J. Phys. Chem. B 2000, 104, 8871-8875
8871
Conformation Change of Poly(dG-dC)•Poly(dG-dC) in Cationic Polyamine Liposome Complexes: Effect of Charge Density and Flexibility of Amine Chains in Headgroups Hiroshi Matsui* and Su Pan UniVersity of Central Florida, Center for DiscoVery of Drugs & Diagnostics and Department of Chemistry, Orlando, Florida 32816 ReceiVed: April 17, 2000
Identification of DNA conformations in delivery cargoes is important to understand the transfection mechanisms. Conformations of poly(dG-dC)•poly(dG-dC), as a model of DNA, were studied in two different cationic liposome complexes prepared from N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmithyl spermine (TM-TPS) with a helper lipid, dioleoyl phosphotidylethanolamine (DOPE) by Raman microscopy. Characteristic spectral changes in guanine peaks and cytosine peaks of the poly(dG-dC)•poly(dG-dC) distinguished between the right-handed B-form and lefthanded Z-form inside the cationic liposome complexes. The poly(dG-dC)•poly(dG-dC) in the TM-TPS/DOPE liposome undergoes the B-Z transition while no conformation change was observed in the poly(dG-dC)•poly(dG-dC) inside the DOTMA/DOPE liposome. Each multi-charged TM-TPS molecule contains four cationic nitrogen atoms in the headgroup. The DOTMA molecule with less cationic nitrogens does not have enough negative charges in the headgroup to cause the B-Z transition. This model was also confirmed by the B-Z transition of the poly(dG-dC)•poly(dG-dC) in another cationic polyamine liposome complex, 2,3-dioleyloxyN-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and the DOPE, whose multi-charged amine chains are more flexible than the amine chains of the TM-TPS. Intermolecular interaction between cationic nitrogen atoms of the DOSPA and oxygen/nitrogen atoms of the guanine sites in poly(dG-dC)•poly(dG-dC) is considered to be one of major driving forces for the B-Z transition. Systematic studies of DNA conformations in various cationic liposome complexes may help developing improved gene delivery system.
I. Introduction Cationic liposome complexes have been proven efficient gene delivery cargoes.1-7 While DNA has been used as a drug in gene therapy, it has been reported that external variables such as pH, lipid/DNA ratio, and temperature affect structures and charge distributions of DNA-cationic liposome complexes significantly.8 More effective gene delivery may be achieved by controlling DNA conformations in cationic liposome complexes for a certain gene disorder therapy.9 Control of DNA conformations is also important since it affects stability of the DNA-cationic liposome structure and release mechanism of DNA.10 Systematic conformation studies of DNA interacting with various cationic liposome complexes are highly desirable to develop improved gene delivery cargoes. For example, structures and dynamics of DNA condensates and DNA toroidal transformation have been studied extensively on various surfaces.11-16 As model studies for DNA-liposome complexes, DNA structure changes were monitored on cationic lipid membranes.17,18 X-ray studies of a DNA-cationic liposome complex structure, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl phosphotidylethanolamine (DOPE), were also recently reported.19,20 Despite extensive studies of DNA-cationic liposome interactions and structures, there are few reports systematically studying conformations of DNA inside cationic liposomes.21,22 DNA conformation changes can be induced by various factors such as pH, salt concentration, and humidity,23,24 while a * Author to whom correspondence should be addressed.
