Sequence specific nonenzymatic ligation of single- and double-stranded DNA by triple helix formation
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 1, sequence specific recognition of double-stranded DNA by triple helix formation is reviewed. In the second part of Chapter 1, 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 these was activation in situ with the condensing agent N-cyanoimidazole with which condensation yields greater than 80% could be obtained. A single mismatch in one of the condensing oligodeoxyribonucleotides resulted in a 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[prime] terminus and one 5[prime] 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[prime] termini was directed by a pyrimidine oligodeoxyribonucleotide of two segments joined 3[prime] to 3[prime] through an abasic linker. The sequence specificity of these reactions is not accessible by enzymatic ligation of double-stranded DNA.
- Research Organization:
- California Inst. of Tech., Pasadena, CA (United States)
- OSTI ID:
- 5709739
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
550400 -- Genetics
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
CATALYSTS
CHEMICAL BONDS
CONFIGURATION
DNA
HELICAL CONFIGURATION
KINETICS
LIGANDS
MOLECULAR BIOLOGY
NUCLEIC ACIDS
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHORUS COMPOUNDS
REACTION KINETICS
SPECIFICITY