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Watching DNA Condensation Induced by Poly(amido amine) Dendrimers with Time-Resolved Cryo-TEM Anna M. Carnerup,* Marie-Louise Ainalem, Viveka Alfredsson, and Tommy Nylander Division of Physical Chemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, 221 00 Lund, Sweden Received August 17, 2009. Revised Manuscript Received September 24, 2009 The condensation of DNA by poly(amido amine) dendrimers of generation 1, 2, and 4 has been followed by timeresolved cryogenic transmission electron microscopy (cryo-TEM). The recorded images show that significant morphological rearrangement occurs for DNA condensed with the lower generation dendrimers leading to the formation of toroidal aggregates. Higher charge density dendrimers, on the other hand, give rise to globular aggregates, where no transient morphologies are observed. We suggest that the dendrimers in this case are kinetically trapped as soon as they bind to the DNA strand.
Introduction Deoxyribonucleic acid (DNA) condensation in vitro has attracted considerable attention due to the possibilities within nonviral gene delivery systems.1-3 In eukaryotic organisms, histone proteins are responsible for the packaging of DNA within the nucleus, and are therefore involved in the control of the genetic activity, processes that are not fully understood.4,5 DNA condensation can also be achieved with a range of different synthetic condensation agents, such as cationic surfactants, polymers, and amine containing histone analogues.6-9 The cationic nature of the condensing agents decreases the repulsion between the anionic phosphate groups of the DNA backbone, allowing condensation to occur. Around 90% of the negative charges on the DNA need to be neutralized in order to overcome the intramolecular electrostatic repulsion between the different segments of the DNA strands.10 In vitro, condensed DNA can adopt a number of different morphologies all depending on the conditions during the condensation process. Rod-like, globular, and toroidal DNA condensates are most commonly observed. Toroidal DNA aggregates have attracted particular attention due to their fascinating structure and because they are observed in both sperm cells *Corresponding author. E-mail:
[email protected]. Current address: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, U.K. (1) Luo, D.; Saltzman, W. M. Nat. Biotechnol. 2000, 18, 33–37. (2) Kubasiak, L. A.; Tomalia, D. A. In Polymeric Gene Delivery: Principles and Applications; Amiji, M. M., Ed.; CRC Press: Boca Raton, FL, 2004; pp 133-157. (3) Kabanov, V. A.; Sergeyev, V. G.; Pyshkina, O. A.; Zinchenko, A. A.; Zezin, A. B.; Joosten, J. G. H.; Brackman, J.; Yoshikawa, K. Macromolecules 2000, 33, 9587–9593. (4) Jenuwein, T.; Allis, C. D. Science 2001, 293, 1074–1080. (5) Bednar, J.; Horowitz, R. A.; Grigoryev, S. A.; Carruthers, L. M.; Hansen, J. C.; Koster, A. J.; Woodcock, C. L. Proc. Natl. Acad. Sci. 1998, 95, 14173–14178. (6) Melnikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 9951–9956. (7) Plum, G. E.; Arscott, P. G.; Bloomfield, V. A. Biopolymers 1990, 30, 631– 643. (8) Dias, R. S.; Pais, A.; Miguel, M. G.; Lindman, B. Colloids Surf., A 2004, 250, 115–131. (9) Chen, W.; Turro, N. J.; Tomalia, D. A. Langmuir 2000, 16, 15–19. (10) Bloomfield, V. A. Biopolymers 1997, 44, 269–282. (11) Klimenko, S. M.; Tikchone, T.; Andreev, V. M. J. Mol. Biol. 1967, 23, 523. (12) Hud, N. V.; Vilfan, I. D. Annu. Rev. Biophys. Biomol. Struct. 2005, 34, 295– 318.
