In the Laboratory
An Undergraduate Investigation into the 10-23 DNA Enzyme That Cleaves RNA: DNA Can Cut It in the Biochemistry Laboratory Amber Flynn-Charlebois* Department of Chemistry and Pharmaceutical Science, Fairleigh Dickinson University, Madison, New Jersey 07940, United States *
[email protected] Jamie Burns, Stephanie Chapelliquen, and Holly Sanmartino Department of Chemistry and Physics, William Paterson University of New Jersey, Wayne, New Jersey 07970, United States
DNA is best known as the carrier of genetic information. RNA, on the other hand, is recognized for its integral role in protein synthesis and for its direct involvement in catalysis. Undergraduate laboratory exercises have been developed that use molecular biology and biochemical techniques and that readily demonstrate catalysis of RNA enzymes (1, 2) as well as RNA structure elucidation (3). However, more recently, DNA has also been shown to be a useful catalyst in vitro (4). To date, there are no known naturally occurring DNA enzymes, only ones that have been engineered and developed in the laboratory. Deoxyribozymes are single-stranded DNA sequences that are capable of catalyzing a variety of different chemical reactions including RNA cleavage and ligation (5-7). Because DNA is considerably more stable than RNA and is much more affordable, it is a practical choice for use in the undergraduate laboratory. This experiment employs the 10-23 DNA enzyme (8, 9) to site-specifically cleave an RNA substrate, offering students a unique opportunity to explore the use of DNA as a catalytic molecule while gaining additional experience in working with detection of nucleic acids following gel electrophoresis. The novel qualitative visualization technique incorporated here is the postelectrophoretic staining with cresyl violet, which can visually differentiate between DNA and RNA in the gel. The major advantages of this experiment include a remarkable cost savings associated with the nonfluorescent visualization of a DNA-enzyme reaction. The savings are twofold. First, a DNA sequence typically can be purchased for an order of magnitude less when compared to an RNA sequence of similar length and quantity (DNA $0.95 per base and RNA $9.50 per base). Although a 20-base RNA substrate is still required, the 35-base DNA enzyme is much less expensive than its RNA analogue. Even if the nucleic acids are purchased at the lowest quantities possible, there are sufficient RNA and DNA for multiple years (more than 10 years) if properly stored in the freezer. Second, this experiment uses this novel postelectrophoretic visible staining protocol that avoids the cost of fluorescent labeling with fluorescein, which is priced at approximately $60 per nucleic acid molecule. Furthermore, specialized instrumentation including a fluorescence spectrophotometer is not required for this experiment. The only pitfalls of the cresyl violet staining method are that it is considerably less sensitive when compared to other detection methods (lower detection limit 226
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Figure 1. The 10-23 DNA enzyme with RNA substrate bound. The DNA enzyme, E (black), binds an RNA substrate, S (gray), through two substrate-recognition domains, each involving Watson-Crick base pairing.
is ∼20 pmol of nucleic acid) and that it is not quantitative using standard methods. Experimental Overview The 10-23 DNA enzyme was isolated using in vitro selection in the presence of Mg2þ (8, 9). It is composed of a 15-nucleotide catalytic core flanked by two substrate-recognition domains (about 8-nucleotides each) that base-pair with the target RNA molecule or substrate (S) (Figure 1). These binding arms provide the specificity to bind only the RNA substrate of interest and provide the binding energy to keep the substrate in the active site so that cleavage can occur. In the presence of the Mg2þ cofactor, the enzyme cleaves the phosphodiester linkage between the unpaired purine (A) and the base-paired pyrimidine (U). The products of the reaction are a 20 -30 cyclic phosphate on the adeninosine and a 50 -hydroxyl group on the uridine, as shown in Figure 2. Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for the separation, purification, and characterization of nucleic acids (RNA and DNA). Visualization of single-stranded nucleic acids usually involves sophisticated and expensive techniques that are not ideal for undergraduate environments. These common approaches include the use of fluorescent labels or radioactive isotopes. Although sensitive and useful, these protocols can be expensive and have important limitations, including the possibility of interference with interactions and reactions of interest, requirement for specialized instrumentation, and possible regulatory and
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In the Laboratory
Figure 2. 10-23 DNA enzyme reaction mechanism of the phosphodiester bond hydrolysis creating a 20 -30 cyclic phosphate and a 50 -hydroxyl.
