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Type of Document	Dissertation
Author	Urbach, Adam Robert
Author's Email Address	aurbach AT trinity.edu
URN	etd-05202002-104244
Persistent URL	http://resolver.caltech.edu/CaltechETD:etd-05202002-104244
Title	One-to-one motif for DNA recognition by beta-alanine-linked polyamides
Degree	PhD
Option	Chemistry
Advisory Committee	Advisor Name Title Professor Robert H. Grubbs Committee Chair Professor John D. Roberts Committee Member Professor Peter B. Dervan Committee Member Professor Richard W. Roberts Committee Member Professor Stephen L. Mayo Committee Member
Keywords	DNA recognition NMR structure beta-alanine minor groove polyamide
Date of Defense	2002-05-17
Availability	unrestricted
Abstract Polyamides composed of N-methylpyrrole (Py), N-methylimidazole (Im), and 3-hydroxypyrrole (Hp) amino acids linked by beta-alanine (beta) bind in the minor groove of DNA in 1:1 and 2:1 ligand:DNA complexes. Although the energetics and structure of the 2:1 motif have been explored extensively, there is remarkably less understood about 1:1 recognition beyond the initial studies on netropsin and distamycin. Laemmli and coworkers used beta-linked polyamides, which bind in a 1:1 motif, to effect phenotypic changes in Drosophila melanogaster. The thesis work described here investigates Laemmli's 1:1 motif in order to further understand and exploit this novel mode of DNA recognition. By selectively replacing Py residues with beta it was found that the Im-beta-Im subunit is important for high-affinity binding in 1:1 and 2:1 modes. This study also demonstrates that a single ligand can target very different DNA sequences based on 1:1 or 2:1 binding. This ambiguity of sequence targeting based on stoichiometry was addressed. It was discovered that hairpin and 1:1 binding modes, which are dependent on ligand conformation, are controlled by changing the linker between polyamide subunits. The possibility of developing a 1:1 recognition code was explored by selectively mutating polyamide residues and DNA base pairs and comparing the association constants for the resulting complexes. It was found that Im residues tolerate all four Watson-Crick base pairs; Py and beta residues are specific for A?T and T?A base pairs; and Hp specifies a single base pair, A?T, in the sequence context 5'-AAAGAGAAGAG-3'. Attempts to improve upon this recognition code using novel heterocyclic amino acids, such as furan, thiophene, thiazole, and hydroxythiophene, are presented. The sequence-dependence of ligand orientation and the effect of ligand size on binding affinity were also explored. The NMR structure of a 1:1 polyamide:DNA complex was determined. It reveals B-form DNA with a narrow minor groove and large negative propeller twist, which are shown to be stabilized by bifurcated hydrogen bonds between polyamide NH groups and purine N3 and pyrimidine O2 atoms. The first direct evidence is provided for hydrogen bond formation between Im-N3 and guanine NH2 in the 1:1 motif, thus confirming the original lexitropsin model.
Files	Filename       Size       Approximate Download Time (Hours:Minutes:Seconds)     28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access    0_Complete_Thesis.pdf 12.42 Mb 00:57:29 00:29:33 00:25:52 00:12:56 00:01:06   1_Title_Ab_TOC.pdf 72.86 Kb 00:00:20 00:00:10 00:00:09 00:00:04 < 00:00:01   2_Introduction.pdf 459.12 Kb 00:02:07 00:01:05 00:00:57 00:00:28 00:00:02   3_Results_and_Discussion.pdf 2.41 Mb 00:11:09 00:05:44 00:05:01 00:02:30 00:00:12   4_Conclusions.pdf 58.51 Kb 00:00:16 00:00:08 00:00:07 00:00:03 < 00:00:01   5_Experimental.pdf 112.78 Kb 00:00:31 00:00:16 00:00:14 00:00:07 < 00:00:01   6_References.pdf 89.34 Kb 00:00:24 00:00:12 00:00:11 00:00:05 < 00:00:01   7_Appendix.pdf 9.65 Mb 00:44:39 00:22:58 00:20:05 00:10:02 00:00:51