dynamic change of DNA conformation is provoked by addition of polyamines.25,26 For example, spermine, a polyamine, induces a left-handed Z-form transition in DNA and one of important driving forces for this transformation is considered to be intermolecular interactions such as electrostatic forces and hydrogen bonds between DNA and spermine.10,27 These interactions compress DNA along the helical axis and shift the base pairs into the major groove to transform the B-form into the Z-form. This B-Z transition could also occur with the condensation in a variety of polyamine concentrations.28-30 Most of cationic liposomes used as DNA delivery cargoes contain cationic amine groups to increase the interaction with anionic DNA phosphate groups. Among various cationic liposomes, polyamine-based liposomes consist of multi-charged cationic amine groups,31 and lipospermines, tetravalent polyamine lipids, showed significant gene transfer to leukemia cells.32 But the lipospermines may potentially induce structure changes of DNA inside the liposome vesicles due to the four cationic nitrogen atoms. In this report, conformations of DNA inside a cationic lipospermine complex, N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmithyl spermine (TM-TPS) and dioleoyl phosphatidylethanolamine (DOPE), were studied in solution by Raman microscopy. Another cationic polyamine liposome complex, 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,Ndimethyl-1-propanaminium trifluoroacetate (DOSPA) and the DOPE, was also examined. Both liposomes contain four positively charged nitrogen atoms, but chains of the nitrogen atoms in the DOSPA liposome are considered to be more
10.1021/jp001439g CCC: $19.00 © 2000 American Chemical Society Published on Web 08/22/2000
8872 J. Phys. Chem. B, Vol. 104, No. 37, 2000
Matsui and Pan
Figure 2. Picture of poly(dG-dC)•poly(dG-dC)-DOTMA/DOPE complex.
Figure 1. Chemical structures of (a) N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium chloride (DOTMA), (b) N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmithyl spermine (TM-TPS), (c) 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), and (d) dioleoyl phosphotidylethanolamine (DOPE).
flexible since each DOSPA molecule has only two long carbon chain as a tail group while chains of nitrogen atoms in the TMTPS liposome are immobilized by long hydrocarbon chains attaching all of the four nitrogen atoms (Figure 1). These results were also compared with DNA conformation study in a cationic liposome with less amine groups, N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium chloride (DOTMA) and the DOPE. Although Raman technique may not be sensitive enough to probe all of weak intermolecular interactions between cationic lipids and DNA molecules, conformation changes of DNA driven by the interactions are expected to give sufficient changes in the Raman spectra33,34 and become detectable due to conformation specific vibrational frequency shifts even inside cationic liposome complexes.35 II. Experiment Raman spectra were acquired with a LabRam (JobinYvon/ Horiba, Edison, NJ) confocal Raman microprobe. The 632.8 nm emission from an integrated air-cooled He-Ne laser is spatially filtered and then injected into an integrated Olympus BX40 microscope by a holographic notch filter. An 80× working distance objective focuses the beam to provide approximately 6 mW in a spot of less than 1 µm in diameter. The Raman scattering is collected at 180° by the focusing objective, and the accompanying reflected laser radiation is rejected by the same notch filter. The filtered radiation is focused at the entrance slit (250 µm) of a 0.3 mm spectrograph. An 1800 gr/ mm grating disperses the spectrum across a 1024 × 256 pixel TE-cooled CCD detector, providing a dispersion of approximately 1 cm-1/pixel. As a model of DNA transformation in a cationic liposome complex, we studied poly(dG-dC)•poly(dG-dC) in a cationic lipospermine complex, N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-
