Electrospray ionization mass spectrometry analysis of covalent and non-covalent DNA complexes

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Title: Electrospray ionization mass spectrometry analysis of covalent and non-covalent DNA complexes
Author: Pierce, Sarah Elizabeth
Abstract: The covalent and non -covalent interactions between DNA and external ligands and between DNA and itself are critical for cellular function . An increased knowledge of these interactions can be used for the development of disease -fighting agents , specifically anti -cancer drugs with improved sensitivity and specificity for tumor cells . Electrospray ionization mass spectrometry (ESI -MS ) is useful in the screening and characterization of the interactions involving nucleic acids given the speed and small sample sizes that can be analyzed . In this dissertation , ESI -MS is used to characterize covalent and non -covalent interactions involving DNA to assist in determining how these interactions can lead to better therapeutics . The non -covalent binding of ligands to quadruplex oligonucleotides is discussed first . Pyrrole inosine ligands , which bind to guanine bases , were found to interact with both quadruplexes and with guanine rich oligonucleotides without a quadruplex structure . While those interactions were specific with guanine , novel platinum complexes were found to form specific interactions with quadruplex structures themselves as the size of the ligands matched the size of a guanine quartet . This allowed the ligands to end -stack with quadruplexes with large thymine -rich loops between guanine -rich regions . The non -covalent and covalent interactions between ligands and other DNA structures were also studied . The non -covalent binding of anthracycline ligands to mismatched DNA hairpins was probed . The analysis of solutions of approximately equimolar ligand and oligonucleotide indicated preferential binding to the mismatched sequences . Diazirdinyl benzoquinone crosslinkers , including the clinically studied RH1 and an analogue of RH1 , were reacted with a variety of duplex oligonucleotides . The complexes were observed by LC -MS and dissociated using both CID and IRMPD to determine the sites of crosslinking . It was determined that both ligands could form interstrand crosslinks in DNA with 5’ -GNC or 5’ -GNNC sequences . The RH1 analogue , with a bulky phenyl group , formed fewer crosslinks than RH1 . In addition to studying DNA /ligand interactions , the interactions between oligonucleotides were also probed . Oligonucleotides containing non -standard isoguanine repeats were annealed in the presence of various cations to determine how those cations would affect the resulting secondary structures . In most cases , isoguanine containing strands formed pentaplexes rather than quadruplexes , which were observed for strands containing guanine bases .
URI: http : / /hdl .handle .net /2152 /ETD -UT -2010 -05 -762
Date: 2010-09-01

Citation

Electrospray ionization mass spectrometry analysis of covalent and non-covalent DNA complexes. Doctoral dissertation, University of Texas at Austin. Available electronically from http : / /hdl .handle .net /2152 /ETD -UT -2010 -05 -762 .

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