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2007, 111, 5542-5544 Published on Web 05/02/2007
Natural DNA Mixed with Trehalose Persists in B-Form Double-Stranding Even in the Dry State Bo Zhu,† Takao Furuki,† Takashi Okuda,‡ and Minoru Sakurai*,† Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259 Nagatsuda-cho, Midori-ku, Yokohama 226-8501, Japan, and Department of Physiology and Genetic Regulation, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba 305-8634, Japan ReceiVed: March 12, 2007
Desiccated calf-thymus DNA has been found to persist in the B-form double-stranding when mixed with trehalose. The stabilization effect on natural DNA depends on the trehalose content, and should basically arise from its ability to tightly hydrogen bond to phosphate groups of DNA, which leads to screening of the large phosphate-phosphate repulsion.
The long-term storage and convenient shipping of cells and tissues are critical to the research and application in clinical medicine and other fields of life science. For the broad dissemination of biomaterials, we need a way to preserve biomaterials without freezing.1 Some small animals, e.g., tardigrades, nematodes, rotifers, embryonic cysts of primitive crustaceans including brine shrimp (Artemia salina), and larvae of the African chironomid (Polypedilum Vanderplanki), can survive long-term dehydration in a suspended animation state known as anhydrobiosis.2 In anhydrobiosis, all metabolic processes are switched off, which, however, upon rehydration can be restored without any irreversible damage. A common feature for them, when in anhydrobiosis, is the presence of large amounts of sugar, particularly trehalose,3 which has been found to most effectively protect biomaterials.4-6 On the basis of this understanding, Crowe et al. succeeded in preparing a recoverable dry platelet, 90% of which can be restored by adding water, even after 2 years of storage.7 However, using trehalose to preserve complex cells with nuclei could be much different. One of the key issues to be resolved is whether trehalose plays the same role in the densely packed nucleus as it does in the cell cytoplasm. Herein, for the first time, we demonstrate the stabilization effect of trehalose (TRH) on the three-dimensional structure of calf-thymus NaDNA (CTDNA) in the dry state through Fourier transform infrared (FTIR) and differential scanning calorimetry (DSC) analyses. The in-plane CdO stretching and NH2 and NsH bending of bases with a frequency from 1800 to 1500 cm-1, as evidenced experimentally8 and theoretically,9 undergo the most profound changes upon base pairing and stacking interaction. The CTDNA at 92% relative humidity (RH) has a Γ value (water molecules per nucleotide) of ∼20 and adopts a double-helix conformation near the Watson-Crick mode, which, as shown * To whom correspondence should be addressed. E-mail: msakurai@ bio.titech.ac.jp. † Tokyo Institute of Technology. ‡ National Institute of Agrobiological Sciences.
10.1021/jp071974h CCC: $37.00
in Figure 1a, has five characteristic bands at 1712, 1665, 1606, 1676, and 1628 cm-1. Particularly, the intensive 1712 cm-1 band is well-known to arise from the double-stranding.8b-d Once dehydrated to Γ ) ∼1 in silica gel, the CT-DNA loses most of its structural integrity10 and thus changes to show six major absorptions at 1696, 1684, 1653, 1602, 1574, and 1531 cm-1. When the mixture of CT-DNA and TRH is similarly desiccated to Γ ) ∼1, the spectral line shape of CT-DNA in the 18001500 cm-1 range, however, strongly resembles that of the hydrated CT-DNA, even at a ΓTRH value (trehalose molecules per nucleotide) as low as 0.125. The amazing similarity, especially in the band wavenumber, as confirmed in Figure 1c, strongly indicates that the CT-DNA bases in TRH are paired and stacked, i.e., double-stranded in a way very similar to the hydrated ones. The stabilization effect, as indicated in Figure 2a by the intensity ratio of 1712-1684 cm-1, is enhanced with increasing TRH content and becomes saturated at ΓTRH ) ∼1. The double-stranded DNA, depending on the environment, takes different conformations via puckering deoxyribose rings and rotating glycosyl bonds,10a,11 which are mainly reflected in the vibrations of the sugar-phosphate backbone from 1000 to 750 cm-1.8 Normally, the DNA, when equilibrated at 92% RH, is assumed to be the B-form. As shown in Figure 1b and d, the B-form marker at 836 cm-1 (mainly C2′-endo puckered sugar)8a,b is clearly observed for the hydrated CT-DNA. The A-form markers at 883, 863, and 807 cm-1 (C3′-endo puckered sugar)8a,b are also detected in our hydrated CT-DNA, which should arise from the small residual A-form. For mixture samples, the absorption of TRH in the 1000-750 cm-1 range seriously interferes with our observation on DNA conformation. When the TRH content is the smallest, i.e., ΓTRH ) 0.125, the overlap from TRH, however, is largely weakened, and could be mostly neglected. In this case, the B-form marker is clearly observed with an intensity comparable to that of the hydrated CT-DNA, while the 883 and 807 cm-1 bands become nearly absent even in the secondary derivative spectra. The feature at 863 cm-1 still persists but is rather weaker than that of the © 2007 American Chemical Society
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J. Phys. Chem. B, Vol. 111, No. 20, 2007 5543
Figure 1. IR spectra in the ranges (a) 1750-1500 cm-1 and (b) 1000-750 cm-1 for the dehydrated (dry) CT-DNA, CT-DNA at 92.0% RH, dry CT-DNA in TRH with ΓTRH from 0.125 to 1.5, and amorphous TRH and their secondary derivatives (parts c and d).
