Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지

Identification of a Deoxyribonuclease I Inhibitor from a Phage-Peptide Library

  • Choi, Suk-Jung (Department of Chemistry, Kangnung National University) ;
  • Sperinde, Jeffrey J. (Department of Biopharmaceutical Sciences, School of Pharmacy, University of California) ;
  • Szoka, Francis C. Jr. (Department of Biopharmaceutical Sciences, School of Pharmacy, University of California)
  • Received : 2004.08.24
  • Accepted : 2004.10.29
  • Published : 2005.02.28
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Abstract

Deoxyribonuclease I (DNase I) is a divalent cation dependent endonuclease and thought to be a significant barrier to effective gene delivery. The only known DNase I-specific inhibitor is monomeric actin which acts by forming a 1:1 complex with DNase I. Its use, however, is restricted because of tendency to polymerize under certain conditions. We screened two random phage peptide libraries of complexity $10^8$ and $10^9$ for DNase I binders as candidates for DNase I inhibitors. A number of DNase I-binding peptide sequences were identified. When these peptides were expressed as fusion proteins with Escherichia coli maltose binding protein, they inhibited the actin-DNase I interaction ($IC_{50}=0.1-0.7{\mu}M$) and DNA degradation by DNase I ($IC_{50}=0.8-8{\mu}M$). Plasmid protection activity in the presence of DNase I was also observed with the fusion proteins. These peptides have the potential to be a useful adjuvant for gene therapy using naked DNA.

Keywords

Acknowledgement

Supported by : Kangnung National University, UC

References

  1. Adey, N. B., Mataragnon, A. H., Rider, J. E., Carter, J. M., and Kay, B. K. (1995) Characterization of phage that bind plastic from phage-displayed random peptide libraries. Gene 156, 27-31 https://doi.org/10.1016/0378-1119(95)00058-E
  2. Barry, M. E., Pinto-Gonzalez, D., Orson, F. M., McKenzie, G. J., Petry, G. R., et al. (1999) Role of endogenous endonucleases and tissue site in transfection and CpG-mediated immune activation after naked DNA injection. Hum. Gene Ther. 10, 2461-2480 https://doi.org/10.1089/10430349950016816
  3. Budker, V., Budker, T., Zhang, G., Subbotin, V., Loomis, A., et al. (2000) Hypothesis: naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process. J. Gene Med. 2, 76-88 https://doi.org/10.1002/(SICI)1521-2254(200003/04)2:2<76::AID-JGM97>3.0.CO;2-4
  4. Choi, S. J., Ahn, M., Lee, J. S., and Jung, W. J. (1997) Selection of a high affinity angiogenin-binding peptide from a peptide library displayed on phage coat protein. Mol. Cells 7, 575-581
  5. Choi, S. J. and Szoka, F. C. (2000) Fluorometric determination of deoxyribonuclease I activity with PicoGreen. Anal. Biochem. 281, 95-97 https://doi.org/10.1006/abio.2000.4529
  6. He, Y., Pimenov, A. A., Nayak, J. V., Plowey, J., Falo, L. D., Jr. et al. (2000) Intravenous injection of naked DNA encoding secreted flt3 ligand dramatically increases the number of dendritic cells and natural killer cells in vivo. Hum. Gene Ther. 11, 547-554 https://doi.org/10.1089/10430340050015734
  7. Hyde-DeRuyscher, R., Paige, L. A., Christensen, D. J., Hyde-DeRuyscher, N., Lim, A., et al. (2000) Detection of smallmolecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries. Chem. Biol. 7, 17-25 https://doi.org/10.1016/S1074-5521(00)00062-4
  8. Kabsch, W., Mannhertz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Atomic structure of the actin: DNase I complex. Nature 347, 37-44 https://doi.org/10.1038/347037a0
  9. Lahm, A. and Suck, D. (1991) DNase I-induced DNA conformation. 2 A structure of a DNase I-octamer complex. J. Mol. Biol. 222, 645-667 https://doi.org/10.1016/0022-2836(91)90502-W
  10. Lazarides, E. and Lindberg, U. (1974) Actin is the naturally occurring inhibitor of deoxyribonuclease I. Proc. Natl. Acad. Sci. USA 71, 4742-4746
  11. Levy, M. Y., Barron, L. G., Meyer, K. B., and Szoka, F. C. (1996) Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood. Gene Ther. 3, 201-211
  12. Malone, J. G. and Malone, R. W. (1999) Marked enhancement of direct respiratory tissue transfection by aurintricarboxylic acid. Hum. Gene Ther. 10, 1703-1713 https://doi.org/10.1089/10430349950017707
  13. Nagata, S. (2000) Apoptotic DNA fragmentation. Exp. Cell Res. 256, 12-18 https://doi.org/10.1006/excr.2000.4834
  14. Rizzuto, G., Cappelletti, M., Maione, D., Savino, R., Lazzaro, D., et al. (1999) Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc. Natl. Acad. Sci. USA 96, 6417-6422
  15. Ross, G. F., Bruno, M. D., Uyeda, M., Suzuki, K., Nagao, K., et al. (1998) Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor. Gene Ther. 5, 1244-1250 https://doi.org/10.1038/sj.gt.3300721
  16. Sinicropi, D., Baker, D. L., Prince, W. S., Shiffer, K., and Shak, S. (1994) Colorimetric determination of DNase I activity with a DNA-methyl green substrate. Anal. Biochem. 222, 351-358 https://doi.org/10.1006/abio.1994.1502
  17. Sperinde, J. J., Choi, S. J., and Szoka, F. C. (2001) Phage display selection of a peptide DNase II inhibitor that enhances gene delivery. J. Gene Med. 3, 101-108 https://doi.org/10.1002/jgm.165
  18. van der Lest, C. H. A., Veerkamp, J. H., and van Kuppevelt, T. H. (1995) ImageCalc: a Microsoft Windows application for quantitative image analysis and comparison. BioTechniques 18, 1050-1055
  19. Zhang, G., Budker, V., and Wolff, J. A. (1999) High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA. Hum. Gene Ther. 10, 1735-1737 https://doi.org/10.1089/10430349950017734
  20. Hum. Gene Ther. v.10 High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA Zhang, G.;Budker, V.;Wolff, J.A.