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Nuclear Localization of Polyamide-Fluorescein Conjugates in Cell Culture


Edelson, Benjamin S. (2005) Nuclear Localization of Polyamide-Fluorescein Conjugates in Cell Culture. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/XQYG-DW76.


Chapter 1: Distamycin A is a crescent-shaped natural product that preferentially binds to (A,T) sequences in the minor grove of DNA as 1:1 and 2:1 ligand-DNA complex [1,2]. Analogs of the N-methylpyrrole (Py) rings of these polyamides afford a set of five-membered heterocycles that can be combined—as unsymmetrical ring pairs—in a modular fashion to recognize predetermined DNA sequences with affinity and specificity comparable to DNA-binding proteins (Figures 1 and 2) [3,4]. We describe here recent advances in the field of DNA-binding polyamides, including structural verification of binding models, new heterocycles for recognition, cellular and nuclear uptake properties, and recent biological applications.

Chapter 2: A series of hairpin pyrrole-imidazole polyamide-fluorescein conjugates were synthesized and assayed for cellular localization. Thirteen cell lines, representing eleven human cancers, one human transformed kidney cell line, and one murine leukemia cell line, were treated with 5 µM polyamide-fluorescein conjugates for 10-14 h, then imaged by confocal laser scanning microscopy. A conjugate containing a [beta]-alanine residue at the C-terminus of the polyamide moiety showed no nuclear localization, while an analogous compound lacking the [beta]-alanine residue was strongly localized in the nuclei of all cell lines tested. The localization profiles of several other conjugates suggest that pyrrole-imidazole sequence and content, dye choice and position, linker composition, and molecular weight are determinants of nuclear localization. The attachment of fluorescein to the C-terminus of a hairpin polyamide results in an approximate 10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.

Chapter 3: A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in thirteen mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks, and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a [beta]-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of this data on the design of polyamide-dye conjugates for use in biological systems.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
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:
  • Unknown, Unknown
Defense Date:1 January 2005
Record Number:CaltechETD:etd-03162007-135225
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:969
Deposited By: Imported from ETD-db
Deposited On:27 Mar 2007
Last Modified:27 May 2020 21:36

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