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Volumn 275, Issue 5301, 1997, Pages 810-814

Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes

Author keywords

[No Author keywords available]

Indexed keywords

DNA; LIPOSOME;

EID: 0031036238     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5301.810     Document Type: Article
Times cited : (1350)

References (50)
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  • 25
    • 14444271448 scopus 로고    scopus 로고
    • note
    • A mixture of DOPE/DOTAP (1:1, wt:wt) was prepared in a 20-mg/ml chloroform stock solution; 500 ml was dried under nitrogen in a narrow glass beaker and desiccated under vacuum for 6 hours. After addition of 2.5 ml of Millipore water and 2 hours incubation at 40°C, the vesicle suspension was sonicated to clarity for 10 min. The resulting solution of liposomes (25 mg/ml) was filtered through 0.2-μm Nucleopore filters. For optical measurements, the concentration of SUV used was between 0.1 and 0.5 mg/ml. All the lipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL).
  • 26
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    • note
    • The liposome and complex sizes were measured by dynamic light scattering (Microtrac UPA 150, Leeds and Northrup).
  • 27
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    • note
    • Purified λ-phage DNA and pBR322 plasmid were purchased from Biolabs, New England. Optical and x-ray data were taken with linear λ prepared in two ways: (i) used as delivered, and (ii) by heating to 65°C and reacting with a surplus of a 12-base oligo complementary to the 3′ COS end. Subsequently, the DNA was ligated (T4 DNA ligase, Fischer). The methods gave the same result. For the optical experiments, the DNA concentration used was between 0.01 and 0.1 mg/ml.
  • 28
    • 14444272873 scopus 로고    scopus 로고
    • note
    • A. Nikon Diaphot 300 equipped for epifluorescence and high-resolution DIC was used.
  • 29
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    • note
    • Sonicated DOPE-DOTAP (1:1) liposomes were prepared at 0.1 mg/ml with 0.2 moi % DHPE-Texas Red fluorescence label. DNA stained by YOYO (Molecular Probes) was added under gentle mixing at different L/Ds.
  • 31
    • 14444285362 scopus 로고    scopus 로고
    • note
    • High-resolution synchrotron x-ray scattering was performed at the Stanford Synchrotron Radiation Laboratory. Lower resolution XRD experiments were performed with a rotating anode source.
  • 32
    • 14444284675 scopus 로고    scopus 로고
    • note
    • The DNA-lipid condensates were prepared from a 25-mg/ml liposome suspension and a 5-mg/ml DNA solution. The solutions were filled in 2-mm-diameter quartz capillaries with different ratios L/D, respectively, and mixed after flame sealing by gentle centrifugation up and down the capillary.
  • 34
    • 14444283729 scopus 로고    scopus 로고
    • note
    • The intercalation of λ-DNA between membranes in CL-DNA complexes was found to protect it against a Hind III restriction enzyme, which cuts naked λ-DNA at seven sites (22).
  • 38
    • 14444287879 scopus 로고    scopus 로고
    • note
    • The multilamellar structure of the complex (with λ-DNA) and the distinct DNA interhelical packing regimes were also found in SAXS data in binary mixtures of cationic lipids that contained DOPE [which has a high transfection efficiency (2)] as the neutral colipid. However, the complexes showed a phase separation into two condensed phases.
  • 41
    • 14444275406 scopus 로고    scopus 로고
    • note
    • 2 each and thus permits near complete neutralization of the complex (Fig. 3A).
  • 42
    • 14444271189 scopus 로고    scopus 로고
    • note
    • w = 25 ± 1.5 Å, close to the spacing for the DNA monolayer (see Fig. 3A).
  • 50
    • 14444271447 scopus 로고    scopus 로고
    • note
    • We acknowledge useful discussions with R. Bruinsma, N. Dan, W. Gelbart, P. Pincus, J. Prost, T. Lubensky, and D. Lasic. Supported in part by NSF grant DMR-9624091, the Petroleum Research Fund (31352-AC7), and a Los Alamos CULAR grant STB/ UC:96-108. J.O.R. and T.S. acknowledge partial support by DFG (Ra 655/1-1) and DAAD scholarships, respectively. The Materials Research Laboratory at Santa Barbara is supported by the NSF under grant DMR-9632716. The synchrotron experiments were carried out at Stanford (SSRL) supported by the U.S. Department of Energy.


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