Details

Oligonucleotide-directed cleavage of single- and double-stranded DNA by double and triple helix formation

by Griffin, Linda Chu-Li

Abstract (Summary)
Part I Oligonucleotide-Directed Cleavage of Single-Stranded DNA by Double Helix Formation Chapter 1: Sequence-Specific Cleavage of Single-Stranded DNA with Oligonucleotide-EDTAâ¢Fe(II): Study of Reaction Conditions The machine synthesis of a DNA hybridization probe 19-nucleotides in length, equipped with the metal chelator EDTA 1 has the sequence 5'-GCAAGGCGAT*TAAGTTGGG-3', which is complementary to a 19-nucleotide sequence in M13mp7 (+) strand phage DNA. In the presence of Fe(II), O2, and dithiothreitol, oligonucleotide 1 affords specific cleavage (0°C, pH 7.4, 20 hours) at its complementary sequence in the 7214 base M13mp7 (+) strand. Cleavage occurs over a range of 17 nucleotides at the site of hybridization of 1. No other cleavage sites are observed in the 7214 base strand. Optimum cleavage conditions occur at concentrations of 0.25-1 µM T* oligonucleotide, 20 µM Fe(II), and 50-1000 mM NaCl. Optimum pH is 7.4 (25 mM tris-acetate buffer). The optimum cleavage time is between 16-24 hours at 0°C. The melting temperature (Tm) for T* oligonucleotide 1 and its 19-nucleotide complement under reaction conditions is 54.5°C, 3.5°C lower than the same duplex without T* (Tm=58°C). Part II Oligonucleotide-Directed Cleavage of Double-Stranded DNA by Triple Helix Formation Chapter 2: Recognition of Thymineâ¢Adenine Base Pairs by Guanine in a Pyrimidine Triple Helix Motif Oligonucleotide recognition offers a powerful chemical approach for the sequence-specific binding of double helical DNA. In the pyrimidine-Hoogsteen model, a binding site size of >15 homopurine base pairs affords >30 discrete sequence-specific hydrogen bonds to duplex DNA. Because pyrimidine oligonucleotides limit triple helix formation to homopurine tracts, it is desirable to determine whether oligonucleotides can be used to bind all four base pairs of DNA. A general solution would allow targeting of oligonucleotides (or their analogs) to any given sequence in the human genome. A study of 20 base triplets reveals that the triple-helix can be extended from homopurine to mixed sequences. Guanine contained within a pyrimidine oligonucleotide specifically recognizes thymineâ¢adenine base pairs in duplex DNA. Such specificity allows binding at mixed sites in SV40 and HIV DNA. Chapter 3: Recognition of All Four Base Pairs of Duplex DNA by Triple Helix Formation ⢠Design of Pyrimidine Specific Bases Oligonucleotide recognition offers a powerful chemical approach for the sequence-specific binding of double helical DNA. In the pyrimidine-Hoogsteen model, a binding site size of >15 homopurine base pairs affords >30 discrete sequence-specific hydrogen bonds to duplex DNA. Because pyrimidine oligonucleotides limit triple helix formation to homopurine tracts, it is desirable to determine whether oligonucleotides can be used to bind all four base pairs of DNA. A general solution would allow targeting of oligonucleotides (or their analogs) to any given sequence in the human genome. The novel base 4-(3-benzamido)phenylimidazole specifically recognizes pyrimidineâ¢purine base pairs over purineâ¢pyrimidine base pairs. Such specificity allows binding at an 18 base pairs site in SV40 DNA (pH 7.4, 40°C) containing all four base pairs.
Bibliographical Information:

Advisor:Peter B. Dervan

School:California Institute of Technology

School Location:USA - California

Source Type:Master's Thesis

Keywords:chemistry

ISBN:

Date of Publication:08/01/1989

© 2009 OpenThesis.org. All Rights Reserved.