Laboratory Experiment pubs.acs.org/jchemeduc
Teaching Upper-Division Biochemistry Students the Role of Noncovalent Interactions and Ligand Structure in DNA Binding Kristen C. Roy* Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, United States S Supporting Information *
ABSTRACT: The experiment described here uses the binding of cyanine dyes to DNA as a model system for demonstrating the role that both ligand structure and noncovalent interactions play in DNA binding. Upper-division biochemistry laboratory students observe changes in absorbance and fluorescence spectra when small molecules bind either in the minor groove of DNA or by intercalation. Students specifically note that ligand charge, substituents, and flexibility greatly influence binding mode. Based on these observations, students then determine the binding mode of an unknown small molecule. This experiment also fosters independent thought and problem-solving abilities. Students must selfdirect binding-mode identification of their unknown without any guidance from the instructor. After completing this experiment, students demonstrate a clear understanding of the factors that influence DNA binding. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Collaborative/Cooperative Learning, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning, Biophysical Chemistry, Dyes/Pigments, Nucleic Acids/DNA/RNA, Fluorescence Spectroscopy
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A significant pedagogical goal of the experiment is to lead students to an appreciation of the role of noncovalent interactions, such as electrostatics, hydrogen bonding, aromatic stacking, van der Waals forces, and hydrophobic interactions, in the binding of various molecules with DNA. DNA-binding proteins, naturally occurring antibiotics, and numerous synthetic molecules used as sensors, therapeutics, and probes for biological functions all use combinations of these forces in their interactions with DNA.1−3 Thus, discussion of these applications as related to the model system emphasizes the realworld applicability of understanding DNA binding. A studentdirected component, in which students determine the binding mode of an unknown without any guidance, is used to foster independent thinking. This experiment also engages students by linking concepts from many different courses such as biochemistry, organic chemistry, and physical chemistry to arrive at a better overall understanding of DNA−small molecule interactions. Only a few previously published experiments have explored the binding of small molecules to DNA. Healy described an experiment to determine binding stoichiometry of DAPI with DNA,4 while Strothkamp and Strothkamp presented one that uses Scatchard plots to describe the binding of ethidium bromide to DNA.5 These experiments teach important concepts using a single binding mode as the focus. In contrast to previous experiments, this module is designed to allow students to observe multiple binding modes of small molecules with DNA within a single laboratory exercise, allowing students
n-depth study of the factors that govern binding of small molecules to DNA is often entirely neglected in undergraduate laboratory courses because the sensitivity of these interactions to environmental conditions and the need for specialized equipment render these experiments impractical. An experiment is described that has been successfully implemented for seven semesters over four years to teach a total of 218 upper-division biochemistry students the importance of various factors in binding of small molecules to DNA. This experiment uses the binding of different cyanine dyes (Figure 1) as a model system for demonstrating the role that both ligand structure and noncovalent interactions play in binding to DNA in the minor groove and by intercalation. Changes in the photophysical properties of these dyes are used to identify the binding mode.
Figure 1. DNA sequences and dyes used in this experiment. © 2013 American Chemical Society and Division of Chemical Education, Inc.
Published: August 28, 2013 1384
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Figure 2. Student-generated absorbance (A) and fluorescence (B) data for DiSC2(5). All samples contained 10 μM DiSC2(5), 10 mM sodium phosphate buffer, pH 7.4, and 20% methanol. In samples containing DNA, 5 μM duplex was used. For fluorescence measurements, DiSC2(5) was excited at 651 nm.
to compare and contrast the properties exhibited by each. Different cyanine dyes can bind to DNA in the minor groove as monomers or aggregates, or by intercalation.6 The binding mode is dependent on structural features of the dye such as flexibility, substituent groups, and heterocycle composition.6 In addition, the dyes bind using combinations of noncovalent interactions that vary between the binding modes.6 Furthermore, minor groove binding (MGB) dyes bind in a sequence-dependent manner, binding preferentially to A-T DNA sequences over G-C sequences because of steric hindrance by the G-C amino group.1,7−10 MGB dyes that bind as aggregates exhibit a stronger sequence dependence than MGB dyes that bind as monomers.8,9 This property adds a critical-thinking component to the experiment by allowing students first to predict, then to observe, variations in dye aggregation based on variations in DNA sequence. Many cyanine dyes are commercially available at low cost, and their binding to DNA is characterized by profound spectroscopic changes that are well characterized and easily observed using standard teaching lab spectrophotometers.2,11,12 In addition, because these spectroscopic changes are diagnostic of the binding mode, using simple techniques, such as UV−vis absorbance and fluorescence spectroscopies, students are able to monitor both the binding of dye to DNA and the effects of factors such as DNA sequence and dye structure. The spectroscopic properties of the dyes are also of interest because they demonstrate interesting photophysical properties that are commonly exploited for biochemical applications.
