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Design and synthesis of ligands for recognition of the major and minor grooves of DNA

Citation

Greenberg, William Anthony (1998) Design and synthesis of ligands for recognition of the major and minor grooves of DNA. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-04282006-114932

Abstract

The chemical approach to the sequence specific recognition of double stranded DNA in this laboratory focuses on two distinct structural motifs. The first is oligonucleotide-directed triple helix formation. Pyrimidine oligonucleotides bind homopurine duplex sequences in the major groove by formation of T•AT and C+GC base triplets in a parallel orientation relative to the purine target strand. Purine rich oligonucleotides also bind homopurine sequences in the major groove, by formation of G•GC and A•AT or T•AT base triplets, in this case oriented antiparallel to the purine target strand. The lack of ability recognize CG and TA base pairs by either pyrimidine or purine oligonucleotides has resulted in efforts to design nonnatural bases which can recognize these base pairs in the context of triple helices. Such bases would greatly expand the generality of triple helix formation by expanding the range of targetable sequences.

The second structural motif consists of pyrrole-imidazole polyamides which form 2:1 side-by-side antiparallel complexes in the minor groove of DNA. A set of "pairing rules" has been elucidated in which an imidazole-pyrrole pair recognizes a GC base pair, a pyrrole-imidazole pair recognizes CG, and a pyrrole-pyrrole pair is degenerate for both AT and TA base pairs.

This thesis describes the application of quantitative DNase I footprinting to the evaluation of the energetics of triple helix formation by purine and pyrimidine oligonucleotides containing designed, nonnatural bases, as well the energetics of complex formation between covalently linked H-pin polyamides and the minor groove of DNA. Chapter Two describes the energetics of formation of sixteen triple helical complexes containing only the natural bases A, G, C, and T, which vary at single position within the purine motif. The values obtained set a basis with which to evaluate the energetics of triple helices containing nonnatural bases, and demonstrate the exquisite sensitivity of triple helices to single base mismatches. Chapter Three describes the energetics of formation of purine motif triple helical complexes which contain single substitutions of the nonnatural purines inosine, 2-aminopurine, nebularine, and isoinosine. The results emphasize that new structural space must be explored in order to design bases which will specifically recognize CG and TA base pairs within the purine motif. Chapter Four describes the design and synthesis of methyl-substituted imidazole nucleosides and evaluation of the energetics of formation of purine motif triple helical complexes containing single substitutions of these nonnatural bases. The results suggest that use of small, fivemembered ring heterocycles is likely a good design toward TA recognition, but that hydrophobic interactions between a designed nonnatural base and the 5-methyl group of thymine is not energetically favorable in the context of a purine motif triple helix. Chapter Five describes the design and synthesis of substituted pyrazole nucleosides and evaluation of the energetics of formation of purine motif triple helical complexes containing single substitutions of these nonnatural bases. The pyrazole substitution showed improved specificity towards TA, but at low affinity. This affinity could not be improved by appropriate substitution of an amino group, which instead improved affinity for GC. This points to the difficulty of forming a hydrogen bond to the 04 carbonyl of thymine relative to the more accessible 06 carbonyl of guanine. Chapter Six describes an evaluation of the energetics of formation of pyrimidine motif triple helical complexes containing a previously published nonnatural base and the discovery that as previously prepared, the base retains a benzoyl protecting group in the pyrimidine oligonucleotide. This benzoylated base shows specificity for CG and TA base pairs, similarly to the previously reported, structurally similar D3, which has been shown to bind through an intercalative mode. Independent synthesis of the unprotected base through a new route proved that this base did not bind specifically to any of the four base pairs in the context of a pyrimidine motif triple helix. Chapter Seven describes an evaluation of the energetics of formation of pyrimidine motif triple helical complexes containing a nonnatural base derived from a model heterocycle which was shown to bind isolated CG base pairs in organic solvent. The results suggest that model studies in organic solvent cannot accurately predict the behavior of nonnatural bases in water, in the context of a triple helix, since this base showed no specificity for any base pair by quantitative DNase I footprinting studies. Chapter Eight describes the design and solid phase synthesis of a series of pyrrole-imidazole polyamides which are covalently linked across central pyrrole nitrogens to increase affinity and specificity towards their DNA target sites according to the 2:1 polyamide:DNA binding model. DNase I footprint titration results show that these H-pin polyamides do bind with increased affinity and specificity relative to unlinked analogues, but that hairpin polyamides show a greater improvement, and are thus more optimal designs.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Unknown, Unknown
Defense Date:24 July 1997
Record Number:CaltechETD:etd-04282006-114932
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-04282006-114932
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1534
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:28 Apr 2006
Last Modified:26 Dec 2012 02:38

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