Nucleic Acids Research Advance Access originally published online on December 14, 2006
Nucleic Acids Research 2007 35(2):363-370; doi:10.1093/nar/gkl1042
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Nucleic Acids Research, 2007, Vol. 35, No. 2 363-370
© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Improved nuclear localization of DNA-binding polyamides
Division of Chemistry and Chemical Engineering, California Institute of Technology Pasadena CA 91125, USA
*To whom correspondence should be addressed. Tel: +1 626 395 6002; Fax: +1 626 683 8753; Email: dervan{at}caltech.edu
Received September 17, 2006. Revised November 2, 2006. Accepted November 3, 2006.
Regulation of endogenous genes by DNA-binding polyamides requires effective nuclear localization. Previous work employing confocal microscopy to study uptake of fluorophore-labeled polyamides has demonstrated the difficulty of predicting a priori the nuclear uptake of a given polyamide. The data suggest that dye identity influences uptake sufficiently such that a dye-conjugate cannot be used as a proxy for unlabeled analogs. Polyamides capable of nuclear localization unaided by fluorescent dyes are desirable due to size and other limitations of fluorophores. Recently, a polyamide-fluorescein conjugate targeted to the hypoxia response element (HRE) was found to inhibit vascular endothelial growth factor (VEGF) expression in cultured HeLa cells. The current study uses inhibition of VEGF expression as a biological read-out for effective nuclear localization of HRE-targeted polyamides. We synthesized a focused library of non-fluorescent, HRE-targeted polyamides in which the C-terminus ‘tail’ has been systematically varied. Members of this library bind the HRE with affinities comparable or superior to that of the fluorescein-labeled analog. Although most library members demonstrate modest or no biological activity, two non-fluorescent polyamides are reported with activity rivaling that of the previously reported fluorescein-labeled polyamide. We also show evidence that promoter occupancy by HIF-1, the transcription factor that binds the HRE, is inhibited by HRE-targeted polyamides.
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