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Localization of DNA-binding polyamides in living cells

Citation

Best, Timothy Patrick (2005) Localization of DNA-binding polyamides in living cells. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-10172004-184202

Abstract

Regulation of the processing of genes into nucleic acids and proteins is a substantial goal in medicine. Small molecules that could enter cells, localize to the nucleus, and bind chromosomal DNA sequence-specifically and with high affinity would be important tools for gene regulation. Pyrrole-imidazole polyamides are small molecules that bind the minor groove of DNA in a sequence-specific fashion according to a set of pairing rules, and with affinities rivaling natural transcription factors. Several in vitro experiments have shown that by directly competing with transcription factors for binding sites in gene promoter regions, polyamides can act to inhibit transcription of those genes. Polyamides bearing transcription activation domains can bind to promoter regions, recruit the transcriptional machinery to the gene, and activate transcription in vitro. Attempts to reproduce these results in vivo were largely unsuccessful, perhaps due to poor cellular trafficking properties of polyamides and polyamide-peptide conjugates.

It was found that polyamides bearing the Bodipy fluorophore localize primarily to the cytoplasm of cells, or were excluded from cells altogether. In attempts to overcome this quality, peptides shown to improve cellular trafficking were appended to the polyamides. These peptides were generally not successful at inducing uptake, and were in many cases toxic to the cells. Small molecules were also appended to polyamides, likewise to improve uptake properties, but met with limited success. Surprisingly, the addition of a fluorescein or fluorescein-like fluorophore to polyamides permit them to localize to the nuclei of all cell lines tested, in a molecular content- and shape-dependent manner. This technology has been applied to several in vivo experiments, including the inhibition of androgen receptor binding to its cognate element in gene promoter regions.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:cell uptake; confocal microscopy; DNA; DNA-recognition; footprinting; imidazole; minor groove; polyamides; pyrrole; transcription activation
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Hsieh-Wilson, Linda C. (chair)
  • Parker, Carl Stevens
  • Dervan, Peter B.
  • Fraser, Scott E.
Defense Date:22 July 2004
Author Email:best (AT) berkeley.edu
Record Number:CaltechETD:etd-10172004-184202
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-10172004-184202
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:4123
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:19 Oct 2004
Last Modified:26 Dec 2012 03:05

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