Liberles, David Alan (1998) Sequence specific DNA recognition and bending. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-04272006-155123
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Intrinsic DNA curvature and protein induced DNA bending play a crucial role in the regulation of many biological processes, including transcription, replication, transposition, viral integration, site-specific recombination, and DNA packaging in nucleosomes. The design of ligands to artifically regulate DNA topology may be important in creating artificial regulators of these processes, with potential utility in biology and human medicine. Much of the work described in this thesis is based upon the ability of a triple helix forming oligonucleotide to recognize double helical DNA. This sequence specific recognition occurs in two motifs and is governed by the formation of specific hydrogen bonds. One limitation of this technique is the inability to recognize pyrimidine bases in the Hoogsteen bonded strand. A novel base, [...], has been previously described with the potential to recognize pyrimidine bases.
Chapter 2 describes a thermodynamic analysis of the binding properties of oligonucleotides bearing single and multiple D3 bases in the context of triple helix formation. An NMR structure has indicated the binding mode of [...] as intercalative and the ability of [...] containing oligonucleotides to bend DNA through this intercalative wedge was examined.
Chapter 3 describes an alternative experimental approach to triple helix mediated DNA bending. Two third strand binding domains are appended by a linker of variable size to target two cognate binding sites separated by 10 base pairs, or one turn of the helix. Using this experimental approach, large bend angles were obtained, as analyzed electrophoretically.
Chapter 4 analyzes the nature of this binding and bending event using the designed third strand ligand. The energetics of bending were examined as were other factors influencing the ability to form a bent structure, including oligonucleotide binding motif, third strand length, sequence composition, pH, ion concentration and valence, target site spacing, and linker composition and orientation to examine the effects of electrostatics and hydrophobicity.
Chapter 5 describes the design of a complementary architectural factor to straighten DNA that has been bent by a triple helical ligand. These complementary polyamide ligands recognize DNA sequence specifically and bind in the minor groove of the linker region to straighten DNA. Such molecules may also be useful as artificial regulators of biological processes.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||16 June 1997|
|Non-Caltech Author Email:||liberles (AT) uwyo.edu|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||27 Apr 2006|
|Last Modified:||26 Dec 2012 02:38|
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