ARTICLE pubs.acs.org/jchemeduc
DNA Structure and Supercoiling: Ribbons and a Yo-Yo Model J. David Van Horn* Department of Chemistry, University of Missouri—Kansas City, Kansas City, Missouri 64110, United States
bS Supporting Information ABSTRACT: The double-helical structure of DNA is a pop cultural icon. Images of the DNA molecule appear in newspapers, popular journals, and advertisements. In addition to scientific instrument sales, the aura surrounding the central molecule of life has been used to sell everything from perfume to beverages and is the inspiration of items ranging from jewelry to staircases and from paintings to sculpture. The 50th anniversary of the discovery of the structure of DNA (19532003) highlighted the dramatic change in both the scientific and public perception of this central biological molecule. The helical structure and physical features of DNA are the topic of classroom instruction from the high school level through post-graduate training. The major structural features of the DNA molecule are presented in a new “model for everyone” based on a printed ribbon. Tertiary and quaternary nucleic acid structure, including supercoiling, are demonstrated using a yo-yo model; the replication fork required for nucleic acid replication and transcription can also be demonstrated using this model. These simple models can be adapted for use in high school classrooms or for university and graduate course instruction. KEYWORDS: High School/Introductory Chemistry, Upper-Division Undergraduate, Biochemistry, Hands-On Learning/ Manipulatives, Descriptive Chemistry, Nucleic Acids/DNA/RNA, Physical Properties, Student-Centered Learning
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he structure of the double helix of DNA has become a pop cultural icon over the last few decades. Images of the DNA molecule appear in newspapers, popular journals, and advertisements. In addition to scientific instruments, the aura surrounding the central molecule of life has been used to sell everything from perfume to beverages. The “double-helix” is the inspiration for items ranging from jewelry to staircases and from toys to art. The 50th anniversary of the discovery of the structure of DNA1 has recently highlighted the dramatic change in both the scientific and public perception of this biological molecule. Beyond the helical representation of DNA that most people have seen, there are subtleties in molecules of DNA (and in nucleic acids in general) that are accessible to high school- and college-level students, or the general public, with simple models that can demonstrate these structural complexities. As part of the development of a bioorganic chemistry class for undergraduates, we were interested in presenting materials and models on the chemical architecture and function of DNA, including primary, secondary, and tertiary structures. For instance, one subtlety in the double helix of DNA is the righthandedness of the common A- and B-form helices. In contrast, examples of inverted, incorrect left-hand forms2,3 have appeared in advertising media and popular serials, and even on the cover of some textbooks and journals. These instances can be easily found on the Internet (search the term “DNA left hand” or similar). Various aspects of the structure and physical features of DNA have been commented on in this Journal.414 Some of the models presented include the use of a cardboard tube from a paper towel roll,5 aluminum rods and tubing for a framework model,6 and stacked boxes7 or plastic canvas8 as the base pairs in helical models. By adding beads or wires, the plastic canvas in the Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.
last model allows for the modification of the chemical groups on the base pairs, the demonstration of H-bonding, and so on. Custom fabricated plastic pieces have also been developed to demonstrate many features of duplex nucleic acid structure including major and minor grooves and the replication fork.9 Cox provided an excellent table reviewing types of models and the features of nucleic acid structure that each model effectively demonstrates.9 The models presented in this article fit under the ribbon category, but also highlight features of local structure and of an additional category, supercoiling. Supercoiling and the topology of DNA have been addressed in educational reviews10,11 and in biochemical experiments with circular DNA,12 as has the physical chemistry of strand melting13 and helix transitions.14 Finally, Rhodes has presented a detailed demonstration of supercoiling using rubber tubing to help explain the features of DNA topology and the action of type-II topoisomerase enzymes.15 This article revisits the major structural features of the DNA molecule, presents a new “model for everyone”,5 and describes a new demonstration of DNA supercoiling and replication using a yo-yo model.a
’ NUCLEIC ACID STRUCTURE REPRESENTATION The double helix of DNA commonly adopts one of two righthanded helical forms, designated “A-form” and “B-form”. The B-form is the more stable structure; A-form is a more tightly wound right-handed helix compared to the B-form. A left-handed helix, the “Z-form” structure of DNA, is found in unusual Published: June 20, 2011 1264
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Figure 3. Comparison of D- and L-ribose versus L-lyxose. D-ribose is the naturally occurring pentose in nucleic acids, and the configuration at C-30 and C-40 are the key stereocenters in the formation of regular righthanded DNA and RNA helices.
