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

Citation

Griffin, Linda Chu-Li (1990) Oligonucleotide-directed cleavage of single- and double-stranded DNA by double and triple helix formation. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06072007-095616

Abstract

Part I

Oligonucleotide-Directed Cleavage of Single-Stranded DNA by Double Helix Formation

Chapter 1: Sequence-Specific Cleavage of Single-Stranded DNA with Oligonucleotide-EDTA•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 Thymine•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 thymine•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 pyrimidine•purine base pairs over purine•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.

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:1 August 1989
Record Number:CaltechETD:etd-06072007-095616
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-06072007-095616
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2504
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:20 Jun 2007
Last Modified:26 Dec 2012 02:52

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