Synthesis and Evaluation of Artificial DNA Scissors: An

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

pubs.acs.org/jchemeduc

Synthesis and Evaluation of Artificial DNA Scissors: An Interdisciplinary Undergraduate Experiment Jan Hormann,† Sabine Streller,‡ and Nora Kulak*,† †

Anorganische Chemie, Institut für Chemie und Biochemie, Freie Universität Berlin, 14195 Berlin, Germany Didaktik der Chemie, Institut für Chemie und Biochemie, Freie Universität Berlin, 14195 Berlin, Germany

‡

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S Supporting Information *

ABSTRACT: Bridging organic and inorganic chemistry as well as molecular biology, this undergraduate experiment deals with the synthesis of ligands for metal complexes that can cleave DNA. Students gain theoretical and experimental expertise about metal−DNA interactions and the mechanisms behind DNA cleavage. This experiment lays a basis for the understanding and the rational design of artificial DNA scissors and provides insight into interdisciplinary research in the field of bioinorganic chemistry.

KEYWORDS: Upper-Division Undergraduate, Interdisciplinary/Multidisciplinary, Synthesis, Coordination Compounds, Molecular Biology, Nucleic Acids/DNA/RNA, Bioinorganic Chemistry

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By comparison of the DNA cleavage activities of [Cu(L1)2]2+ and [Cu(L2)2]6+, the Cu(II) complexes of 2,2′-bipyridine (L1) and 5,5′-bis[1-(triethylammonio)methyl]-2,2′-bipyridine (L2) (Figure 1), under hydrolytic and oxidative conditions, knowledge of different DNA cleavage mechanisms, DNA binding, and metal complex−DNA interactions can be communicated. Complex [Cu(L2)2]6+ is reported to cleave DNA in a hydrolytic fashion.5 A metal-bound water molecule is deprotonated to yield a hydroxido species of the form [Cu(L2)2OH]5+ that can cleave the phosphodiester bond. As the distance between the quaternary ammonium groups and the coordinated hydroxido ligand is about 5.5−5.7 Å, which is in the same range as the distance between adjacent phosphorus atoms (6 Å) in the DNA backbone, the electrostatic interaction between the ammonium and phosphate groups allows orientation of the nucleophilic OH− toward the phosphodiester bond to be cleaved (Figure 2).5 [Cu(L1)2]2+, which has no possibility to interact in this fashion with DNA, is thus expected to show a lower DNA cleavage activity. Under oxidative conditions, however, where the redox activity of the Cu(II) center is the principal mechanism, the two complexes are expected to show comparable cleavage activities.

BACKGROUND Highly specialized enzymes are able to cleave DNA inside the body for several purposes. Within the limits of a laboratory experiment, small metal complexes, so-called artificial metallonucleases ormore descriptivelyDNA scissors, can also conduct DNA cleavage. Such DNA scissors have several applications in biochemistry and biomedicine: they can help to reveal the mechanism and structure of natural enzymes (functional and structural model complexes), and they can be applied as tools in molecular biology. DNA scissors can be used in medicine, for example, as anticancer drugs. With regard to these two different applications, two different mechanisms for the cleavage of DNA are conceivable: hydrolytic and oxidative DNA cleavage. Hydrolytically cleaving artificial nucleases catalyze the attack of metal-bound hydroxide ions toward the phosphate backbone in the fashion of an SN2 reaction involving a five-coordinate phosphorus intermediate1 (Scheme 1). While the design of hydrolytically cleaving complexes should provide a Lewis acidic metal center, oxidatively cleaving scissors involve metals that are redox-active in order to generate DNAdamaging reactive oxygen species (ROS). Cu(II), as a metal ion exhibiting Lewis acidity as well as redox activity, can build the basis for both classes of artificial metallonucleases.2 Accordingly, oxidative DNA cleavage based on Cu(II) requires a reducing agent such as ascorbate and molecular oxygen3 (Scheme 2). The formed ROS (superoxide radical anions, hydrogen peroxide, hydroxyl radicals) damage the DNA bases or the sugar moiety by hydrogen abstraction.4 Subsequent reactions and rearrangements then lead to DNA strand breakage (Scheme 3). © XXXX American Chemical Society and Division of Chemical Education, Inc.