designed to be performed during two three- to four-hour lab periods followed by a day of presentations. Briefly, students work in groups of three to four students throughout the experiment. During the first lab period each student group prepares 5 μM dilutions of each DNA duplex in 10 mM sodium phosphate buffer (NaPi), pH 7.4 containing 20% methanol. 3,3Diethylthiadicarbocyanine iodide [DiSC2(5)] or thiazole orange (TO) is then added to a final concentration of 10 μM. The absorbance of all samples is measured. Subsequently, fluorescence emission spectra of the same samples are measured. The excitation wavelengths for DiSC2(5) and TO are 651 and 470 nm, respectively. During the next lab period, each lab group is given a unique, unknown small molecule for which they must determine its binding mode. Determination of binding mode is made entirely by the students with no directions provided. Unknowns are intentionally selected to be ambiguous compared to the model system. This fosters further discussion of the multiple factors that affect binding mode. Additional details on the unknowns can be found in the Supporting Information. Once the binding mode has been identified by a student group, the chemical name of the unknown is given. Students are then required to prepare a twelve- to fifteen-minute presentation in which they first present background information about the unknown, such as its structure and practical uses. The remainder of the presentation is about the self-guided portion of the experiment, describing the analytical process through which they arrived at the binding mode of the unknown. Students should present the data collected and explain how it supports the binding mode. Presentations are given one week following identification of the unknown binding mode.
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PROCEDURE AND INSTRUMENTATION A thorough prelab lecture must be given for students to be able to correlate observed data with the theoretical concepts. An outline of the instructor lecture notes is available in the Supporting Information. The author dedicates one full hour to prelab lecture on the outlined topics. Although students will likely be familiar with the possible binding modes with DNA, it is unlikely that they have enough knowledge of the factors that govern binding. Furthermore, cyanine dyes exhibit many interesting optical properties that are used in this experiment, but not generally discussed in undergraduate courses. Thus, details about the esoteric theory related both to cyanine dyes and to DNA binding should be given. Protocols, reagents, and instrumentation are described in the Supporting Information, where explicit instructions for both students and instructors are detailed. This experiment was
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HAZARDS Methanol is flammable, is toxic by inhalation, and can cause blindness. DiSC2(5) is extremely hazardous and may be fatal if swallowed and is an irritant to skin, eyes, and the respiratory system. TO is harmful by ingestion and an irritant by inhalation and skin contact. The hazards for each of the unknowns are similar to those of both DiSC2(5) and TO. To minimize these hazards, all dyes should be provided to students in solution. DNA sequences have no known hazardous effects, but may be an irritant to the skin, eyes, and respiratory tract. Students should be required to wear goggles and gloves for all experimentation. During the prelab lecture the dangerous nature of the reagents is emphasized. 1385
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Figure 3. Student-generated absorbance (A) and fluorescence (B) data for TO. All samples contained 10 μM TO, 10 mM sodium phosphate buffer, pH 7.4, and 20% methanol. In samples containing DNA, 5 μM duplex was used. For fluorescence measurements, TO was excited at 470 nm. Fluorescence intensity for TO in both MeOH and buffer is negligible and, thus, overlays with the baseline (B).