form (a rope, an electrical cord, a garden hose, a tree trunk, etc.) or induced into a helix by twisting (a ribbon, flexible tubing, etc.). Even monofilament and “single-stranded” objects (copper wire, fishing line, coaxial cable, etc.) are affected by twisting and can become supercoiled, sometimes to the consternation of the user.
Figure 1. Ribbon model of DNA showing left- and right-handed helices, respectively. DNA is found in right-handed A- and B-forms and less commonly in Z-form—a left-handed helix, but not the structural opposite (enantiomer) of the right-handed helices.
Figure 2. A ribbon may be used as a model of DNA. The edges represent the phosphodiester backbone and the printing represents the base pairs, H-bonding, and π-stacking.
circumstances or conditions. As a result of the widespread use of widely available graphic generation and manipulation software, it is now quite commonplace to reproduce images of biomolecules, including nucleic acids. This same software has the capability to reverse images that may lead to the misrepresentation of nucleic acids in helices that are impossible to adopt in the naturally occurring molecules. Reversal of an image takes an image of regular A- or B-form DNA and turns it into its left-handed enantiomer—an unnatural structure! The publication and production of such mirror images of nucleic acid helices is quite commonplace;2,3 the reader may find such instances upon a detailed inspection of their favorite periodical or newspaper. The helices formed by nucleic acids and the handedness of these structures are a fundamental facet of these molecules in particular and of helical objects in general. For instance, any ribbon or multi-stranded object may be inherently helical in its
’ A RIBBON MODEL OF NUCLEIC ACIDS A ribbon model that is accessible, inexpensive, and easily used to demonstrate a number of features of double-helical nucleic acid structures is presented9 (a printable sheet for demonstration use is available in the Supporting Information). This model developed out of an exercise with a strip of paper to demonstrate left-handed versus right-handed orientations of a duplex DNA strand and is illustrated in Figures 1 and 2. Initially, one can form a B-form right-hand helix and demonstrate how the first stages of supercoiling tightens the helix to give A-form DNA. The instructor can also describe how the sequence, tertiary and quaternary interactions, or solution conditions give rise to additional structures in a given strand of DNA (e.g., AT-rich regions). The ribbon may also model the difference between the major groove and the minor groove of DNA, also illustrated in Figure 1. The paper strip indicates, if somewhat imperfectly, the “minor groove” and the gap between the loops of the paper act as the “major groove”. The model is enhanced by the addition of printed markings to denote the phosphodiester backbone of DNA along the edges of the ribbon. These are connected with markings for the H-bonded base pairs in a stepped fashion to the next phosphate above, rather than its neighbor directly across, to properly give the base pairs a nearly perpendicular orientation to the right-handed helical axis, analogous to A- or B-form DNA. Turning the ribbon into a left-handed helix arrives at a model that presents a number of difficulties in comparison to the known helices in DNA. A blank ribbon would give rise to two enantiomers when wound in the two directions; the inclusion of printing makes these two particular ribbon conformations into diastereomers, as the base pairs are aligned almost parallel to the axis of the left-handed helix. Finally, it should be noted that this lefthand helix is not a model of Z-form DNA. These observations, and the orientation of the base pairs in the left-handed helix ribbon model (parallel to the helix axis), suggest that it is geometrically impossible for the structure of nucleic acids consisting of D-ribose units to form a “regular” left-hand helix. Thus, the Z-form appears to represent the limiting lefthand helix that could be formed with D-ribose nucleotides. There are three stereocenters in the ribose unit that may specifically give rise to the right-hand helical formation of A- and B-form nucleic acids, not including the C-20 position of ribonucleotides (see Figure 3 for a comparison of the relevant 20 -deoxypentose units). 1265
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Figure 4. The yo-yo model of DNA: a double-helical structure (bottom) that exhibits supercoiling (top).