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PEDAGOGY Herein, an interdisciplinary experiment bridging the areas of organic chemistry, coordination chemistry, and molecular Received: August 31, 2017 Revised: July 10, 2018

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DOI: 10.1021/acs.jchemed.7b00662 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

Scheme 1. Mechanism of Hydrolytic DNA Cleavage

• Problem solving skills: What kind of equipment do I need for the organic synthesis? How do I manage a multistep synthesis in order to prepare enough pure material for biological applications in the end? • Chemical literature and information management skills: What is already known about the application of artificial nucleases in the literature? • Team skills: It is recommended that the bioinorganic/ molecular biology part of the experiment be done in groups of two. Such an interaction with peers will lead to more productive completion of the experiment due to the sharing of tasks, mutual control, and support. • Laboratory safety skills: The experiment utilizes two particularly hazardous chemicals: tetrachloromethane (organic synthesis) and ethidium bromide (molecular biology). Students are required to inform themselves about the hazardous properties and follow the safety precautions accordingly. There are substitutes for both chemicals, though. The planning of the experiment should thus be based on the importance of experience (future chemists have to learn how to work with hazardous chemicals), practicability (tetrachloromethane gives the highest yields in the reaction to be carried out), and costs (substitutes for ethidium bromide are usually very expensive; tetrachloromethane is also expensive and hard to obtain). The products are also potential hazards, as there is no proof to the contrary. The experiment is clearly inquiry-based: students evolve “their understanding through their own investigation” and execute tasks “similar to those in which scientists engage in developing understanding”.10 In contrast to other experiments in the same field,6 this experiment can be easily accomplished within a synthetic laboratory and does not need elaborate additional equipment (the equipment for gel electrophoresis is affordable or can be used in collaboration with a research lab; see the Supporting Information A-2,3). As an innovation, this experiment spans three subdisciplines instead of only two, including the respective experimental techniques.

Scheme 2. Copper-Catalyzed Generation of ROS (Asc = Ascorbate)a

a

Adapted and modified from ref 3. Published by the Royal Society of Chemistry under a Creative Commons Attribution-NonCommercial 3.0 Unported License.

biology is presented that is suitable for undergraduate chemistry students who have already acquired basic knowledge and some practical experience in the aforementioned areas of chemistry and biology. After the first half of the undergraduate curriculum usually covering the traditional subdisciplines, upper-division undergraduate students can thus gain exposure to interdisciplinary research for the first time. Such interdisciplinary experimental approaches are barely covered in the chemistry educational literature (see, e.g., Rabago Smith et al.6), even though the American Chemical Society (ACS) has recognized and presented in its guidelines for Bachelor’s programs that the subdisciplines are more and more overlapping.7 Concordantly, the German Chemical Society (GDCh) asserts the importance of chemistry subdisciplines and especially related laboratory work, but at the same time the GDCh recommends supporting students in German Bachelor’s programs8 in acquiring interdisciplinary capabilities with a basis for developing and conducting future research projects on their own.9 Independent and critical thinking abilities, as requested by the ACS,7 are supported in different ways within the experiment presented herein. For example, the students are required to discuss the results of UV/VIS spectroscopy, suggest substitutes for hazardous substances, and be creative in finding potential applications of artificial nucleases. Several skills can be improved by completing this experiment:

Scheme 3. Oxidative DNA Damage through Hydrogen Abstraction or Base Damage

B




5.5 mmol) was added, and the resulting solution was stirred for 6−24 h (see the Results and Discussion) at room temperature. The precipitate that formed was collected by filtration, washed with chloroform, and dried under reduced pressure.

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BIOINORGANIC/MOLECULAR BIOLOGY SECTION The second part of the experiment involved the preparation of the Cu(II) complexes and the evaluation of their DNA cleaving abilities in the presence and absence of ascorbate (oxidative/ hydrolytic cleavage) by agarose gel electrophoresis. This part was carried out in teams of two. Two lab periods of 4 h were needed for the preparation and characterization of the complexes and the gel runs. A third lab period of 4 h might be needed for evaluation of the results. The information for instructors (Supporting Information, A-2) provides details on complex synthesis, analysis, and DNA cleavage experiments; experimental procedures for students are described in the handout (Supporting Information, B-2).

Figure 1. Structures of ligands L1 and L2.

