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Sequence specific nonenzymatic ligation of single- and double-stranded DNA by triple helix formation


Luebke, Kevin J. (1992) Sequence specific nonenzymatic ligation of single- and double-stranded DNA by triple helix formation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/60d2-7x44.


Phosphodiesters link the units of chemical information within nucleic acids. The formation of a phosphodiester linkage by condensation of phosphate and hydroxyl termini of DNA requires activation of the phosphate toward nucleophilic displacement and proper placement of the hydroxyl for nucleophilic attack on the activated phosphate in competition with water. Sequence information is transferred when a nucleic acid template promotes this condensation reaction by the sequence specific formation of a complex in which condensing functionalities are juxtaposed. This thesis describes investigations of the sequence specific formation of phosphodiester linkages by the assembly of triple-helical complexes. In the first part of Chapter I, sequence specific recognition of double stranded DNA by triple helix formation is reviewed. Structural features of nucleic acid triple helices, the sequence specificity of their formation, and functions associated with them are considered in this review. In the second part of Chapter I, literature regarding template-directed formation of phosphodiesters in aqueous solution is reviewed. Chapter 2 describes investigations of the sequence specific formation of a phosphodiester linkage between pyrimidine oligodeoxyribonucleotides using a double-stranded DNA template to juxtapose their termini in a triple helix. Several approaches to activation of the condensing phosphate were explored. The most effective of these was activation in situ with the condensing agent N -cyanoimidazole with which condensation yields greater than 80% could be obtained. The reaction was directed by the double stranded template and could be shown to form a 3',5' phosphodiester linkage between the two oligodeoxyribonucleotides. A single mismatch in one of the condensing oligodeoxyribonucleotides at the condensing terminus resulted in at least a 25 to 60 fold decrease in the rate of the reaction. The relevance of this reaction to the detection of sequences, nucleic acid catalysts, and the evolution of template-directed information transfer is discussed. Chapter 3 describes the nonenzymatic sequence specific ligation of blunt-ended duplex DNA by triple helix formation. Using a pyrimidine oligodeoxyribonucleotide as a template and N-cyanoimidazole as a condensing agent, a double-stranded plasmid with homopurine tracts at one 3' terminus and one 5' terminus could be covalently circularized In yields exceeding 50%. Ligation on both strands was demonstrated in some of the circularized product. Ligation of duplexes with homopurine tracts at their 3' termini was directed by a pyrimidine oligodeoxyribonucleotide of two segments joined 3' to 3' through an abasic linker, creating a duplex of the sequence type 5'-(purine)_m(pyrimidine)_n-3'. The linkages formed in the ligation reaction were demonstrated to be substrates for a restriction endonuclease, identifying them as phosphodiesters. The sequence specificity of these reactions is not accessible by enzymatic ligation of double-stranded DNA.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Unknown, Unknown
Defense Date:17 April 1992
Record Number:CaltechTHESIS:09072011-081106540
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6652
Deposited By: Dan Anguka
Deposited On:07 Sep 2011 16:31
Last Modified:09 Nov 2022 19:20

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