Figure 3. Structure of cresyl violet acetate.
safety issues. This laboratory experiment provides the student with a basic and qualitative visualization of the kinetics of the enzyme cleavage without the use of any radioactive or fluorescent labeling. This exercise utilizes a unique and inexpensive visualization technique, the postelectrophoretic staining with cresyl violet (CV, Figure 3). CV is a visible oxazine dye that is commonly used as a histological stain as it binds tightly to DNA and RNA-rich cell compounds (10, 11). CV has interesting spectroscopic properties. Its absorption maximum is in the visible region centered around 600 nm (11, 12). Although not employed in this experiment, CV has also been proposed as a standard for relative fluorescence with an excitation wavelength of 610 nm and mean fluorescence wavelength of 623 nm in water (13). In this experiment, we use the visible aspect of CV and have shown that CV does bind to and therefore stain single-stranded nucleic acids following PAGE: DNA stains blue and RNA stains purple. There is a short (30 min) destain required that provides a low level of background stain. This color distinction and low level of background stain allows for straightforward visual analysis of the gel results. Experimental Procedure A mini-gel apparatus (Mini-PROTEAN III from Bio-Rad), power supplies, heat block (or water bath), rocker, UV-vis spectrophotometer, and camera for photographic documentation are required. The DNA enzyme and the RNA substrate (100 nmol synthesis for RNA and 250 nmol synthesis for DNA sequences are recommended) can be obtained from Integrated DNA Technologies (IDT). Other requirements are 300 mM MgCl2, 5 reaction buffer (200 M Tris 3 HCl, pH 8.0, 750 mM NaCl), electrophoresis buffer of 10 TBE (890 mM Tris, 890 mM boric acid, 2 mM EDTA, pH 8.0), and 80% stop solution and loading buffer (80% formamide, 1 TBE, 50 mM EDTA, 0.025% Bromophenol blue (BPB)). Typically, xylene cylenol (XC) would be added to the BPB loading-running dye; however, in this experiment the XC is not used because it is not completely removed during destaining. CV binds to XC, as well as to the DNA and RNA, giving misleading results. Fifteen-well urea PAGE precast
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gels (12% or 15%) are used for this separation and can be purchased (Bio-Rad). The cost for these precast gels is approximately $10/gel. (This purchase is recommended as it is much easier for the student to accomplish the experiment in two laboratory sessions and it avoids exposure to unpolymerized acrylamide, which is a possible human carcinogen and a possible teratogen.) The gel-staining solution is an aqueous 0.02% CV solution and the destaining solution is an aqueous 0.5% acetic acid. These reagents are available from SigmaAldrich. The DNA enzyme (625 pmol, 42 μM) and the RNA substrate (2.5 nmol, 167 μM) are placed in a total volume of 15.0 μL with a final concentration of 40 mM Tris 3 HCl, pH 8.0, 150 mM NaCl. For annealing, the reaction tube is placed in a dry bath incubator at 95 °C for 2 min, followed by 5 min in an ice bath, and is finally allowed to equilibrate in a dry bath incubator at 37 °C for at least 2 min before the reaction is initiated. To initiate the reaction, 3.0 μL of 300 mM MgCl2 is added and the reaction mixture is then quickly mixed using a vortex, spun down, and incubated at 37 °C. Aliquots, 2.0 μL, are removed at the following time points: 0.5, 1, 3, 5, 10, and 30 min. The reaction aliquots are each quenched by quickly dispensing into a labeled Eppendorf tube with 8 μL of 80% stop solution, which doubles as the gel-loading dye. The resulting six quenched aliquots, alongside the two RNA standards, are separated using a 12% or 15% urea PAGE, run at 180 W for 30 min. Following electrophoresis, the gels are stained on a rocker in aqueous 0.02% CV for 30 min and destained three times in 5% acetic acid for 10 min each. A gel documentation station or a digital camera can be used to capture the gel image. Hazards Avoid RNases. Wear gloves at all times to avoid contamination of your RNA samples with RNases. Rinse pipets with ethanol to remove RNases. Gloves should be worn when handling all of the reagents required for this reaction. Formamide is a teratogen and is highly corrosive and should never be handled without gloves and goggles. Concentrated acetic acid is also corrosive and must be handled with care. Nitrile rubber gloves should be worn when handling acetic acid, as they provide the best protection. In addition, urea can be irritating to skin, eyes, and the respiratory tract. It should also be noted that gel electrophoresis equipment can pose significant electrical hazards, and precautions should be taken to operate equipment according to the safety recommendations. The health hazards of cresyl violet are unknown according to the MSDS, so it must be handled with caution; therefore, gloves should be worn at all time during the staining and destaining protocol. Much caution should be used when using a 95 °C dry bath incubator. Because this lab suggests that the precast gels be purchased, there is less likely exposure to unpolymerized acrylamide, which is a possible human carcinogen and a possible teratogen. Discussion This two-session laboratory experiment has been performed a number of times and reliably gives reproducible results. A representative gel is shown in Figure 4. The DNA enzyme, RNA substrate, and RNA cleaved product are separated using polyacrylamide gel electrophoresis and visualized using a cresyl
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Acknowledgment I thank Anita Brandolini and Christine Chow for helpful discussions, advice, and comments on the manuscript. Literature Cited
Figure 4. Cleavage of the 20-nucleotide (nt) RNA substrate by the 10-23 DNA enzyme. Cresyl violet stained gel reveals DNA and RNA bands in lanes 2-7. Standards are RNA substrate, std-S (lane 1), and RNA product, std-P (lane 8). Reaction times are 0.5, 1, 3, 5, 10, and 30 min. Although not shown here, no cleavage was observed when Mg2þ was not added to the reaction.