tetrapalmithyl spermine (TM-TPS) and dioleoyl phosphatidylethanolamine (DOPE) (1:1.5 (m/m)) by the Raman microscope. Conformations of the poly(dG-dC)•poly(dG-dC) were also examined in another cationic polyamine liposome complex, 2,3dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1propanaminium trifluoroacetate (DOSPA) and the DOPE (3:1 (w/w)), and in a less amine-containing cationic liposome, N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and the DOPE (1:1 (w/w)) using the Raman microscope. All of those lipids were obtained from Life Technologies in membrane-filtered water, and their chemical structures are shown in Figure 1. Poly(dG-dC)•poly(dG-dC) was purchased from Sigma. These liposomes were sonicated for 30 min to clarity in closed centrifuge tubes. An amount of 5 µL of the poly(dG-dC)•poly(dG-dC) (1.47 mg/mL) was mixed with 40 µL of the DOTMA/DOPE (1 mg/mL), the DOSPA/DOPE (2 mg/mL), and the TM-TPS/DOPE (1 mg/mL), respectively. The poly(dG-dC)•poly(dG-dC) was then allowed to incubate at 4 °C overnight. The neat poly(dG-dC)•poly(dG-dC) sample was confirmed to be in B-form using vibrational analysis of the Raman spectrum. III. Results Figure 2 is a micrograph of the poly(dG-dC)•poly(dG-dC)DOTMA/DOPE complex whose size varies between 500 nm and 5 µm in diameter. The poly(dG-dC)•poly(dG-dC)-liposome complexes were assembled in an average diameter of 300 nm, and no characteristic difference in assembled structures was detected among three cationic liposome complexes in light microscopic observation. An excitation laser was focused onto the center of the vesicle to obtain conformational information of DNA inside the vesicle. Figure 3a shows the Raman spectrum of the poly(dG-dC)•poly(dG-dC) in the DOTMA/DOPE liposome. A majority of vibrational modes in this spectrum are also observed in the Raman spectrum of the neat poly(dG-dC)•poly(dG-dC) (Figure 3b) without vibrational frequency shifts. Peaks at 681, 1175, 1319, 1362, 1483, and 1577 cm-1 are from guanine vibrations, while a 1097 cm-1 peak is assigned as a PO2- symmetric stretch in the backbone and peaks at 594, 781, 1240, and 1260 cm-1 belong to cytosine vibrations. The vibrational frequencies of the poly(dG-dC)•poly(dG-dC) in the DOTMA/DOPE liposome are summarized in Table 1. Figure 3c is the Raman spectrum of the neat DOTMA/DOPE liposome. Strong transitions at 1084, 1442, and 1654 cm-1 in Figure 3c are also found in the spectrum of the poly(dG-dC)•poly(dG-dC) in the DOTMA/DOPE liposome (Figure 3a) without frequency shifts. This comparison indicates that the DNA does not undergo major conformation changes inside the DOTMA/DOPE liposome complex. Figure 4a,b shows the Raman spectra of the poly(dGdC)•poly(dG-dC) in the TM-TPS/DOPE liposome and the neat poly(dG-dC)•poly(dG-dC), respectively. The B-Z transition of poly(dG-dC)•poly(dG-dC) induces a characteristic guanine peak shift from 680 to 625 cm-1.36 A 1260 cm-1 cytosine peak was
Poly(dG-dC)•Poly(dG-dC) in Liposome Complexes
Figure 3. Raman spectra of (a) poly(dG-dC)•poly(dG-dC) in DOTMA/ DOPE liposome, (b) neat poly(dG-dC)•poly(dG-dC), and (c) neat DOTMA/DOPE liposome.
J. Phys. Chem. B, Vol. 104, No. 37, 2000 8873
Figure 4. Raman spectra of (a) poly(dG-dC)•poly(dG-dC) in TMTPS/DOPE liposome, (b) neat poly(dG-dC)•poly(dG-dC), (c) neat TMTPS/DOPE liposome.
TABLE 1: Frequencies and Vibrational Assignments of Raman Spectra of Poly(dG-dC)•Poly(dG-dC) in Various Cationic Liposomes. An Error for the Band Positions of Poly(dGdC) Is (0.5 cm-1 a band position of poly(dGdC) (cm-1) neat (B-form) in DOTMA in TM-TPS in DOSPA 594
594
681 781 1097 1175 1240 1260 1319 1362 1483 1577
681 781 1097 1175 1240 1260 1319 1362 1483 1577
a
594 625
594 625
781 1097 1175 1246 1265 1319 1355 1483 1581
781 1097 1183 1246 1265 1319 1355 1486 1577
assignment C G(C3′-endo/syn) G (C2′-endo/anti) C/PO2- backbone PO2- backbone G C C G G G G
G and C designate guanine and cytosine, respectively.