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of why the monomeric dye is fluorescent whereas the Haggregate is not.11 Thus, students were able to correlate the increasing amount of aggregation with an attenuated fluorescence. Students also observed a hypsochromic shift in the fluorescence maximum that is attributed to a solvatochromic effect in which the dye emission shifts with respect to the polarity of the environment.7 Student-generated data for TO measurements are shown in Figure 3. Here, students noted that, in contrast to the MGB dyes, the absorbance spectrum of the intercalator did not change significantly under any of the conditions (Figure 3A). Furthermore, students contrasted the approximate 100-fold increase in fluorescence intensity in the presence of DNA with the approximate 10-fold decrease observed for the MGB dyes (Figures 3B and 2B, respectively). It should be noted that TO is not fluorescent in either buffer or methanol. Thus, the fluorescence spectra for these two samples overlay the baseline. The increase in fluorescence in the intercalated state due to restriction of relaxation pathways involving bond rotation and vibrational relaxation was explained during the prelab lecture.13 In addition, the students observed that, typical of intercalators, the fluorescence intensity of TO exhibited a different sequence dependence than the MGB dyes, preferring G-C sequences due to the greater dipole moment of G-C compared to A-T.14 Finally, students contrasted the DiSC2(5) structure with that of TO, observing that the short inflexible linker in TO precluded minor groove binding, but the aromaticity provided strong π stacking interactions that stabilized intercalation. It should be noted that, because of time and instrument constraints, students did not carry out experiments with MGB dyes that bind as monomers. Rather this binding mode and sample data were discussed during the prelab lecture. This discussion is key because several of the unknowns used during the second lab period bind in this manner. Subsequently, student understanding of the concepts presented above was evaluated by a student-driven component in which each student group was provided an unknown molecule for which they were required to determine the binding mode without any guidance provided. The only information supplied included the knowledge that all unknowns have been previously shown to bind to DNA, concentration of the unknown, and the information on the possible binding modes being MGB dye aggregate, MGB dye monomer, and intercalation. A typical student plan usually included initial absorbance measurements using the unknown first in buffer, then with AT5 or AT15 and also with GC15. The resulting
RESULTS AND DISCUSSION Initially, students recorded prescribed absorbance and fluorescence measurements on DiSC2(5) and TO in the absence and presence of various DNA duplexes (Figure 1). The DNA sequences were designed to accommodate varying degrees of aggregation. For example, as the A-T content increases from AT5 to AT15, students observed an increase in aggregation of the MGB dye. GC15 accommodates almost no aggregation due to the absence of A-T base pairs. Finally, MS, which is composed of a mixture of G-C and A-T base pairs in a randomized order, demonstrates the necessity of consecutive AT stretches for efficient aggregation. Thus, rational design of DNA sequence allows aggregation of the MGB dye to be finely tuned. The dyes were chosen because they are representative of MGB dyes and intercalators with characteristic optical properties. Student-generated data for DiSC2(5) are shown in Figure 2. As shown in Figure 2A, students observed a hypsochromic shift of the absorbance maximum from 651 to 590 nm for DiSC2(5) in the presence of AT-rich DNA, which represents DNA-templated H-aggregation of DiSC2(5) in the DNA minor groove.7 Absorbance characteristics of the dye were deliberately noted with an explanation during the prelab lecture of both why the shift occurs and how the flexible linker and cationic nature are essential structural features for the minor groove binding mode. Noncovalent interactions such as electrostatics, hydrophobics, and van der Waals forces, which stabilize binding, were also discussed. Students also observed that the change in intensity of the peak at 590 nm was dependent on DNA sequence. The sequences containing stretches of A-T base pairs (AT5 and AT15) accommodated aggregation, whereas GC15 did not. The exocyclic amino group of guanine sterically hinders aggregation in the minor groove.9,10 Students observed that as the G-C content increased, aggregation decreased. Aggregation propensity corresponded to AT15 > AT5 > MS > GC15. Furthermore, the intensity of the peak at 651 nm, which corresponds to the monomer dye, was inversely related to the extent of aggregation on DNA. As the aggregate peak at 590 nm increased, the peak at 651 nm decreased. Finally, students noted that aggregation occurred to a significant extent only in the presence of a DNA template. In its absence, only a small aggregate peak was observed for DiSC2(5) in aqueous buffer. Subsequently, students performed fluorescence measurements (Figure 2B). The prelab lecture included an explanation 1386
