DNA G-quadruplexes are alternative DNA secondary structures formed from guanine-rich nucleotide sequences. Telomeric quadruplexed DNA (qDNA) is known to inhibit elevated telomerase activity, a key enzyme in tumorigenesis. Because of this anticancer implication, the development of quadruplex-stabilizing ligands for use as potential chemotherapeutic agents is of great interest. N-methyl mesoporphyrin IX (NMM) is an anionic porphyrin quadruplex-stabilizing ligand that is selective for binding parallel and mixed-hybrid solution conformations of G-quadruplexes. NMM is therefore a potential conformational probe for quadruplex formation and folding studies. However, little is known about the nature of NMM binding interactions with quadruplex solution conformations. Our studies seek to map the NMM binding site(s) using a series of telomeric DNA sequences labeled with a fluorescent guanine analog (6-Methylisoxanthopterin, 6MI) substituted at various guanine positions along the quadruplex backbone. Decreased 6MI fluorescence intensity signals are sensitive to quadruplex folding and NMM binding through a static-quenching mechanism. Circular dichroism and fluorescence spectroscopy studies reveal that 6MI replacement of specific guanine sites within qDNA results in hindrance of the mixed hybrid to parallel conformational switch required for NMM binding. In contrast, qDNA labeled within the G7-6MI loop region allows NMM binding associated with the mixed hybrid to parallel conformational switch. Analysis of time-resolved 6MI-qDNA fluorescence quenching data in the presence of NMM suggests guanine locations that actively participate in NMM binding through modulation of the mixed hybrid/parallel conformational switch. These insights can ultimately further aid in the design of quadruplex-targeting anticancer drugs.

Conformational binding of N-methyl mesoporphyrin IX with model telomeric DNA G-quadruplex forming sequences

ACS Fall 2021

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