Figure 2. (a) Melting enthalpy (∆H) and temperature (Tm), glass transition temperature (Tg), and (b) intensity ratio of 1712-1684 cm-1 for the dehydrated mixtures of CT-DNA with TRH as a function of ΓTRH.
hydrated CT-DNA. The previous works point out the fact that the weak band at 863 cm-1 is also observable for the 100% B-form DNA.8c,d Furthermore, another famous A-form marker band at 1188 cm-1 (sugar-phosphate vibration with a fairly high contribution from the C3′-endo puckered sugar),12 as shown in Figure 3b, is not observed at all. Considering all of the above, it is reasonable to suggest that the CT-DNA dehydrated with TRH at ΓTRH ) 0.125 is even more inclined to adopt the B-form than that equilibrated at 92% RH. With further increase of ΓTRH, no additional conformational arrangement could be detected by the secondary derivative spectra. In general, the high similarity in the vibrations sensitive to base stacking and pairing and
conformation indicates the high uniformity in spatial arrangement between the CT-DNA in TRH and that at 92% RH. The double-stranding of dry CT-DNA in dehydrated mixtures is further confirmed by observing its thermal denaturation (melting) during DSC heating scans.14 In Figure 2b, the melting enthalpy (∆H) and temperature (Tm) are shown to evaluate the dependence of the double-stranding and thermal stability of CTDNA on the TRH content. As noted, the ∆H value has an initial positive dependence on ΓTRH but subsequently becomes constant at ΓTRH ) 1, which agrees well with the change for the intensity of the 1712 cm-1 band and possibly indicates that the base stacking and paring become uniform and perfect at ΓTRH ) 1.
5544 J. Phys. Chem. B, Vol. 111, No. 20, 2007
Letters
Figure 3. IR spectra in the ranges (a) 3700-2700 cm-1 and (b) 1300-1150 cm-1 for the dehydrated (dry) CT-DNA in TRH with ΓTRH from 0 to 1.5 and amorphous TRH.
The ∆H value for the CT-DNA saturated with TRH is ∼12.5 kJ‚mol-1, which is smaller than but comparable to that (16.7 kJ‚mol-1) for the fully double-stranded CT-DNA in water as measured by us. On the other hand, the thermal stability, i.e., Tm, of CT-DNA, as expected, presents a negative dependence on ΓTRH, as the glass transition temperature (Tg) increases with decreasing TRH content. Nevertheless, the CT-DNA saturated by TRH is much more thermally stable than that at 92% RH (Tm ) 103 °C). It is of our interest to investigate the origin of the stabilization effect of TRH on the DNA structure. Indeed, by IR measurements, TRH molecules are found to be restrained from crystallization in dehydration and to be amorphous in the final products, strongly indicating that there is some strong interaction between CT-DNA and TRH. As shown in Figure 3a, with decreasing ΓTRH to 0.5 (i.e., increasing CT-DNA content), the OH stretching of TRH is gradually red-shifted from 3367 to 3342 cm-1, indicating that hydroxyl groups of TRH are strongly hydrogenbonded with DNA.13 It should be noted that the red-shift of OH stretching does not arise from the enhanced overlap from the NH stretching of CT-DNA at all. As shown in Figure 3a, with increasing ΓTRH from 0 only to 0.125, the major absorption of CT-DNA is distinctively blue-shifted from 3349 to 3396 cm-1, which, with consideration of the double-stranding of CTDNA in the presence of TRH, should be attributed to the doublestranded CT-DNA. Obviously, the overlaps at 3396 cm-1 indeed seemingly blue-shift the OH stretching of TRH instead. The strong variation in absorption wavenumber is also found for phosphate groups of CT-DNA, which are known to be preferential binding sites for hydrophilic residues. As shown in Figure 3b, the antisymmetric PO2- stretching is red-shifted from 1240 to 1221 cm-1 upon an