also observed to shift to 1265 cm-1 accompanied with the intensity depletion while a 1362 cm-1 guanine peak shifts to 1355 cm-1 with the intensity enhancement via the B-Z transition.37,38 In Figure 4a,b, a guanine peak at 681 cm-1 is shifted to 625 cm-1, as depicted by the dotted lines. A cytosine peak of the neat poly(dGdC)•poly(dG-dC) at 1260 cm-1 (Figure 4b) increases its intensity and shifts to 1265 cm-1 as the poly(dGdC)•poly(dG-dC) is contained by the TM-TPS/DOPE liposome (Figure 4a), while a guanine peak of the neat poly(dGdC)•poly(dG-dC) at 1362 cm-1 (Figure 4b) shifts to 1355 cm-1 in the TM-TPS/DOPE liposome with weaker Raman intensity (Figure 4a). The frequency shifts of the poly(dGdC)•poly(dG-dC) in the TM-TPS/DOPE liposome are summarized in Table 1. These spectral changes indicate that the poly(dG-dC)•poly(dG-dC) undergoes a structural transformation from the B-form to the Z-form inside the TM-TPS/DOPE liposome. Figure 4c is the Raman spectrum of the neat TMTPS/DOPE liposome. Strong transitions at 1061, 1099, 1435, 1457, and 1654 cm-1 in Figure 4c appear in the spectrum of the poly(dG-dC)•poly(dG-dC)-TM-TPS/DOPE complex (Figure 4a).
Figure 5. Raman spectra of (a) poly(dG-dC)•poly(dG-dC) in DOSPA/ DOPE liposome, (b) neat poly(dG-dC)•poly(dG-dC), (c) neat DOSPA/ DOPE liposome.
Raman spectra of the poly(dG-dC)•poly(dG-dC) in the DOSPA/DOPE liposome and the neat poly(dG-dC)•poly(dGdC) are shown in Figure 5, parts a and b, respectively. The characteristic frequency shifts and intensity changes in guanine and cytosine vibrations due to the B-Z transition of the poly(dG-dC)•poly(dG-dC) are also observed in these figures and summarized in Table 1. The guanine peak shift from 681 to 625 cm-1 is depicted by the dotted lines. The characteristic spectral changes of guanine and cytosine peaks indicate that the DNA also undergoes the structural transformation from the B-form to the Z-form inside the DOSPA/DOPE liposome as well as the TM-TPS/DOPE liposome. The guanine peak shifts from 1175 to 1183 cm-1 and from 1483 to 1486 cm-1 are due to the interaction with the headgroup of DOSPA. Figure 5c is the Raman spectrum of the neat DOSPA/DOPE liposome. A C-N stretching mode of the O-C-C-N+ backbone of the
8874 J. Phys. Chem. B, Vol. 104, No. 37, 2000
Matsui and Pan
Figure 6. Proposed arrangements of DNA and (a) DOTMA/DOPE liposome, (b) TM-TPS/DOPE liposome, (c) DOSPA/DOPE liposome. Nitrogen atoms in the TM-TPS are highlighted in blue and oxygen atoms in red. The helper lipids, DOPE, and water molecules are not depicted between the lipid layers and sizes of DNA and the lipid molecules are not scaled.
DOSPA appears at 719 cm-1, and a C-O-C vibration of the DOSPA is also observed at 854 cm-1 in Figure 5c.39,40 The stretching mode of O-C-C-N+ backbone shifts to 727 cm-1 and a C-O-C vibration shifts to 843 cm-1 in the Raman spectrum of poly(dG-dC)•poly(dG-dC)-DOSPA/DOPE complex (Figure 5a). Other strong transitions at 1080, 1434, and 1650 cm-1 in Figure 5c also appear in the spectrum of the poly(dG-dC)•poly(dG-dC)-DOSPA/DOPE complex in Figure 5a without frequency shifts.
molecule is 4+ per four hydrocarbon chains while the DOTMA molecule has a 2+ charge per four hydrocarbon chains. Therefore, the charge density on the surface of the TM-TPS/ DOPE liposome is higher than the DOTMA/DOPE liposome. This model can be considered to be comparable to the saltDNA transition mechanism. Thus, it is reasonable that the highly negatively charged TM-TPS surfaces undergo the B-Z DNA conformation change, as seen in the B-Z transition DNA in high salt solutions.