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(2) Armitage, B. A. Cyanine Dye-DNA Interactions: Intercalation, Groove Binding, and Aggregation. Top. Curr. Chem. 2005, 253, 55−76. (3) Geierstanger, B. H.; Wemmer, D. E. Complexes of the Minor Groove of DNA. Annu. Rev. Biophys. Biomol. Struct. 1995, 24, 463− 493. (4) Healy, E. F. Quantitative Determination of DNA−Ligand Binding Using Fluorescence Spectroscopy. J. Chem. Educ. 2007, 84, 1304−1307. (5) Strothkamp, K. G.; Strothkamp, R. E. Fluorescence Measurements of Ethidium Binding to DNA. J. Chem. Educ. 1994, 71, 77−79. (6) Mishra, A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera, G. B. Cyanines During the 1990s: A Review. Chem. Rev. 2000, 100, 1973−2011. (7) Seifert, J. L.; Connor, R. E.; Kushon, S. A.; Wang, M.; Armitage, B. A. DNA-Templated Supramolecular Assemblies of Cyanine Dyes. J. Am. Chem. Soc. 1999, 121, 2987−2995. (8) Hannah, K. C.; Armitage, B. A. DNA-templated Assembly of Helical Cyanine Dye Aggregates: A Supramolecular Chain Polymerization. Acc. Chem. Res. 2004, 37, 845−853. (9) Sehlstedt, U.; Kim, S. K.; Nordén, B. Binding of 4′,6-diamidino-2phenylindole to [poly(dI-dC)]2 and [poly(dG-dC)]2: the exocyclic amino group of guanine prevents minor groove binding. J. Am. Chem. Soc. 1993, 115, 12258−12263. (10) Hannah, K.; Gil, R. R.; Armitage, B. A. 1HNMR and Optical Spectroscopic Investigation of the Sequence-Dependent Dimerization of a Symmetrical Cyanine Dye in the DNA Minor Groove. Biochemistry 2005, 44, 15924−15929. (11) Eisfeld, A.; Briggs, J. S. The J- and H-bands of Organic Dye Aggregates. Chem. Phys. 2006, 324, 376−384. (12) Jelley, E. E. Spectral Absorption and Fluorescence of Dyes in the Molecular State. Nature 1936, 138, 1009−1010. (13) Silva, G. L.; Ediz, V.; Yaron, D.; Armitage, B. A. Experimental and Computational Investigation of Unsymmetrical Cyanine Dyes: Understanding Torsionally Responsive Fluorogenic Dyes. J. Am. Chem. Soc. 2007, 129, 5710−5718. (14) Müller, W.; Crothers, D. Interactions of Heteroaromatic Compounds with Nucleic Acids. 1. The Influence of Heteroatoms and Polarizability on the Base Specificity of Intercalating Ligands. Eur. J. Biochem. 1975, 54, 267−277.
measurements can reveal a MGB dye aggregate with no further experimentation. They cannot, however, distinguish between MGB dyes binding as monomers and intercalators. Thus, the majority of students also collected fluorescence data. Sample student data can be found in the Supporting Information. At least four of six student groups regularly correctly identify the binding mode of their unknown. The remaining two groups occasionally require some guidance from the instructor to correctly interpret their data. Ideally, students will perform this experiment after they have completed discussions concerning DNA structure and noncovalent interactions in their lecture courses. Thus, this experiment expands upon and reinforces recently learned theoretical concepts. The experiment can also stand alone with some additional details given during the prelab lecture, but it has been found that students appreciate how the lecture course complements the lab when the experiment is performed with this consideration. Furthermore, if lecture topics complement the lab at the end of the semester, most students have learned to think intuitively about experimental results, asking themselves what comes next and providing logical answers to that question. These are critical skills for this experiment, which relies heavily on a student’s ability to interpret data and to decide the next step independently.
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OUTCOMES AND CONCLUSIONS The experiment described effectively taught upper-division students about the multiple factors that affect DNA binding, such as noncovalent interactions and ligand structure. It also fostered independent thought by requiring students to selfdirect part of the experiment. Students’ success in learning the desired concepts is judged both by their responses to questions asked during the presentation and by their performance on exam questions related to DNA binding. Upon completing the experiment, students acquired a clear understanding of the factors that affect DNA binding by small molecules.
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ASSOCIATED CONTENT
S Supporting Information *
Outline of the instructor lecture notes; protocols, reagents, and instrumentation; instructions for students and instructors; details on the unknowns; sample student data. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The author thanks CU students in Chem 4761 for testing this project. The author also gratefully acknowledges Elissa Guralnick (CU Boulder Faculty Teaching Excellence Program), Robert Batey (CU Boulder), and Jennifer Kugel (CU Boulder) for assistance in manuscript preparation.
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REFERENCES
(1) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E. The Molecular Recognition of DNA-drug Specificity in Netropsin and Distamycin. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376−1380. 1387
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