violet staining protocol. Standards are in lane 1 (std-S, uncleaved substrate 20-nt), and lane 8 (std-P, cleaved product 11-nt). Reaction products are in lanes 2-7: the top band (blue) is the DNA enzyme and is found in all lanes; the middle band (purple) is the RNA substrate and decreases in intensity as the reaction progresses; and the lower band, the cleaved RNA product, increases in intensity as time increases. After 30 min, most of the substrate is cleaved as can be seen in lane 7. It is difficult to acquire a zero time point sample because after mixing and spinning down enough time has passed that the reaction is well underway. It should be noted that the std-S in lane 1 could also be considered a negative control because it contains everything from the reactions except the catalyst, the DNA enzyme. A nice addition to this gel would be an additional negative control of the reaction with no magnesium added. Although it is not included in the gel in Figure 4, it has been included in the supporting information. Because this reaction is straightforward and inexpensive, it is possible to take it further and use it as a springboard for future investigations including variation of the metal ion (and concentration) required for cleavage and exploration of the effect of pH on its activity. Because the only requirements for the cleavage site is a purine base followed by a pyrimidine base and sufficiently long binding arms on each side of the cleavage site, it is possible for students to incorporate modifications in the binding arms of the DNA enzyme (as long as the substrate complement is also modified to follow WatsonCrick base pairing). Students can take ownership of the 10-23 DNA enzyme experiment and also become comfortable with the concept that DNA can catalyze cleavage of RNA in the laboratory.
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1. Chow, C. S.; Somne, S. Synthesis and purification of a hammerhead ribozyme and a fluorescein-labeled RNA substrate. J. Chem. Educ. 1999, 76, 648–650. 2. Chow, C. S.; Somne, S.; Llano-Sotelo, B. Monitoring hammerhead ribozyme-catalyzed cleavage with a fluorescein-labeled substrate: Effects of magnesium ions and antibiotic inhibitors. J. Chem. Educ. 1999, 76, 651–652. 3. Kirk, S. R.; Silverstein, T. P.; Holman, K. L. M.; Taylor, B. L. H. Probing changes in the conformation of tRNAPhe: An integrated biochemistry laboratory course. J. Chem. Educ. 2008, 85, 666–673. 4. Breaker, R. R.; Joyce, G. F. A DNA enzyme that cleaves RNA. Chem. Biol. 1994, 1, 223–229. 5. Baum, D. A.; Silverman, S. K. Deoxyribozymes: useful DNA catalysts in vitro and in vivo. Cell. Mol. Life Sci. 2008, 65, 2156–2174. 6. Flynn-Charlebois, A.; Wang, Y.; Prior, T. K.; Rashid, I.; Hoadly, K. A.; Coppins, R. L.; Wolf, A. C.; Silverman, S. K. Deoxyribozymes with 20 -50 RNA ligase activity. J. Am. Chem. Soc. 2003, 125, 2444–2454. 7. Silverman, S. K. Deoxyribozymes: DNA catalysts for bioorganic chemistry. Org. Biomol. Chem. 2004, 2, 2701–2706. 8. Santoro, S. W.; Joyce, G. F. A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4262–4266. 9. Santoro, S. W.; Joyce, G. F. Mechanism and Utility of an RNAcleaving DNA enzyme. Biochemistry 1998, 37, 13330–13342. 10. Dutt, M. K. Aqueous solution of cresyl violet-- its use in the staining of DNA in fixed mammalian tissue sections. Microscopia Acta 1981, 84, 87–90. 11. Vogel, E.; Gbureck, A.; Kiefer, W. Vibrational spectroscopic studies on the dyes cresyl violet and coumarin 152. J. Mol. Struct. 2000, 550-551, 177–190. 12. Dutt, M. K. Basic dyes for the staining of DNA in mammalian tissues and absorption spectra of stained nuclei in the visible light. Microsc. Acta 1981, 86, 59–68. 13. Isak, S. J.; Eyring, E. M. Fluorescence quantum yield of cresyl violet in methanol and water as a function of concentration. J. Phy. Chem. 1992, 94, 1738–1742.
Supporting Information Available Student handout; notes for the instructor. This material is available via the Internet at http://pubs.acs.org.
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