IV. Discussion
This mechanism is also consistent with the observation that the Z-conformation of the DNA in the TM-TPS liposome reverses to the B-form as excess amount of the neutral helper lipid DOPE is added to the complex due to the reduction of surface charge density of the liposome.43
(a) Effect of Charge Density of Cationic Liposome Amine Chains. Z-DNA was first observed in poly(dG-dC) when the salt concentration was raised.41 A high salt concentration is considered to stabilize Z-DNA by reducing the electrostatic repulsions between phosphate groups on the opposite strands. Based on X-ray study of the DNA-liposome complex,19 configurations of the DNA-DOTMA/DOPE and the DNATM-TPS/DOPE complexes are proposed as Figure 6b. The helper lipids, DOPE, and water molecules are not depicted between cationic lipid layers and sizes of the DNA and the cationic lipid molecules are not scaled. The cationic lipid layers may not be completely flat as drawn since the lipids could also change their conformations due to the interactions with the DNA.42 The DOTMA molecule contains one cationic nitrogen atom in the headgroup and two hydrocarbon chains in the tail group while the TM-TPS molecule has four cationic nitrogen atoms in the headgroup and four hydrocarbon chains in the tail group (Figure 1). In other words, a charge of the TM-TPS
(b) Effect of Flexibility of Multi-charged Amine Chains. Polyamines are known to provoke structural and conformational transitions in duplex B-form DNA, and intermolecular interactions between DNA and polyamines such as electrostatic forces and hydrogen bonds are considered as the most important driving forces for DNA transition.10 The region of the major groove has a very high negative charge density, which forms a tight binding site for positively charged polyamines. This charge neutralization induces and stabilizes the left-handed Z-form DNA.44 Structural analysis of the transformed Z-form DNA showed that the helix axis is compressed, and the base pairs are shifted into the major groove.27 The Z-form of DNA is stabilized by polyamines since one polyamine molecule interacts and forms a complex with multiple DNA helices.27
Poly(dG-dC)•Poly(dG-dC) in Liposome Complexes Chains of the amine groups in the DOSPA liposome are more flexible since each DOSPA molecule has only two long carbon chain as a tail group attached to one carbon atom at the end of headgroup while chains of amine groups in TM-TPS liposomes are immobilized by four long hydrocarbon chains attaching all of the four nitrogen atoms (Figure 1). This flexibility allows the amine groups of the DOSPA liposome to penetrate toward the DNA layer located between the lipid layers. As a result, the flexible amine chains of the cationic lipid molecules increase intermolecular interaction with oxygen and nitrogen atoms of the guanine in DNA.27 Therefore, it is reasonable to observe the Z-form transition of poly(dG-dC)•poly(dG-dC) in the DOSPA/DOPE liposome complex as well as in the TM-TPS/ DOPE liposome complex. This model is illustrated in Figure 6c. In Figure 6c, the helper lipids, DOPE, and water molecules are not depicted between DOSPA lipid layers and sizes of DNA and the DOSPA molecules are not scaled. This model is supported by the observation that the amine-guanine interaction affects ring vibrations in the five-membered ring and causes the frequency shifts at 1183 cm-1 (ν53) and 1486 cm-1 (ν51)45 as shown in Table 1. Flexibility and penetration of the amine chains of DOSPA liposome also affect vibrations of the O-C-C-N+ and C-O-C groups of DOSPA, which consist of the junction between the head and tail groups. As the amine chains penetrate into the DNA layer, geometry change of the O-C-C-N+, such as gauche to anti, should induce frequency shifts of the O-CC-N+ and C-O-C vibrations. Thus, it is reasonable to observe the frequency shifts of the DOSPA for these vibrational modes (a 8 cm-1 blue shift for O-C-C-N+ vibration and a 11 cm-1 red shift for C-O-C vibration) between the neat condition and the complexation with the DNA in Figure 5, parts a and c.