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and viruses.11-13 One intriguing aspect of toroidal aggregates is that the DNA adopts hexagonal rod-packing as evidenced by X-ray diffraction and cryo-TEM.12,14 The interhelical spacing between DNA strands within toroidal aggregates varies depending on the level of hydration and the condensation agent used. Hud et al. found that hexammine cobalt(III) condensed DNA toroids have an interhelical spacing of 2.8 nm.14 In addition, toroid dimensions seem to be universal irrespective of the length of the DNA and condensation agent used. DNA toroids are typically 100 nm in diameter and have a 25 nm hole.12 However, larger toroids have been observed.15,16 Several studies have shown that DNA toroid dimensions are governed by ionic strength, the kind of multivalent ions present, and the size of the nucleation loop.17,18 It has therefore become apparent that the toroid formation is a nucleation and growth phenomenon, akin to crystal growth.12,19 Although considerable advances have been made in terms of understanding the condensation behavior of DNA, very few timeresolved microscopy studies have been conducted. Stopped-flow fluorescence spectroscopy20 and light scattering,21 atomic force microscopy,22 and electron microscopy investigations of negatively stained samples23 have provided insights into the temporal condensation process. For example, it has been shown that the condensation of DNA using hexammine cobalt(III) is a multistep process occurring over different time scales:23,24 (1) initial nucleation of rods and toroids (ms), (2) a shift in particle morphology and subsequent growth (min), and (3) aggregation of individual particles (days). It was further shown that the relative rod/toroid (13) Hud, N. V.; Allen, M. J.; Downing, K. H.; Lee, J.; Balhorn, R. Biochem. Biophys. Res. Commun. 1993, 193, 1347–1354. (14) Hud, N. V.; Downing, K. H. Proc. Natl. Acad. Sci. 2001, 98, 14925–14930. (15) Allison, S. A.; Herr, J. C.; Schurr, J. M. Biopolymers 1981, 20, 469–488. (16) Yoshikawa, Y.; Yoshikawa, K.; Kanbe, T. Langmuir 1999, 15, 4085–4088. (17) Conwell, C. C.; Hud, N. V. Biochemistry 2004, 43, 5380–5387. (18) Conwell, C. C.; Vilfan, I. D.; Hud, N. V. Proc. Natl. Acad. Sci. 2003, 100, 9296–9301. (19) Yoshikawa, K.; Matsuzawa, Y. J. Am. Chem. Soc. 1996, 118, 929–930. (20) Braun, C. S.; Fisher, M. T.; Tomalia, D. A.; Koe, G. S.; Koe, J. G.; Middaugh, C. R. Biophys. J. 2005, 88, 4146–4158. (21) Porschke, D. Biochemistry 1984, 23, 4821–4828. (22) Fang, Y.; Hoh, J. H. J. Am. Chem. Soc. 1998, 120, 8903–8909. (23) Vilfan, I. D.; Conwell, C. C.; Sarkar, T.; Hud, N. V. Biochemistry 2006, 45, 8174–8183. (24) He, S. Q.; Arscott, P. G.; Bloomfield, V. A. Biopolymers 2000, 53, 329–341.
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population changes with time, which suggested that rods are kinetically favored during the initial nucleation step. Both kinetic and thermodynamic factors can as such control the DNA condensate morphology.23 Poly(amido amine) (PAMAM) dendrimers are star-like polymers synthesized using an iterative process from a central core (in this case ethylenediamine). The resulting core-shell type architecture has primary amine groups at their branch ends and tertiary amine groups at each branch point.25,26 This design makes them highly cationic at neutral pH. The dendrimer size linearly increases with every addition of a new branch point (generation, G), and the number of functional primary amine groups (Z) increases exponentially by Z=NcNbG, where Nc represents the core multiplicity (for ethylenediamine, Nc=4), Nb is the branchcell multiplicity, and G is the generation.25 (Generation 2 (G2) has for instance Z=4 3 22 =16 surface groups.) Generations 1 (G1), 2 (G2), and 4 (G4) have a diameter of 2.2, 2.9, and 4.5 nm, respectively, based on a spherical geometry.27 However, the lower generation dendrimers are expected to have a more disk-like shape, compared to the higher charge density dendrimers such as G8.28,29 Dendrimers are efficient DNA condensation agents which thereby suppress genetic expression as well as provide protection against nuclease activity.30 In addition, dendrimer/ DNA complexes have proven to be highly efficient in in vitro transfection studies.30-33 In previous studies, employing a combination of dynamic light scattering, steady state fluorescence spectroscopy, and cryoTEM, we showed that the morphology of the condensed DNA aggregates can be controlled by choosing the appropriate dendrimer generation.34,35 The lower dendrimer generations (G1, G2, and G4) tend to form toroidal aggregates, whereas the higher generations (G6 and G8) form globular to more disordered and polydisperse aggregates. This morphological transition is coupled to the charge density of the dendrimers, and uniform toroidal aggregates are only formed when the attractive electrostatic forces between the cationic dendrimer and the anionic DNA are balanced by the repulsive forces.35 In the present study, we used cryo-TEM to investigate how the morphology of PAMAM dendrimer condensed DNA changes with time, which to our knowledge has not been previously reported. Our aim is to better understand the condensation process.