In Situ Synthesis of the Cu(II) Complexes

Ligands L1 and L2 (1 mL of a 20 mM solution of each), respectively, were mixed with copper(II) nitrate (1 mL, 10 mM solution), and the mixtures were left undisturbed for at least 30 min. The formation of complexes [Cu(L1)2]2+ and [Cu(L2)2]6+ was monitored by UV/VIS spectroscopy. For DNA cleavage experiments, 50 μM and 500 μM stock solutions of each complex were prepared.

Figure 2. Proposed intermediate in the mechanism of hydrolytic DNA cleavage by [Cu(L2)2]6+. Adapted with permission from ref 5. Copyright 2006 Royal Society of Chemistry.

Students are provided with a handout and the “before the experiment” assessment form a couple of days before starting in the laboratory, with the task to read and inform themselves about the background of the experiment. They then meet with their instructor for about 30 min to talk about the experiment (scientifically and technically) and safety issues.

DNA Cleavage Studies

In Eppendorf tubes, water, HEPES buffer (20 mM, pH 7.4), the corresponding complex solution (50 μM for oxidative cleavage and 500 μM for hydrolytic cleavage), ascorbate (for oxidative cleavage only), and pBR322 plasmid DNA were mixed according to the pipetting schemes (Supporting Information, Tables A-1 and A-2) and incubated for 2 h at 37 °C.

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ORGANIC CHEMISTRY SECTION In the first part of the experiment, students synthesized the 2,2′bipyridine-based ligand L2 in a two-step procedure involving column chromatography for purification and NMR spectroscopy for characterization. For this section, students needed four lab periods of 4−6 h. The information for instructors (Supporting Information, A-1) provides details on experimental procedures and analytical data, and the experimental procedures for the students are described within the handout (Supporting Information, B-1).

Gel Electrophoresis

The samples were loaded onto a 1% agarose gel stained with ethidium bromide or GelRed and run at 40 V for 2 h.

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HAZARDS Students must follow general safety precautions in chemistry laboratories at all times. Eye protection and lab coats must be worn during the entire experiment, as well as gloves when appropriate. The steps of the organic chemistry section must be performed under a fume hood. Students are required to identify independently the hazards of the chemicals used and should be tested on their knowledge of the hazards prior to performing the experiments (see Pedagogy above). Especially when working with DNA-intercalating substances and DNA scissors, safety precautions have to be followed strictly, as these substances are potentially mutagenic.11 As ethidium bromide is regarded to be teratogenic and carcinogenic, working with alternative DNA stains such as SYBR Green I or GelRed might be considered,12 although even these chemicals are potentially carcinogenic because of their high affinity for DNA.13

Synthesis of 5,5′-Dibromomethyl-2,2′-bipyridine

Under inert conditions, 5,5′-dimethyl-2,2′-bipyridine (0.93 g, 5 mmol), N-bromosuccinimide (NBS) (1.84 g, 10 mmol), and azobis(isobutyronitrile) (AIBN) (0.08 g, 0.5 mmol) were dissolved in tetrachloromethane or ethyl acetate (25 mL) in a 50 mL Schlenk flask with a reflux cooler and stirring bar. The solution was heated to 80 °C and irradiated with a 300 W sunlight lamp for 72 h (e.g., over a weekend). The formed precipitate was filtered off and washed with chloroform (100 mL). The combined solvents were removed under reduced pressure, and the resulting solid was purified by column chromatography with silica gel and a mixture of dichloromethane and methanol (25:1 v/v) (Rf = 0.8).

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Synthesis of 5,5′-Bis[1-(triethylammonio)methyl]-2,2′-bipyridine Dibromide (L2Br2)

RESULTS AND DISCUSSION

Organic Chemistry Section

On the basis of the yield of the previous step, 5,5′-dibromomethyl2,2′-bipyridine (0.55 g, 1.6 mmol) was added to a 100 mL flask and dissolved in chloroform (45 mL). Triethylamine (0.56 g,