V. Conclusion DNA conformations between the B-form and the Z-form in a cationic liposome complex can be determined by monitoring characteristic spectral changes of guanine and cytosine peaks using a Raman microscope. DNA in the TM-TPS/DOPE liposome undergoes the B-Z transition while no conformation change was observed in the DNA inside the DOTMA/DOPE liposome. The TM-TPS contains four cationic nitrogen atoms in the headgroup and the high charge density induces the B-Z transition. The DOTMA with less cationic nitrogen atoms does not have enough negative charges in the headgroup to cause the B-Z transition in DNA. The DOSPA/DOPE liposome complex, which contains more flexible chains of four cationic nitrogen atoms, also induces the Z-form DNA transformation by stronger interactions with the DNA due to penetration of the flexible amine chains toward the DNA layer. Monitoring DNA conformations inside liposome carriers within living cells would be very informative, and would aid in the design of improved DNA-liposome complexes, which could enhance the efficiency of gene delivery. Acknowledgment. This work was supported by University of Central Florida, Office of the Vice President for Research and Graduate Studies, I-4 Matching Fund.
J. Phys. Chem. B, Vol. 104, No. 37, 2000 8875 References and Notes (1) Behr, J. P.; Demeneix, B.; Loeffler, J. P.; Mutul, J. P. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6982. (2) Caplen, N. J.; Alton, E. W. F. W.; Middleton, P. G.; Dorin, J. R.; Stevenson, B. J.; Gao, X.; Durham, S. R.; Jeffery, P. K.; Hodson, M. E.; Coutelle, C.; Huang, L.; Porteous, D. J.; Williamson, R.; Geddes, D. M. Nat. Med. 1995, 1, 39. (3) Felgner, P. L.; Ringold, G. M. Nature 1989, 337, 387. (4) Nabel, G. J.; Nabel, E. G.; Yang, Z. Y.; Fox, B. A.; Plautz, G. E.; Gao, X.; Huang, L.; Shu, S.; Gordon, D.; Chang, A. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11307. (5) Rui, Y.; Wang, S.; Low, P. S.; Thompson, D. H. J. Am. Chem. Soc. 1998, 120, 11213. (6) Smith, J. G.; Walzen, R. L.; German, J. B. Biochim. Biophys. Acta 1993, 1154, 327. (7) Zhu, N.; Liggitt, D.; Liu, Y.; Debs, R. Science 1993, 261, 209. (8) Farhood, H.; Bottenga, R.; Epand, R. M.; Huang, L. Biochim. Biophys. Acta 1992, 1111, 239. (9) Bhattacharya, S.; Mandal, S. S. Biochim. Biophys. Acta 1997, 1323, 29. (10) Bloomfield, V. A.; Ma, C.; Arscott, P. G. Z trans by polyamine reView; American Chemical Society: Washington, DC, 1994; Vol. 548, p 195. (11) Ma, C.; Bloomfield, V. A. Biophys. J. 1994, 67, 1678. (12) Fang, Y.; Hoh, J. H. J. Am. Chem. Soc. 1998, 120, 8903. (13) Hansma, H. G.; Golan, R.; Hsieh, W.; Lollo, C. P.; Mullen-Ley, P.; Kwoh, D. Nucleic