Mrksich, Milan (1994) Design of peptides for sequence-specific recognition of the minor groove of DNA. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-09212005-075404
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Distamycin A and netropsin are natural products which bind in the minor groove of DNA at four and five A,T base pair sites, respectively. While the peptides pyridine-2-carboxamide-netropsin (2-PyN) and 1-methylimidazole-2-carboxamide-netropsin (2-ImN) were designed to bind [...] sequences in a single orientation, they bind 5'-TGTCA-3' with no orientation preference. Direct characterization of the [...] complex by NMR reveals that the peptide binds in the minor groove as a side-by-side dimer.
Covalent peptide dimers wherein the nitrogens of the central pyrroles of 2-PyN are connected with propyl, butyl, pentyl and hexyl linkers bind the 5'-TGTCA-3' site with affinities ten-fold greater than that of the non-linked peptides. The ratio of binding affinities of 2-PyN for 5'-TGTCA-3' and 5'-TTTTT-3' sites have been altered from 1:1 to 20:1.
Footprinting and affinity cleaving experiments demonstrate that the two peptides distamycin and 2-ImN simultaneously bind the five-base pair sequence 5'-TGTTA-3' as a side-by-side heterodimer in the minor groove. The specific affinity for this site was enhanced with the design and synthesis of a covalent peptide heterodimer.
Four hexapeptides were synthesized wherein the terminal amine and carboxyl groups of distamycin and 2-ImN, respectively, are connected with amino acids. 2-ImN-GABA-P3 binds the 5'-TGTTA-3' site with similar binding affinity as the first generation covalent peptide dimer. The general and efficient synthetic methodology for preparation of GABA linked peptides may allow the design of a new class of hairpin peptide-turn-peptides for specific recognition of many different sequences in the minor groove of DNA.
Footprinting and affinity cleaving experiments demonstrate that the designed peptide ImPImP specifically binds the designated six-base-pair sites 5'-AGCGCT-3' and 5'-TGCGCA-3' in two equal orientations, consistent with a side-by-side antiparallel arrangement of peptides in the minor groove. This example underscores the utility of 2:1 peptide-DNA models for the design of ligands for sequence-specific recognition of designated DNA sites. Binding of this designed peptide to a pure four base pair GC-core sequence represents an absolute reversal of the specificity of the natural products distamycin and netropsin.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||8 March 1994|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||22 Sep 2005|
|Last Modified:||26 Dec 2012 03:02|
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