The compound 2,2′-bipyridine (L1) can be obtained commercially; L25 was synthesized according to Scheme 4. 5,5′-Dimethyl2,2′-bipyridine was brominated in tetrachloromethane or ethyl C




within 6 h of reaction time; however, reaction times longer than 24 h led to a decrease in yield). The precipitated product was collected by filtration. The average yield was 65%. Ligand L2 and its precursor 5,5′-dibromomethyl-2,2′bipyridine were characterized by 1H NMR spectroscopy at 43 MHz. Examples of student spectra are shown in Figure 3; spectra measured at 400 MHz, additional electrospray ionization mass spectrometry (ESI-MS) and elemental analysis (EA) data can be found in the Supporting Information. As both compounds are highly symmetrical, their NMR spectra are rather easy to interpret. The spectrum of the precursor consists of four signals (Figure 3A). Because of the deshielding effect of the bromine atoms, the singlet signal of the methylene groups (4.53 ppm) is shifted downfield in comparison with the starting material (2.37 ppm for the methyl groups14). The signals of the aromatic protons appear between 7.72 and 8.70 ppm. They are of higher multiplicity; however, second-order effects cannot be resolved at 43 MHz (at 400 MHz, the C and D protons show a doublet signal, while the B protons show a doublet of doublets; see the Supporting Information A-1,4). The spectrum of L2 was measured in D2O, as the salt is easily soluble in water (Figure 3B). In addition to the only slightly shifted signals of the dimethylbipyridine core, two signals for the triethylammonium groups can be found: a triplet at 1.48 ppm for the methyl groups and a quartet at 3.38 ppm for the methylene groups. Despite the use of column chromatography, the precursor obtained by students was oftentimes not that pure. However, L2

Scheme 4. Synthesis of 5,5′-Bis[1-(triethylammonio)methyl]2,2′-bipyridine Dibromide (L2Br2)

acetate under irradiation using NBS as bromide source and AIBN as the radical starter within a Wohl−Ziegler reaction (radical substitution reaction). The resulting crude product, which was first checked by 1H NMR spectroscopy, was purified by column chromatography. The average yield was 56% with tetrachloromethane; however, when ethyl acetate was used as the solvent, the yield dropped to 13%. As radical halogenation reactions have a broad range of application in organic synthesis and column chromatography is the most commonly used purification method, students thus had a chance to practice these common methods in organic chemistry. The quaternary amine functions were then introduced by the reaction of triethylamine with the brominated bipyridine starting material in chloroform (nucleophilic substitution reaction) for up to 24 h at room temperature (good yields were also achieved

Figure 3. 1H NMR spectra (43 MHz) of (A) 5,5′-dibromomethyl-2,2′-bipyridine in CDCl3 and (B) 5,5′-bis[1-(triethylammonio)methyl]-2,2′bipyridine dibromide (L2Br2) in D2O. D




precipitates in chloroform and the contaminants stay in solution, guaranteeing high purity of the final product. Bioinorganic/Molecular Biology Section

This part was carried out in teams of two. As the isolation and solid-state characterization of the copper complex of L2 is timeintensive and not manageable within the time frame of a lab class, the ML2 complexes of L1 and L2 were prepared in situ by mixing solutions of L1 and L2, respectively, with a solution of copper(II) nitrate. The formed complexes were characterized by UV/VIS spectroscopy (complex formation was also visible by the naked eye because of the intense color change upon addition of the copper salt to the ligand; see the Supporting Information for additional ESI-MS data). The copper(II) complexes show d−d bands with a maximum absorption wavelength (λmax) of around 730 nm (Figure 4). In contrast, the hexaaquacopper(II) Figure 5. Graphical representations of (A) DNA cleavage during incubation and (B) the corresponding agarose gel.

the presence of ascorbate were compared (Figure 6). The two complexes showed similar concentration-dependent cleavage profiles. At high concentrations form III is observed, even with a smear (or complete disappearance of the DNA band; see the Supporting Information, A-2,8) indicating cleavage into smaller fragments. The substitution of 2,2′-bipyridine with the quaternary ammonium functions had no effect on the generation of reactive oxygen species, which is initiated by the Cu(II) metal center. Optionally, to prove that the cleavage is indeed oxidative in nature, ROS quenching experiments could also be conducted to show that hydroxyl radicals, peroxo species, and superoxide radical anions (see Scheme 2) as well as singlet oxygen caused the cleavage reaction, as it was quenched by the addition of the quenchers DMSO, NaN3, pyruvate, and tiron (see the Supporting Information, A-2,8). For the examination of hydrolytic DNA cleavage, higher complex concentrations were used (up to 75 μM) and ascorbate was omitted from the incubation solution (Figure 7). A comparison of the cleavage activities of [Cu(L1)2]2+ and [Cu(L2)2]6+ revealed a higher activity for the copper complex of the positively charged ligand L2. Student data reproducibly followed this trend (with only one exception due to pipetting errors). Whereas the highest concentration of [Cu(L1)2]2+ yielded only form II DNA, [Cu(L2)2]6+ performed complete cleavage of form I plasmid DNA and even generated form III DNA. The concept of custom-designed artificial nucleases was thus conveyed to students by the comparison of the bipyridine complex [Cu(L1)2]2+ with [Cu(L2)2]6+. Whereas the first complex presumably interacts electrostatically with the phosphate backbone of the DNA through the positively charged metal center only, the latter one features additional electrostatic interactions with two phosphate groups due to its quaternary ammonium groups (Figure 2).

