Dissertations, Theses, and Capstone Projects

Date of Degree

9-2023

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Lesley Davenport

Committee Members

Nicolas Biais

Tevye Celius

Richard Magliozzo

Brian Williams

Subject Categories

Biochemistry

Keywords

G-quadruplex, spectroscopy, 6-MI, NMM

Abstract

DNA G-quadruplexes are alternative secondary structures formed from guanine rich DNA sequences. G-quadruplexes are found most abundantly at the ends of chromosomes called telomeres, where they can ultimately prevent further tumor growth and progression. Thus, quadruplex interactive agents, or ligands that can bind to and stabilize DNA G-quadruplex structures, are of high interest for their potential chemotherapeutic abilities. One quadruplex interactive agent is the porphyrin N-methyl mesoporphyrin IX (NMM), which has high binding selectivity for quadruplex structures. While the NMM-quadruplex complex in crystallized form has been greatly studied, this complex in solution form is not as well studied. Through circular dichroism (CD), fluorescence spectroscopy, and fluorescence lifetime studies, we were able to examine the structural rearrangements and folding of the quadruplex involved with NMM binding in solution, as well as the NMM binding site on the parallel quadruplex. This has allowed us to evaluate NMM as a conformational probe for quadruplex folding and formation and uncover information about folding pathways of telomeric DNA. For our studies, we have used a series of model human telomeric HT4 oligonucleotides, (TTAGGG)4, where the fluorophore, 6-methylisoxanthopterin (6-MI), is strategically introduced at specific locations along the model telomeric DNA sequence through replacement of key guanosine residues. CD spectroscopic studies show that, upon addition of NMM, a switch of the telomeric conformation, from the mixed-hybrid strand solution conformation promoted in 100 mM KCl, to a parallel strand arrangement, occurs. CD studies also allowed us to identify two groups of 6-MI substitutions that either ‘tolerate’ the conformational switch (Group 1), resulting in NMM binding, or ‘lock’ the conformation in the mixed-hybrid (Group 2), with little NMM interaction. Interestingly, Group 1 (G1-, G4, G7-, and G-10- and G11-6MI-qDNA) members exist predominantly in the syn-rotameric base orientation in the mixed-hybrid conformation. In contrast, guanine bases in locations that form members of Group 2 (G5-, G6-, G9- and G-12-6MI-qDNA) are in the more usual anti-rotameric form. Examination of fluorescence signals associated with NMM binding confirms that Group 1 guanine substitutions allow formation of the required parallel conformation, while Group 2 substitutions show little or weak associations of NMM with the “locked” mixed-hybrid conformation. Examination of 6-MI fluorescence signals with addition of NMM showed that all guanine 6-MI substitutions (Group 1 or 2), with addition of low [NMM], resulted in small enhancements of fluorescence emission intensity. Knowing that the fluorescence of 6-MI is significantly quenched because of base stacking interactions, relief of intensity quenching suggests that the mixed-hybrid participates in a conformational backbone rearrangement that results in a relief of base interactions. Addition of high [NMM] resulted in varying decreases of fluorescence quenching for Group 1 members. Determined Stern-Volmer Constant (Ks) values for these 6-MI quadruplex sequences that allow for the switch suggest that NMM likely has a distribution of guanine residues that contribute to the binding site, likely involving diagonal side loops on the parallel conformation, although 5’-end-stacking in the solution phase may not be ruled out. Fluorescence lifetime studies clearly provided evidence for a static ground state quenching process. Here the 6-MI and NMM must be in physical contact to form a non-fluorescent ground-state complex. In conclusion, studies described here provide more about the nature of the NMM-qDNA complex in solution. First, NMM binds to the parallel conformation. Second, structural rearrangements (strand relaxation/opening) prior to the main mixed hybrid to parallel conformation transition are evident. Third, formation of the parallel conformation requires strands 3 and 4 to effectively unfold from the mixed hybrid quadruplex, change direction and re-fold, now forming new stabilizing interactions, including Hoogsteen base pairing. The parallel form of the quadruplex appears to provide a more condensed, crystal-like structure for NMM binding.

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