Figure 5. Illustration of a DNA replication fork in the yo-yo model of DNA. This demonstration is not recommended for your favorite yo-yo...
Obviously, using the enantiomer of D-ribose, L-ribose, should give rise to a regular left-hand A- or B-form duplex—the enantiomer of “normal” DNA—assuming that the nucleobase at C-10 is also epimeric. Alternatively, switching only the stereocenter at C-40 would lead to a nucleic acid composed of L-lyxose units, which should give rise to yet another type of helical structure, if a 50 -to-30 phosphodiester polymer backbone were to be maintained in such a structure. These considerations might be an interesting avenue for further student research and model building, and are certainly of interest in the areas of novel nucleic acid therapeutics16 and speculation about prebiotic chemistry.17
illustrated in Figure 4. The string may be used to illustrate the topological problem that exists when the DNA double helix needs to be unraveled by the helicase enzyme for transcription or replication. If one end of the yo-yo string is not free, similar to natural systems, attempting to relax one end of the helix will lead to supercoiling up the string, as is the case for circular DNA. This can be illustrated holding both ends of the string, and then “pushing” the supercoiled part to one end. In biological systems, this topological problem is alleviated by the topoisomerase (type 1 and type 2) enzymes; the recognition of supercoiled DNA handedness by viral versions of these enzymes, among other biochemical interactions, is a significant area of research interest.18 Finally, a fork in the DNA duplex is required for biological replication or transcription; this rather important biochemical transformation may be illustrated by gently prying apart the two strands of a yo-yo string (use an old string!), as illustrated in Figure 5. With this illustration, an instructor can discuss the action of helicase at the actual fork of the duplex, the action of topoisomerase in advance of the replication fork and other components in the biological machinery of replication and transcription. Of further interest, and shown in Figure 5, is the occurrence of localized supercoiling in the “single-stranded” region of the fork and the occurrence of additional supercoiling in advance of the replication fork caused by separating the strands. This feature is reminiscent of loop structures commonly formed in RNA and are interesting when considering localized structure formation in single-stranded DNA (recall that there are singlestrand DNA binding proteins in the replication machinery). The additional supercoiling in the model reemphasizes the need for topoisomerase enzymes and the potential energy that is stored up in a supercoiled structure.
’ SUPERCOILING AND A YO-YO MODEL OF NUCLEIC ACIDS Any two-stranded object may form a double helix and included in this broad class is the string of a yo-yo. This toy offers instructors a compact and flexible model of the DNA helix and more complex features of nucleic acid structure and function. To demonstrate higher-order structural aspects of DNA (supercoiling, 2°, 3°, and 4° structures), a yo-yo model can be used to illustrate supercoiling, the relaxation of strands by topoisomerase enzymes, or the unwinding of the double helix by helicase in the replication or transcription of DNA. The (sometimes annoying) occurrence of supercoiling is not only found in this toy, but can arise in other facets of life. For instance, small appliance power cords (e.g., coffee grinder), microphone cords, and electrical extension cords all may become supercoiled with use and upon being wound for storage. Practically, this leads to specific strategies for the winding of these macro objects (see movies S1 and S2 in the Supporting Information). The yo-yo string can be used to indicate supercoiling in a double-helical structure; this is obvious in a yo-yo string that has been overwound after some extensive manipulation and tricks (Figure 4). Typically, as one