Figure 4. Absorption spectra of Cu(NO3)2, [Cu(L1)2]2+, and [Cu(L2)2]6+ (5 mM) in water (representative student data).

species, which is easily generated by dissolving the nitrate salt in water, weakly absorbs at 775 nm. Students were asked to explain the origin of the UV/VIS band in their laboratory journal and protocol. Because they had no further information in their handout and UV/VIS spectroscopy of metal complexes was not further explained here, students had to become active, looking into the literature and into textbooks to interpret their recorded spectra. Students compared the DNA cleavage activities of complexes [Cu(L1)2]2+ and [Cu(L2)2]6+ by incubation with pBR322 plasmid DNA and subsequent detection of the cleavage products by agarose gel electrophoresis. To convey a deeper understanding regarding the cleavage mechanism, both oxidative and hydrolytic DNA cleavage experiments were conducted by adding or omitting ascorbate as a reducing agent, respectively. Initially, uncleaved plasmid DNA exists in the supercoiled form I. When incubated with an artificial metallonuclease, it is cleaved into the open circular form II DNA. Another cut on the opposing strand in close proximity to the first one converts the DNA into its linear form III (Figure 5). In an agarose gel, the three different plasmid DNA forms can be separated on the basis of their different conformations and topologies, which result in more or less frictional resistance within the gel and thus different velocities of migration when a voltage is applied. Because of the negative charge of the phosphate backbone, the DNA migrates from the negative pole to the positive pole. Visualization of DNA in the gel can be achieved by UV excitation of the intercalated DNA stain (ethidium bromide or, preferably, the less hazardous GelRed). The relative quantities of the DNA forms are directly proportional to the DNA cleavage activities of the complexesthe more form II or III DNA is present, the more active the complex is. In a first experiment, the oxidative DNA cleavage activities of [Cu(L1)2]2+ and [Cu(L2)2]6+ at concentrations up to 20 μM in

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ASSESSMENT The experiment was carried out by two student groups (13 students total) within the laboratory “Organic and Inorganic Synthetic Chemistry” in 2017 and 2018. An assessment was done using multiple methods: 1. In an objective manner by the protocol written by students to check the gain in knowledge about DNA scissors E




Figure 6. Representative student gel showing the cleavage activities of [Cu(L1)2]2+ and [Cu(L2)2]6+ toward pBR322 plasmid DNA (0.025 μg μL−1) in HEPES buffer (20 mM; pH 7.4) after incubation at 37 °C for 2 h with addition of ascorbate (250 μM). Lane 1: DNA ladder. Lanes 2−4: 5, 10, and 20 μM [Cu(L1)2]2+. Lanes 5−7: 5, 10, and 20 μM [Cu(L2)2]6+. Lane 8: DNA control. All of the other gels prepared by the students as well as idealized triplicate data can be found in the Supporting Information (A-2,8).

Figure 7. Representative student gel showing the cleavage activities of [Cu(L1)2]2+ and [Cu(L2)2]6+ toward pBR322 plasmid DNA (0.025 μg μL−1) in HEPES buffer (20 mM; pH 7.4) after incubation at 37 °C for 2 h. Lane 1: DNA ladder. Lanes 2−4: 25, 50, and 75 μM [Cu(L1)2]2+. Lanes 5−7: 25, 50, and 75 μM [Cu(L2)2]6+. Lane 8: DNA control. All of the other gels prepared by the students as well as idealized triplicate data can be found in the Supporting Information (A-2,8).

