Arrays of mobile tethered vesicles on supported lipid bilayers

Journal of the American Chemical Society

Volumn 125, Issue 13, 2003, Pages 3696-3697

  Yoshina Ishii, Chiaki ^a   Boxer, Steven G ^a  

^a STANFORD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANTISENSE OLIGONUCLEOTIDE; LIPID;

ARTICLE; BINDING AFFINITY; BIOTECHNOLOGY; COLLOID; FLUORESCENCE MICROSCOPY; LIPID BILAYER; MEMBRANE BIOLOGY; TRANSPORT VESICLE;

FLUORESCENT DYES; LIPID BILAYERS; LIPOSOMES; OLIGONUCLEOTIDES; OLIGONUCLEOTIDES, ANTISENSE; PHOSPHATIDYLCHOLINES;

EID: 0037414314     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja029783+     Document Type: Article

References (20)

1. Uses of oligonucleotides in nanotechnology: (a) Heller, J. J. Annu. Rev. Biomed. Eng. 2002, 4, 129-53. (b) Niemeyer, C. M. Curr. Opin. Chem. Bio. 2002, 4, 609-618.
2. Uses of oligonucleotides in nanotechnology: (a) Heller, J. J. Annu. Rev. Biomed. Eng. 2002, 4, 129-53. (b) Niemeyer, C. M. Curr. Opin. Chem. Bio. 2002, 4, 609-618.
3. An example preparation of oligonucleotide-modified vesicles. A mixture of lipids containing egg phosphatidylcholine and 0.5 mol% of reactive lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3- (2-pyridyldithio)propionate] (sodium salt) (N-PDP-PE, Avanti Polar Lipids), and fluorescently labeled lipid. Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR DHPE, Molecular Probes) in chloroform, is dried to a film, reconstituted in buffer (100 mM borate, 50 mM citrate, 100 mM NaCl, 2 mM EDTA, pH 8.0) to 25 mM and extruded through a 50 nm polycarbonate membrane (Avanti) to form vesicles. An oligonucleotide modified with a disulfide group on the 5′-end (IDT DNA technologies) is first reduced to expose a free sulfhydril functionality with 10 molar excess tris(2-carboxyethyl)phosphine (TCEP), then added to the vesicle solution to a final DNA concentration of 50 μM and lipid concentration of 13 mM. The DNA attaches to the outside surface by a disulfide exchange reaction on average 1-2 per vesicle, estimated using an assay based on the fluorescence of a labeled antisense oligonucleotide. Vesicles are isolated on a Sepharse CL-4B gel filtration column with the same buffer as eluant. Supported bilayers displaying oligonucleotides are formed on a cleaned glass coverslip by vesicle fusion as described earlier for simple lipids (Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 14773-81), and excess vesicles are rinsed away with copious amounts of buffer. Vesicles, at approximately 70 μM in lipids, displaying the complementary oligonucleotide, are incubated with this bilayer at room temperature for 30 min followed by further rinsing with buffer to remove free, unattached vesicles. Similar procedures were used to prepare vesicles varying in size from 30 to 200 nm and oligonucleotide lengths of 16 to 24 bases.
4. Intact, tethered vesicles have been previously reported in the literature. These systems do not to our knowledge show lateral mobility, and because a single linkage type was used, multiple types of vesicles encoded by the linkage could not be displayed. Systems include the following: Biotin/Avidin - monolayer on Gold: (a) Jung, L. S.; Shumaker-Parry, J. S.; Campbell, C. T.; Yee, S. S.; Gelb, M. H. J. Am. Chem. Soc. 2000, 122, 4177-84. Biotin/Avidin - supported bilayer: (b) Boukobza, E.; Sonnenfeld, A.; Haran, G. J. Phys. Chem. B 2001, 105, 12165-70. Chemisorption of vesicles onto gold films: (c) Stanish, I.; Santos, J. P.; Singh, A. J. Am. Chem. Soc. 2001, 123, 1008-9.