Experimental Section Sample Preparation. We used linearized Luciferase T7 Control DNA plasmid (4331 base pair, bp) to study the condensation induced by [ethylenediamine core]-PAMAM dendrimers of G= 1, 2, and 4 in solution (10 mM NaBr). Luciferase plasmid DNA (25) Tomalia, D. A. Prog. Polym. Sci. 2005, 30, 294–324. (26) Tomalia, D. A.; Naylor, A. M.; Goddard, W. A., III. Angew. Chem., Int. Ed. Engl. 1990, 29, 138–175. (27) Knecht, M. R.; Wright, D. W. Langmuir 2004, 20, 4728–4732. (28) Lee, I.; Athey, B. D.; Wetzel, A. W.; Meixner, W.; Baker, J. R. Macromolecules 2002, 35, 4510–4520. (29) Paulo, P. M. R.; Lopes, J. N. C.; Costa, S. M. B. J. Phys. Chem. B 2007, 111, 10651–10664. (30) Dufes, C.; Uchegbu, I. F.; Schatzlein, A. G. Adv. Drug Delivery Rev. 2005, 57, 2177–2202. (31) Bielinska, A. U.; KukowskaLatallo, J. F.; Baker, J. R. Biochim. Biophys. Acta, Gene Struct. Expression 1997, 1353, 180–190. (32) Braun, C. S.; Vetro, J. A.; Tomalia, D. A.; Koe, G. S.; Koe, J. G.; Middaugh, C. R. J. Pharm. Sci. 2005, 94, 423–436. (33) Fant, K.; Esbjorner, E. K.; Lincoln, P.; Norden, B. Biochemistry 2008, 47, 1732–1740. (34) Orberg, M. L.; Schillen, K.; Nylander, T. Biomacromolecules 2007, 8, 1557– 1563. (35) Ainalem, M. L.; Carnerup, A. M.; Janiak, J.; Alfredsson, V.; Nylander, T.; Schillen, K. Soft Matter 2009, 5, 2310–2320.
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Figure 1. Time-resolved cryo-TEM images of G1/DNA at rcharge = 0.5 showing different stages of condensation: (a) 45 s (free DNA), (b) 6 min (condensation initiated with aggregated DNA strands), (c, d) 40 min (defined rods appear), and (e, f) 1 h (toroids form). Scale bars are 100 nm except in part a, where the scale bar is 50 nm. White stars indicate the carbon support film, and the arrow in part e indicates frost.
(Promega) was amplified, linearized, and purified as described in detail by Ainalem et al.35 PAMAM dendrimers were purchased from Sigma as a 10 wt % solution in methanol. Before use, the methanol was evaporated under reduced pressure at room temperature and the dendrimers were resolubilized in 10 mM NaBr (Aldrich). Samples were prepared by adding dendrimer solutions of appropriate concentration into equal volumes of DNA solutions, giving a charge ratio (rcharge =NH3þ/PO4-) of 0.5, and a final DNA concentration of 0.1 mg/mL. Cryogenic Transmission Electron Microscopy. CryoTEM is an invaluable technique to image structures and morphologies in soft matter in aqueous dispersions, including compacted DNA,36,37 as it allows imaging of fully hydrated samples without the use of a stain to increase contrast.38 Time-resolved cryo-TEM requires sampling and vitrification of the sample during the time of condensation.39 It is worth mentioning that (36) Adrian, M.; Tenheggelerbordier, B.; Wahli, W.; Stasiak, A. Z.; Stasiak, A.; Dubochet, J. EMBO J. 1990, 9, 4551–4554. (37) Alfredsson, V. Curr. Opin. Colloid Interface Sci. 2005, 10, 269–273. (38) Danino, D.; Bernheim-Groswasser, A.; Talmon, Y. Colloids Surf., A 2001, 183, 113–122. (39) Frederik, P. M.; Sommerdijk, N. Curr. Opin. Colloid Interface Sci. 2005, 10, 245–249.
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Figure 2. Time-resolved cryo-TEM images of G2/DNA at rcharge = 0.5 showing different stages of condensation: (a-d) 1 min, (e, f) 15 min, and (g, h) 35 min. Scale bars are 100 nm except in part a, where the scale bar is 50 nm. The white stars indicate the carbon support film, and the black arrows indicate supercoiled DNA.
we found the sample preparation technique on the EM grids to be important in order to not disturb the condensation process. All results presented here are of samples premixed in Eppendorf tubes and then vitrified on holey carbon coated copper grids (not treated with glow discharge). TEM micrographs were digitally recorded using a Philips CM120 Bio TWIN electron microscope, operated at 120 kV, equipped with a Gatan MSC791 cooled-CCD camera system. To minimize beam damage, all samples were imaged under minimal electron dose conditions.