plays, supercoiling is introduced by the repetitive turning (1/2 turn each time) of the yo-yo body in the hand with successive throws. Over time, the supercoiling increases in the string as it is overwound in this fashion; if left unattended, the supercoiling can sometimes lead to the yo-yo spinning out of control in a lateral fashion. After throwing the yoyo for a short while, a supercoiled string is generally “relaxed” by stopping play, unwinding the string by stretching it out, and finally looping the string back around the body of the yo-yo. One last point: loosening the helix is critical for allowing the yo-yo to “sleep” at the bottom of a throw for certain tricks. The helical nature of the yo-yo string (bottom) and the supercoiling that exists in an overwound yo-yo string (top) are
’ SUMMARY The two simple models presented here, the ribbon and the yoyo, effectively demonstrate various aspects of DNA. The ribbon model of DNA may be used to demonstrate (i) left- and righthandedness in helices, (ii) the major and minor grooves of a duplex, (iii) the winding of a helix as in the transition of B- to A-form DNA, and (iv) the stacking of base pairs perpendicular to the helix axis in the correct right-hand helix of DNA. A yo-yo string is an effective, compact model of the double helix of DNA or RNA that can be adapted to classroom use as a model of supercoiling and the features of biological replication forks. Finally, the utility of the discussion of supercoiling and helix structure is of interest to artists and designers, as well as engineers and inventors in considering the design and the fabrication of 1266
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helical structures. The innate structures and beauty of helices are part of the reason for the general aura surrounding the twists and turns of the DNA molecule.
’ ASSOCIATED CONTENT
bS
Supporting Information Movies S1 and S2 demonstrating small appliance cord coiling that avoids or promotes supercoiling, respectively. A PDF file of printable DNA ribbon models (six per page) for legal-sized paper. This material is available via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ADDITIONAL NOTE a In practice, the demonstrations using the models in this article are preceded with illustrations from a text, or in a computer presentation, to introduce students to the chemical structure of nucleic acids, sequence notation, double-helical structures, major and minor grooves of DNA, etc. Illustrations from advertising, scientific papers, and from computational and structural data are also presented. Students are introduced to the known A-, B-, and Z-forms of DNA, given a ribbon, and shown how to form right- or left-handed helices. The handedness of the helices is demonstrated, and participants are shown how to generally determine the “handedness” of any helix; the discovery of helices in everyday life (e.g., artworks, helical slides in parks, etc.) is also encouraged. ’ REFERENCES (1) Watson, J. D.; Crick, F. H. C. Nature 1953, 171, 737–738. (2) Schneider, F.; Droege, P.; Mueller-Hill, B. Trends Genet. 1999, 15, 95. (3) Eisen, M. Nature 2010, 467, 401. (4) Wolbarst, A. B. J. Chem. Educ. 1979, 56, 733. (5) Wilbraham, A. C. J. Chem. Educ. 1987, 64, 806. (6) Anderson, J. A. J. Chem. Educ. 1972, 49, 329. (7) Bruist, M. F. J. Chem. Educ. 1998, 75, 53–55. (8) Cady, S. G. J. Chem. Educ. 2005, 82, 79–84. (9) Cox, J. P. L. J. Chem. Educ. 2006, 83, 1319–1321. (10) Scovell, W. M. J. Chem. Educ. 1986, 63, 562–565. (11) Sinden, R. R. J. Chem. Educ. 1987, 64, 294–297. (12) Keck, M. V. J. Chem. Educ. 2000, 77, 1471–1473. (13) Howard, K. P. J. Chem. Educ. 2000, 77, 1469–1470. (14) Steinert, R.; Hudson, B. J. Chem. Educ. 1973, 50, 129–130. (15) Rhodes, G. Chem. Educator 1997, 2, 1–8. (16) Urata, H. Yakuga Zasshi 1999, 119, 689–709. (17) Springsteen, G.; Joyce, G. F. J. Am. Chem. Soc. 2004, 126, 9578–9583. (18) McClendon, A. K.; Dickey, J. S.; Osheroff, N. Biochemistry 2006, 45, 11674–11680.
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