A series of studies from our lab have investigated the threading polyintercalator approach to sequence specific DNA binding using a 1,4,5,8-naphthalene tetracarboxylic diimide (NDI) intercalating unit connected by flexible peptide linkers. Herein is a report of the sequence specificity, as well as a detailed kinetic analysis, of a threading NDI tetraintercalator. DNase I footprinting using two ~500 base pair DNA fragments containing one designed binding site for the tetraintercalator confirmed highly sequence specific binding. Kinetic analyses include 1H NMR, gel mobility-shift assays, and stopped-flow UV measurements to reveal a polyintercalation binding mode that demonstrates significant similarities between association rate profiles and rate constants for the tetraintercalator binding to its preferred versus a random oligonucleotide sequence. Sequence specificity was found to derive almost entirely from large differences in dissociation rates from the preferred versus random oligonucleotide sequences. Interestingly, the dissociation rate constant of the tetraintercalator complex dissociating from its preferred binding site was extremely slow, corresponding to a 16 day half-life at a benchmark 100 mM [Na+]. This dissociation result for the tetraintercalator is one of the longest bound half-lives yet measured, and to the best of our knowledge, the longest for a DNA binding small molecule. Such a long-lived complex raises the possibility of using threading polyintercalators to disrupt biological processes for extended periods.
Current focus is given to deciphering a mechanism for the molecular recognition of the tetraintercalator preferred binding site within a long sequence of DNA. Initial DNase I footprinting results on an approximate 500mer DNA sequence containing three sequential preferred binding sites reveal that the tetraintercalator likely locates its designed binding site by a macro- or microscopic dissociation/re-association type of mechanism. Cooperativity is a possible ally to binding, leaving future studies to distinguish the mechanism for molecular recognition in a manner that is capable of circumventing cooperative binding. Taken together, the threading polyintercalation binding mode presents an interesting topology to sequence specific DNA binding. Extraordinarily long dissociation rates from preferred binding sites offers many future possibilities to disrupt biological processes in vivo. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/ETD-UT-2011-08-3863 |
| Date | 22 September 2011 |
| Creators | Holman, Garen Gilman |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | thesis |
| Format | application/pdf |
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