inhibits the ribozyme cleavage reaction at intermediate concentrations (5 mM, Fig. 2, lane 6), and to a lesser extent at lower concentrations (lanes 7–9). This experiment is an excellent way of introducing the concept of RNA as an enzyme and the cleavage event can be efficiently monitored by the use of a fluorescent label on the substrate. If the necessary equipment is available, the substrate and product bands can be quantitated and the students can perform kinetics experiments. In addition, mutant ribozymes or substrates can be used to demonstrate the concept of deleterious mutations. The design of such experiments is the subject of future work. The experiment described here enables students to visualize nucleic acids at the picomole level and also demonstrates contemporary concepts in biochemistry, in particular the use of ribozymes as potential agents for drug therapy.
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Figure 2. Cleavage of a 17-nucleotide substrate RNA by a 38-nucleotide hammerhead ribozyme. Only the fluorescently labeled RNAs are visible by UV light on the 15% polyacrylamide gel. All lanes contain 30 pmol of ribozyme and 100 pmol of 5′-fluorescein-labeled substrate and were heated at 37 °C for 90 min (lanes 1 and 5–9) or less (15, 30, and 60 min for lanes 2, 3, and 4, respectively). The RNA in lane 1 contains no magnesium. RNA in lanes 2–9 contain 20 mM MgCl2. Neomycin has been added to RNAs in lanes 6–9 (5 mM, 0.5 mM, 50 µM, and 5 µM, respectively).
Note that RNase contamination can be a problem in this experiment. Care must be taken to autoclave all pipet tips and microcentrifuge tubes. The gel apparatus and gel plates, combs, and spacers must be rinsed thoroughly with distilled deionized water (ddH2O) and then rinsed with ethanol. For any apparatus that has been used for protein work, an acid wash (10% hydrochloric acid or 5% nitric acid) and base wash (10% NaOH) should precede the ddH2O wash. CAUTION : Students should rinse pipets with ethanol and wear gloves at all times. All solutions should be prepared with RNase-free water (ddH2O) in glass containers that have been baked overnight at 200 °C. Samples containing fluorescein should be loaded in buffer without dye because the presence of bromophenol blue of xylene cyanol may lead to quenching of the fluorescence. The samples will have a slight yellow or green color from the fluorescein and will be somewhat viscous because of the glycerol, which should aid in the loading process. Note W Supplementary materials for this article are available on JCE Online at http://jchemed.chem.wisc.edu/Journal/issues/1999/May/ abs651.html.
Literature Cited 1. Fedor, M. J.; Uhlenbeck, O. C. Biochemistry 1992, 31, 12042– 12054. 2. Stage, T. K.; Hertel, K. J.; Uhlenbeck, O. C. RNA 1995, 1, 95– 101. 3. Athota, R. R.; Radhakrishnan, T. M. Biochem. Educ. 1991, 19, 72–73. 4. McCorkle, G. M.; Altman, S. J. Chem. Educ. 1987, 64, 221– 226. 5. Ohkawa, J.; Koguma, T.; Kohda, T.; Taira, K. J. Biochem. 1995, 118, 251–258. 6. Scott, W. G.; Klug, A. Trends Biochem. Sci. 1996, 21, 220–223. 7. Palfner, K.; Kneba, M.; Hiddemann, W.; Bertram, J. BioTechniques 1995, 19, 926–929. 8. Chow, C. S.; Somne, S. J. Chem. Educ. 1999, 76, 648–650.
Journal of Chemical Education • Vol. 76 No. 5 May 1999 • JChemEd.chem.wisc.edu
