Design of novel bases for recognition of GC base pairs by oligonucleotide-directed triple helix formation

Citation

Koh, Jong Sung (1991) Design of novel bases for recognition of GC base pairs by oligonucleotide-directed triple helix formation. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/9655-mf61. https://resolver.caltech.edu/CaltechETD:etd-11292005-133156

Abstract

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Part I: Triple Helix Formation by Oligonucleotide Analogs on Double-Stranded DNA

Chapter 1: Design of Novel Bases for pH-Independent Recognition of GC Base Pairs by Triple Helix Formation

The ability to design synthetic molecules that bind sequence-specifically to unique sites on human DNA could have major applications in the treatment of genetic, neoplastic, and viral diseases. One powerful approach to sequence-specific binding of double helical DNA is oligonucleotide-directed triple helix formation. Specificity arises from the base triplets ([...] and C+GC) formed by Hoogsteen base pairing of the second pyrimidine strand with the purine strand of the double helix. Because protonation of the N3 is required for cytosine, triple helix formation at rich sequences is limited to a narrow pH range. The novel base 3-methyl-5-amino-7H-pyrazolo(4, 3-d)pyrimidine-7-one (P1) specifically recognizes GC base pairs as selectively and strongly as C+, but with greater affinity and over an extended pH range. Such selectivities allow binding at a 15 base pair site in pDMAG10 DNA (pH 7.8) containing [...] and at a 16 base pair site in pHIV-CAT DNA (pH 7.4, 37[degrees]C) containing [...].

Chapter 2: Extension of Triple Helix Formation. Design of Novel Bases for Recognition of CG Base Pairs

Oligonucleotide recognition offers a powerful chemical approach for the sequence-specific binding of double-helical DNA. Because pyrimidine oligonucleotides limit triple helix formation to homopurine tracts containing AT and GC base pairs, it is desirable to study whether oligonucleotides can be designed to bind to all four base pairs. A general solution would allow targeting of oligonucleotides to any sequence. The novel base 3-methyl-5-amino-7H-pyrazolo(4, 3-d)pyrimidine-7-one (P1) and cytosine (C) moderately recognize CG base pairs. Such specificities allow binding at an 18 base pair site in SV 40 (pH 7.0, 37[degrees]C) DNA containing all four base pairs.

Part II: Design of DNA Cleaving Groups

Chapter 3: Design of New DNA Cleaving Fuctional Groups and Studies of Their DNA Cleaving Mechanisms

The utility of the DNA cleaving molecules is enormous, ranging from the creation of synthetic restriction enzymes for use by molecular biologists to the development of chemotherapeutic agents which may be effective against a variety of neoplastic diseases. We synthesized three compounds: 12 (P3-Ga-His), 19 (P3-Ga-PYML), and 23 (P3-Ga-Phe). Compound 12 shows sequence-specific cleavage in the presence of Cu(II) and dioxygen, while compound 19 shows sequence-specific cleavage in the presence of dithiothreitol and dioxygen. Interestingly, compound 23 shows strong cleavage at a single site in 167 base pair fragment (EcoRI/RsaI) from plasmid pBR322 in the presence of UV light and [beta]-carbonato(trien)cobalt(III)perchlorate complex. The end product analysis of the cleaved oligonucleotide shows 5'-phosphate, 3'-phosphate, and an unknown 3'-product.

Thesis Files