Acids Res. 1998, 26, 2481. (14) Allen, M. J.; Bradbury, E. M.; Balhorn, R. Nucleic Acids Res. 1997, 25, 2221. (15) Ono, M. Y.; Spain, E. M. J. Am. Chem. Soc. 1999, 121, 7330. (16) Fang, Y.; Hoh, J. H. J. Am. Chem. Soc. 1998, 120, 8903. (17) Fang, Y.; Yang, J. J. Phys. Chem. B 1997, 101, 441. (18) Zantle, R.; Baicu, L.; Artzner, F.; Sprenger, I.; Rapp, G.; Radler, J. O. J. Phys. Chem. B 1999, 103, 10300. (19) Radler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810. (20) Koltover, I.; Salditt, T.; Radler, J. O.; Safinya, C. R. Science 1998, 281, 78. (21) Dan, N. Biophys. J. 1996, 71, 1267. (22) Bandyopadhyay, S.; Tarek, M.; Klein, M. L. J. Phys. Chem. B 1999, 103, 10075. (23) Matthew, J. B.; Richard, F. M. Biopolymers 1984, 23, 2743. (24) Moller, A.; Nordheim, A.; Kozlowski, S. A.; Patel, D. J.; Rich, A. Biochemistry 1984, 23, 54. (25) Takahashi, M.; Yoshikawa, K.; Vasilevskaya, V. V.; Khokhlov, A. R. J. Phys. Chem. B 1997, 101, 9396. (26) Pelta, J.; Livolant, F.; Sikorav, J.-L. J. Bio. Chem. 1996, 271, 5656. (27) Egli, M.; Williams, L. D.; Gao, Q.; Rich, A. Biochemistry 1991, 30, 11388. (28) Revet, B.; Delain, E.; Dante, R.; Niveleau, A. J. J. Biomol. Struct. Dyn. 1983, 1, 857. (29) Castleman, H.; Specthrie, L.; Makowski, L.; Erlanger, B. F. J. Biomol. Struct. Dyn. 1984, 2, 271. (30) Thomas, T. J.; Bloomfield, V. A. Biochemistry 1985, 24, 713. (31) Remy, J.-S.; Abdallah, B.; Zanta, M. A.; Boussif, O.; Behr, P.; Demeneix, B. AdV. Drug DeliV. ReV. 1998, 30, 86. (32) Mascarenhas, L.; Stripecke, R.; Case, S. S.; Xu, D. K.; Weinberg, K. I.; Kohn, D. B. Blood 1998, 92, 3537. (33) Twardowski, J.; Anzenbacher, P. Raman and IR Spectroscopy in Biology and Biochemistry; Ellis Horwood: New York, 1994; Chapter 6. (34) Benevides, J. M.; Thomas, G. J., Jr. Nucl. Acids. Res. 1983, 11, 5747. (35) Matsui, H.; Pan, S. Langmuir, manuscript submitted. (36) Peticolas, W. L.; Evertsz, E. Methods Enzymol. 1992, 211, 335. (37) Segers-Nolten, G. M. J.; Sijtsema, N. M.; Otto, C. Biochemistry 1997, 36, 13241. (38) Peticolas, W. L. Methods Enzymol. 1995, 246, 389. (39) Atsuki, H. Biochemistry 1981, 20, 7359. (40) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: New York, 1991; Chapters 5 and 10. (41) Pohl, F. M.; Jovin, T. M. J. Mol. Biol. 1972, 67, 375. (42) Hawirth, I. S.; Rodger, A.; Richards, W. G. J. Biomol. Struct. Dyn. 1992, 10, 195. (43) Matsui, H.; Pan, S. unpublished result. (44) Thomas, T. J.; Ashley, C.; Thomas, T.; Shirahata, A.; Sigal, L. H.; Lee, J. S. Biochem. Cell Biol. 1997, 75, 207. (45) Spiro, T. G. Biological Applications of Raman Spectroscopy; John Wiley & Sons: New York, 1987; Vol. 2, Chapter 3.
