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Improved nuclear localization of DNA-binding polyamides

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 sug...

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Autores principales: Nickols, Nicholas G., Jacobs, Claire S., Farkas, Michelle E., Dervan, Peter B.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1802595/
https://www.ncbi.nlm.nih.gov/pubmed/17175539
http://dx.doi.org/10.1093/nar/gkl1042
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author Nickols, Nicholas G.
Jacobs, Claire S.
Farkas, Michelle E.
Dervan, Peter B.
author_facet Nickols, Nicholas G.
Jacobs, Claire S.
Farkas, Michelle E.
Dervan, Peter B.
author_sort Nickols, Nicholas G.
collection PubMed
description 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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spelling pubmed-18025952007-03-01 Improved nuclear localization of DNA-binding polyamides Nickols, Nicholas G. Jacobs, Claire S. Farkas, Michelle E. Dervan, Peter B. Nucleic Acids Res Molecular Biology 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. Oxford University Press 2007-01 2006-12-14 /pmc/articles/PMC1802595/ /pubmed/17175539 http://dx.doi.org/10.1093/nar/gkl1042 Text en © 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.
spellingShingle Molecular Biology
Nickols, Nicholas G.
Jacobs, Claire S.
Farkas, Michelle E.
Dervan, Peter B.
Improved nuclear localization of DNA-binding polyamides
title Improved nuclear localization of DNA-binding polyamides
title_full Improved nuclear localization of DNA-binding polyamides
title_fullStr Improved nuclear localization of DNA-binding polyamides
title_full_unstemmed Improved nuclear localization of DNA-binding polyamides
title_short Improved nuclear localization of DNA-binding polyamides
title_sort improved nuclear localization of dna-binding polyamides
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1802595/
https://www.ncbi.nlm.nih.gov/pubmed/17175539
http://dx.doi.org/10.1093/nar/gkl1042
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