increase of ΓTRH from 0 to 1.5. It could not be attributed to the weak and broad overlap from the CH deformation of TRH, as evidenced by the simple combination of dry CT-DNA with TRH, but it indicates the participation of phosphate groups in the hydrogen bond.5a,13 It is also noted that the wavenumber shift of antisymmetric PO2- stretching is not monotonic at all, which is similar to what occurs in the hydrated CT-DNA and should be related to the change in the spatial arrangement of DNA. In summary, the CT-DNA has been found to persist in the Bform double-stranding in trehalose when dehydrated to Γ ) ∼1. The effect, as described by the intensity of 1712 cm-1 and the enthalpy of thermal denaturation, is found to be dependent on the TRH content, and to be saturated at ΓTRH ) 1. The stabilization effect of TRH on natural DNA should basically arise
from its ability to tightly hydrogen bond to phosphate groups of DNA, which leads to screening of the large phosphatephosphate repulsion. TRH also probably interacts with other polar groups of DNA, which, combined with hydrogen bonds to phosphate groups, makes TRH a waterlike solvent for DNA, and stabilizes the base stacking during and after dehydration. Acknowledgment. We gratefully acknowledge the kind help from Prof. Yoshio Inoue and the support by the PROBRAIN program and Grants-in-Aid for Scientific Research on Priority Areas (No. 18031012) from the Japanese Government. Supporting Information Available: Experimental procedures, water content of DNA, and IR on the solid state of trehalose. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Brumfiel, G. Nature 2004, 428, 14. (2) (a) Crowe, J. H.; Crowe, L. M. Nat. Biotechnol. 2000, 18, 145. (b) Watanabe, M.; Kikawada, T.; Minagawa, N.; Yukuhiro, F.; Okuda, T. J. Exp. Biol. 2002, 205, 2799. (c) Tunnacliffe, A.; Lapinski, J. Philos. Trans. R. Soc. London, Ser. B 2003, 358, 1755. (d) Clegg, J. S. Comp. Biochem. Physiol. 1965, 14, 135. (3) (a) Kilburn, D.; Townrow, S.; Meunier, V.; Richardson, R.; Alam, A.; Ubbink, J Nat. Mater. 2006, 5, 632. (b) Crowe, L. M.; Reid, D. S.; Crowe, J. H. Biophys. J. 1996, 71, 2087. (4) Crowe, J. H.; Carpenter, J. F.; Crowe, L. M. Annu. ReV. Physiol. 1998, 60, 73. (5) (a) Crowe, J. H.; Crowe, L. M.; Chapman, D. Science 1984, 223, 701. (b) Carpenter, J. F.; Crowe, J. H. Biochemistry 1989, 28, 3916. (6) (a) Massari, A. M.; Finkelstein, I. J.; McClain, B. L.; Goj, A.; Wen, X.; Bren, K. L.; Loring, R. F.; Fayer, M. D. J. Am. Chem. Soc. 2005, 127, 14279. (b) Sun, W. Q.; Leopold, A. C.; Crowe, L. M.; Crowe, J. H. Biophys. J. 1996, 70, 1769. (7) Wolkers, W. F.; Walker, N. J.; Tamari, Y.; Tablin, F.; Crowe, J. H. Cell PreserV. Technol. 2003, 1, 175. (8) (a) Banyay, M.; Sarkar, M.; Gra¨slund, A. Biophys. Chem. 2003, 104, 477. (b) Liquier, J.; Taillandier, E. In Infrared Spectroscopy of Biomolecules; Mantsch, H. H., Chapman, D., Ed.; Wiley-Liss: New York, 1996; p 131. (c) Pichler, A.; Rudisser, S.; Winger, R. H.; Liedl, K. R.; Hallbrucker, A.; Mayer, E. J. Am. Chem. Soc. 2000, 122, 716. (d) Kang, H.; Johnson, W. C., Jr. Biochemistry 1994, 33, 8330. (9) Lee, C.; Park, K.-H.; Cho, M. J. Chem. Phys. 2006, 125, 114508/ 1. (10) (a) Franklin, R. E.; Gosling, R. G. Acta. Crystallogr. 1953, 6, 673. (b) Falk, M.; Hartman, K. A.; Lord, R. C. J. Am. Chem. Soc. 1963, 85, 387. (c) Lee, C.-H.; Mizusawa, H.; Kakefuda, T. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 2838. (11) Saenger, W.; Hunter, W. N.; Kennard, O. Nature 1986, 324, 385. (12) Pohle, W.; Fritzsche, H. Nucleic Acids Res. 1980, 8, 2527. (13) Cacela, C.; Hincha, D. K. Biophys. J. 2006, 90, 2831. (14) Lee, S. L.; Debenedetti, P. G.; Errington, J. R.; Pethica, B. A.; Moore, D. J. J. Phys. Chem. B 2004, 108, 3098.