5. Intact, tethered vesicles have been previously reported in the literature. These systems do not to our knowledge show lateral mobility, and because a single linkage type was used, multiple types of vesicles encoded by the linkage could not be displayed. Systems include the following: Biotin/Avidin - monolayer on Gold: (a) Jung, L. S.; Shumaker-Parry, J. S.; Campbell, C. T.; Yee, S. S.; Gelb, M. H. J. Am. Chem. Soc. 2000, 122, 4177-84. Biotin/Avidin - supported bilayer: (b) Boukobza, E.; Sonnenfeld, A.; Haran, G. J. Phys. Chem. B 2001, 105, 12165-70. Chemisorption of vesicles onto gold films: (c) Stanish, I.; Santos, J. P.; Singh, A. J. Am. Chem. Soc. 2001, 123, 1008-9.
6. Intact, tethered vesicles have been previously reported in the literature. These systems do not to our knowledge show lateral mobility, and because a single linkage type was used, multiple types of vesicles encoded by the linkage could not be displayed. Systems include the following: Biotin/Avidin - monolayer on Gold: (a) Jung, L. S.; Shumaker-Parry, J. S.; Campbell, C. T.; Yee, S. S.; Gelb, M. H. J. Am. Chem. Soc. 2000, 122, 4177-84. Biotin/Avidin - supported bilayer: (b) Boukobza, E.; Sonnenfeld, A.; Haran, G. J. Phys. Chem. B 2001, 105, 12165-70. Chemisorption of vesicles onto gold films: (c) Stanish, I.; Santos, J. P.; Singh, A. J. Am. Chem. Soc. 2001, 123, 1008-9.
7. A typical lipid labeled vesicle preparation includes 1 mol% Texas Red DHPE, so a single 100 nm diameter vesicle contains approximately 1000 fluorophores. A 100 nm vesicle entrapping 10 mM Oregon Green 488 carboxylic acid contains approximately 2300 dye moledules.
8. Groves, J. T.; Boxer, S. G. Acc. Chem Res. 2002, 35, 149-57.
9. Kung, L. A.; Kam, L.; Hovis, J. S.; Boxer, S. G. Langmuir 1999, 16, 6773-6.
10. See Supporting Information for a video of diffusing vesicles, Movie 1.
11. Qian, H.; Sheetz, M. P.; Elson, E. L. Biophys. J. 1991, 60, 910-21.
12. Saxton, M. J.; Jacobson, K. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 373-99.
13. Lee, G. M.; Jacobson, K. Current Top. Membr. 1994, 40, 111-42.
14. Lee, G. M.; Ishihara, A.; Jacobson, K. A. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6274-8.
15. The lipid compositions of the supported bilayer and the vesicles are independent. Fluorescently labeled lipids in the underlying supported bilayer are observed by FRAP to diffuse in the presence of tethered vesicles as in their absence.
16. Single and double stranded oligonucleotide lipid headgroups in the supported bilayer subjected to a lateral electric field move as expected towards the positive electrode. By contrast, tethered vesicles move rapidly in the direction of the field (see Supporting Information for a movie demonstrating this, Movie 2). This is opposite to the direction expected based on their net negative charge, suggesting that they respond to electroosmotic flow as has been observed for some tethered proteins (Groves, J. T.; Wülfing, C.; and Boxer, S. G. Biophys. J. 1996, 71, 2716- 2723). Note that the double helical oligonucleotide tether extends less than 10 nm from the supported bilayer surface, whereas a tethered vesicle extends out further by its diameter. Because electrophoretic drift is very rapid, it is possible to concentrate tethered vesicles along corral boundaries (see Supporting Information). Even when highly concentrated, tethered vesicles are observed to retain their contents and no lipid leaflet mixing is observed. When the field is turned off, the vesicles relax back to randomness by lateral diffusion or they can be moved in the opposite direction by reversing the bias of the field.
17. Groves, J. T.; Boxer, S. G. Biophys. J. 1995, 69, 1972-75.
18. Hovis, J. S.; Boxer, S. G. Langmuir 2000, 16, 894-7.
19. Kam, L.; Boxer, S. G. J. Am. Chem. Soc. 2000, 122, 12901-2.
20. Saxton, M. J. Biophys. J. 1997, 72, 1744-5.