2. I am able to explain the functional principle of column chromatography. 3. I am able to interpret the 1H NMR spectrum of a heteroaromatic compound. 4. I am able to explain the origin of bands in a UV/VIS spectrum of a transition metal complex. 5. I am able to explain the functional principle of gel electrophoresis. 6. I am aware of the hazardous properties of chemicals such as tetrachloromethane and ethidium bromide. 7. I know how to look for substituents for hazardous chemicals and to evaluate their applicability. The student response mean scores provide a rough overview (Figure 8). It is evident that all of the postexperimental values are higher than the pre-experimental ones. The former ones are above the theoretical mean of 3.5, and thus, the tendency for the postexperimental knowledge is toward “(very) good”. As expected from the students’ experiences in previous laboratories, knowledge of column chromatography, multistep organic synthesis, and interpretation of 1H NMR spectra did not increase that much. The largest gains of knowledge were in the fields of gel electrophoresis and UV/VIS spectroscopy. For method 3, the postexperiment questionnaire was complemented with a survey containing open-ended questions regarding students’ opinions about the experiment to elicit general feedback. This was planned for internal evaluation use; nevertheless, the questionnaire is provided in Supporting Information (Part C), as it might be of interest for readers. The student survey indicated that students gained their first experiences working with small amounts of substances and volumes. They experienced gel electrophoresis as a new and interesting technique and enjoyed being in a research laboratory

2. In a subjective manner by using pre- and postexperiment questionnaires 3. By using open-ended questions regarding the overall experience with that experiment and for providing feedback. These multiple approaches were used to gain insight into the learning outcomes as well as the quality and impact of the experiment from each student’s point of view. Using method 1, students were able to show that they had acquired knowledge about artificial DNA cleavage agents, a topic previously unknown to them, as had become obvious during the pre-experiment meeting. Within the discussion part of their protocol, students were even able to develop their own ideas for potential applications (consulting and exploiting current scientific literature also), which showed that the topic was understood and classified within a larger context. For example, students proposed the following applications: the development of hydrolytic DNA cleaving agents for the specific removal of disease-related DNA sequences and the development of artificial enzymes that mimic and overcome the limits of naturally occurring enzymes. In method 2, assessment questionnaires were administered to students both before and after the experiment (see the Supporting Information, Part C). Students subjectively evaluated pre- and postexperimentally (1 = no knowledge, 6 = very good knowledge) the following areas: problem solving skills (question 1), chemical literature and information management skills (questions 2−5), and laboratory safety skills (questions 6 and 7) touching on different aspects and techniques of the experiment.15 The statements for students’ response are as follows: 1. I am able to carry out multistep organic syntheses (considering time and material resources). F




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and all student results); optional quench experiments; student handout material; assessment forms (PDF, DOCX)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Nora Kulak: 0000-0002-8347-4046 Author Contributions

All of the authors contributed to writing the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Petra Skiebe-Corrette (NatLab, Freie Universität Berlin) for initiating a fruitful collaboration with our lab by supporting the development of interdisciplinary experiments for young pupils and students. Jelena Wiecko, Reinhold Zimmer, and Alexander Oehrl are acknowledged for supporting this experiment within the laboratory “Organische und Anorganische Synthesechemie”, as are their students for showing so much interest in our experiment and providing constructive feedback. J.H. thanks Studienstiftung des deutschen Volkes and Dahlem Research School for fellowships. All members of the Kulak group are acknowledged for proofreading the manuscript. This project was supported by the DFG (German Research Foundation), GR 3585/3.

Figure 8. Bar diagram showing the means of the students’ answers (N = 13) concerning their prelab (blue bars) vs. postlab knowledge (green bars), with 1 indicating no knowledge and 6 indicating very good knowledge. Error bars represent standard deviations. Statements 1−7 for students’ response are given in the text.

working side-by-side with their instructor outside of the lab classroom, for example, when UV/VIS spectra were measured. Overall, the experiment was experienced as complex in a positive sense, and students appreciated how much they learned. Students appreciated the suitable mixture of the disciplines and the balance of working alone and in teams.