Results and Discussion Figure 1 displays a series of cryo-TEM micrographs showing the condensation behavior of G1/DNA aggregates. The G1 dendrimer, which has eight primary amine groups at the surface of the dendrimer, is known to induce toroidal aggregates.35 Figure 1a shows that only free, uncondensed DNA is observed after 45 s. After 6 min, a network of semicondensed DNA is observed (Figure 1b). This network has an ill-defined morphology, extending up to micrometers in size and is composed of what seems to be a thin layer of interconnected DNA strands. This kind of semicondensed DNA is a transient morphology, as it is not observed at later stages (>40 min) of the condensation process. In Figure 1c and d, the cryo-TEM micrographs show the characteristic morphologies observed after 40 min. Here, entangled elongated structures (Figure 1c) and rods (Figure 1d) are present in addition to a network of semicondensed DNA. Only very few toroidal aggregates are observed in this sample (see the inset of Figure 1d). The dimensions of the elongated rod-like aggregates vary dramatically from very uniform short rods with L ≈ 25 nm and a length of ca. 300 nm to longer entangled structures with a diameter of up to 100 nm. The toroidal aggregates present have L ≈ 80 nm and are around 20 nm thick, and are therefore smaller than the toroids observed at 24 h after mixing.35 After 1 h (Figure 1e and f), the elongated entangled morphologies become more uniform in thickness, and more toroidal aggregates are observed. Here, toroid dimensions range from very thin (L ≈ 60 nm, 12468 DOI: 10.1021/la903068v
ca. 10 nm thick) to larger and thicker aggregates with L ≈ 85 nm and around 30 nm thick. In addition, some semicondensed DNA is still visible (Figure 1f). Akin to what has been observed in hexammine cobalt(III) condensed DNA,23 G1-condensed rod-like morphologies seem to have a kinetic advantage over toroids during the early stages of condensation. For this particular sample, the recorded number of images, which clearly show specific morphologies, does unfortunately not allow us to determine the relative populations of the different morphologies with good statistics. However, all of the described morphologies are observed after 24 h and, as such, are believed to represent the equilibrium end state. Considering that the DNA contains 4331 bp, and that a universally sized toroid has been shown to contain around 50 kbp DNA,12,40-42 the toroids produced by G2 are composed of multiple DNA molecules. At an rcharge of 1.0, the toroidal nucleation event is considerably faster for G1 induced condensation where complete toroids are formed within 20 min. Cryo-TEM micrographs of the evolution of morphologies in the G2/DNA system are shown in Figure 2. Even though the G2 dendrimer has 16 primary amine surface groups and is only marginally larger compared to G1 (L = 2.9 nm compared to 2.2 nm for G1), the condensation process proceeds very differently. As shown in Figure 2, toroidal aggregates are formed early on and continue to develop over the course of 30 min. The condensation process for the G1 system, presented in Figure 1, is as such considerably slower compared to the G2 case at the same rcharge. Within 1 min, complete individual toroids are formed that have the same dimensions as one would expect after 24 h (see Figure S2 in the Supporting Information and Figure 6 in Ainalem et al.35), and which agrees well with the “universal” size of toroidal DNA. In addition to complete toroids, other kinds of (40) Bloomfield, V. A. Biopolymers 1991, 31, 1471–1481. (41) Gelbart, W. M.; Bruinsma, R. F.; Pincus, P. A.; Parsegian, V. A. Phys. Today 2000, 53, 38–44. (42) Bloomfield, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334–341.
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Figure 4. Dimensions of the different morphologies observed for G4 after 7 min: globules (9), rods (]), and toroids (b), where the short diameter (thickness) is plotted against the long diameter (length). In the toroid case, the thickness of the toroid is plotted against the “rod-equivalent length” (REL), defined as indicated in the inset figure. Figure 3. Cryo-TEM images of G4/DNA at rcharge = 0.5, 7 min after mixing. The scale bar in part a is 100 nm, and that in parts b-d is 50 nm.