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(1) Mancin, F.; Scrimin, P.; Tecilla, P.; Tonellato, U. Artificial Metallonucleases. Chem. Commun. 2005, No. 20, 2540−2548. (2) Wende, C.; Lüdtke, C.; Kulak, N. Copper Complexes of N-Donor Ligands as Artificial Nucleases. Eur. J. Inorg. Chem. 2014, 2014 (16), 2597−2612. (3) Cheignon, C.; Faller, P.; Testemale, D.; Hureau, C.; Collin, F. Metal-Catalyzed Oxidation of Aβ and the Resulting Reorganization of Cu Binding Sites Promote ROS Production. Metallomics 2016, 8 (10), 1081−1089. (4) Burrows, C. J.; Muller, J. G. Oxidative Nucleobase Modifications Leading to Strand Scission. Chem. Rev. 1998, 98 (3), 1109−1152. (5) An, Y.; Tong, M.-L.; Ji, L.-N.; Mao, Z.-W. Double-Strand DNA Cleavage by Copper Complexes of 2,2′-Dipyridyl with Electropositive Pendants. Dalton Trans. 2006, No. 17, 2066−2071. (6) Rabago Smith, M.; McAllister, R.; Newkirk, K.; Basing, A.; Wang, L. Development of an Interdisciplinary Experimental Series for the Laboratory Courses of Cell and Molecular Biology and Advance Inorganic Chemistry. J. Chem. Educ. 2012, 89 (1), 150−155. (7) American Chemical Society, Committee on Professional Training. Undergraduate Professional Education in Chemistry: ACS Guidelines and Evaluation Procedures for Bachelor’s Degree Programs; American Chemical Society: Washington, DC, 2015; pp 1−38. (8) The German bachelor program equals 3 years of undergraduate studies. (9) Gesellschaft Deutscher Chemiker e.V. (GDCh). Empfehlungen der GDCh-Studienkommission zum Bachelor-Studium Chemie an Universitäten; GDCh: Frankfurt, Germany, 2015; pp 1−52. (10) Harlen, W.; Bell, D.; Devés, R.; Dyasi, H.; Fernández de la Garza, G.; Léna, P.; Millar, R.; Reiss, M.; Rowell, P.; Yu, W. Principles and Big Ideas of Science Education; Association for Science Education: Hatfield, U.K., 2010; p 45. (11) Shafirovich, V.; Singh, C.; Geacintov, N. E. Photoinduced Oxidative DNA Damage Revealed by an Agarose Gel Nicking Assay: A

CONCLUSIONS This interdisciplinary experiment comprised the preparation and characterization of a rationally designed artificial metallonuclease. Its DNA cleavage activity was juxtaposed with that of its nonfunctionalized parent compound, thus also providing insight into the different mechanisms for DNA cleavage. Within this scope, students glimpsed research in the field of bioinorganic chemistry. Overall, students engaged with theoretical background and experimental skills that are essential in interdisciplinary research at the interface between chemistry and biology. The experiment was supposed to dismantle the perceptions of the different subdisciplines. From the responses to the objective questions (e.g., potential applications of the experiment) and the subjective assessment, students enhanced their knowledge and became familiar with interdisciplinary approaches at the intersection of chemistry and biology. In bioinorganic research groups as well as in the pharmaceutical industry, interdisciplinary projects are part of daily working life. Students thus benefited by becoming familiarized as early as possible with such approaches within their curricula.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00662. Detailed instructor information with experimental procedures; elemental analysis, NMR, UV/VIS and mass spectra; pipetting schemes; agarose gel photographs for oxidative and hydrolytic DNA cleavage (both idealized G




Biophysical Chemistry Laboratory Experiment. J. Chem. Educ. 2003, 80 (11), 1297−1299. (12) Bourzac, K. M.; LaVine, L. J.; Rice, M. S. Analysis of DAPI and SYBR Green I as Alternatives to Ethidium Bromide for Nucleic Acid Staining in Agarose Gel Electrophoresis. J. Chem. Educ. 2003, 80 (11), 1292−1296. (13) Saeidnia, S.; Abdollahi, M. Are Other Fluorescent Tags Used Instead of Ethidium Bromide Safer? DARU, J. Pharm. Sci. 2013, 21 (1), 71. (14) Schubert, U. S.; Eschbaumer, C.; Hochwimmer, G. High Yield Synthesis of 5,5′-Dimethyl-2,2′-Bipyridine and 5,5″-Dimethyl2,2′:6′,2″-Terpyridine and Some Bisfunctionalization Reactions Using N-Bromosuccinimide. Synthesis 1999, 1999 (5), 779−782. (15) Team skills were interrogated only in assessment method 3 because they are difficult to quantify.

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Synthesis and Evaluation of Artificial DNA Scissors: An

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