toroidal aggregates representing different stages of condensation are observed (Figure 2b-d). It is evident that toroids can grow from multiple DNA strands simultaneously. In Figure 2b, a toroidal aggregate is associated with bundles of DNA, which seem to be supercoiled (see arrows in Figure 2b and c). Moreover, interconnected toroidal aggregates are also observed (Figure 2d). All of these morphologies continue to develop over time. After 15 min (Figure 2e and f), a lesser amount of semicondensed DNA is observed compared to the 1 min sample, and after 35 min (Figure 2g and h), only fully condensed toroidal morphologies, both individual toroids and interconnected toroidal aggregates, are observed. Similar “rings-on-a-string” aggregates have been observed when compacting single DNA molecules using diammonium salts,43 gemini surfactant,44 as well as in Mg2þ-assisted DNA condensation using hexammine cobalt(III).17 In the latter case, the presence of interconnected toroids was explained as being caused by the intra- and intermolecular stabilizing effect of the added Mg2þ ions. In the present case, cationic polyions are present. The presence of these aggregates can also be related to the relatively high concentration of DNA in the present study. Some degree of DNA entanglement prior to condensation would increase the chance of intermolecular condensation. It is also interesting to note that none of the observed toroidal aggregates showed any evidence of order, except the toroid viewed on the edge in Figure 2f. Here, the hexagonal cross section indicates that the DNA, at least in this segment, is organized in a hexagonal close packed lattice of rods. It should be noted that toroids can still be locally ordered even though no lattice fringes are observed.14 It is possible that the multistep condensation of bundles of DNA poses a constraint for optimal hexagonal packing within toroidal aggregates. If so, the increased packing error that the bundles would bring could account for the absence of visible lattice fringes. Locally, some order is still expected, but (43) Zinchenko, A. A.; Sergeyev, V. G.; Murata, S.; Yoshikawa, K. J. Am. Chem. Soc. 2003, 125, 4414–4415. (44) Miyazawa, N.; Sakaue, T.; Yoshikawa, K.; Zana, R. J. Chem. Phys. 2005, 122, 044902.
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no long-range order would appear. On the other hand, it may be so that long-range order evolves with time as significant DNA rearrangement evidently occurs. The larger G4, for which Z=64, is known to induce a mixture of toroidal, globular, and rod-like morphologies at rcharge=0.5.35 Figure 3a shows that all of these morphologies are formed within 7 min of mixing. Most of the aggregates have a globular morphology, whereas the toroidal aggregates are the least common form of morphology (30 nm). The size of the toroidal aggregates ranges between 60 and 90 nm in diameter and has an almost constant thickness of 15 nm (Figure 3a and b). In addition, toroids of 4-8 nm in thickness having a diameter of around 50-80 nm are also observed (Figure 3c). These thinner toroids, called proto-toroids, are believed to be the first condensation loops that with time develop into complete toroids.17 In Figure 4, the dimensions of the various G4/DNA morphologies observed after 7 min are compared. The rod population is generally shorter than the equivalent length of the toroids, and we also note that the toroid thickness is only marginally thinner than the ones observed after 24 h (∼20 nm) (cf. Figure 6 in Ainalem et al.35) but much thinner than G2 condensed toroids. We conclude that, for G4, the formation of toroidal aggregates seems kinetically hindered compared to globular and rod-like morphologies. Considering the greater dimensions of globular aggregates (see Figure 4), they contain more DNA molecules compared to the toroidal aggregates, and are as such more likely to have nucleated at an earlier time. It may be so that the effective charge density of G4 under these conditions is just at the limit where toroid formation can occur. As globular aggregates are also observed for higher generation dendrimers (G6 and G8), these morphologies are a result of strong electrostatic attractive interaction, where the DNA is able to wrap around the dendrimer, or stick on first contact.9,35 This tight binding would not allow any morphological rearrangement, as observed for both G1 and G2. DOI: 10.1021/la903068v
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Conclusion We can conclude that transient morphologies play an important role during the condensation process when using G1 and G2. It is clear that both thermodynamic and kinetic factors influence the morphology of dendrimer/DNA aggregates. The nucleation is dependent on the degree of binding of dendrimer to the DNA, and the successive condensation process depends on the mobility of the dendrimer on the DNA strand. The smaller and less charged G1, and to some extent G2, evidently gives a greater mobility than G4 as morphological rearrangements occur during the condensation and growth. In order for toroidal aggregates to form, the electrostatic attraction has to be moderate; that is, a balance between mobility and high affinity binding of the DNA to the dendrimer has to exist. In such a system, the condensed DNA chains will be able to arrange into a toroid. If the charge density is too high, the DNA will wrap around the dendrimer, forming
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globular aggregates. As it is the effective charge density that controls the condensation process, it lends itself to the possibility of controlling the morphology using increased ionic strength. Such studies are currently underway. Acknowledgment. The authors thank the sixth EU framework program, as part of an EU-STREP project with NEST program (NEONUCLEI, Contract 12967l) for financial support. Supporting Information Available: Examples of cryoTEM micrographs showing the effect of cryo-TEM sample preparation (Figure S1). Figure S2 displays a chart comparing toroid size with respect to time for G2. This material is available free of charge via the Internet at http://pubs.acs. org.
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