Specific Photocleavage Activity of N‐Methylmesoporphyrin IX on G‐Quadruplex DNA Structures

Abstract

G‐rich regions, at which G‐quadruplexes (G4) are proposed to form, are associated with promoter regions within proto‐oncogenes and may play a role in cell proliferation and carcinogenesis. The ability to identify the formation of G4 structures in these regions, including the temporal aspects of G‐quadruplex formation, is essential to establish the role of these structures in transcriptional regulation. We have focused on identifying highly selective G4 photocleavage agents as molecular probes for establishing the location and timing of G4 formation. N‐methylmesoporphyrin IX (NMM) has long been known as a G4 ligand; however, there has been little investigation into its DNA photocleavage activity. Here we report the ability of NMM to act as a highly selective photocleavage agent for specific G4 structures. We first studied the interaction of NMM with the G4 structures formed by DNA oligonucleotides containing the c‐MYC promoter or human telomeric (hTel) sequences formed in the presence of either Na+ or K+‐containing buffers using a FRET‐based Tm assay and CD spectroscopy. We employed a fluorescence‐based solution assay for G4 cleavage to establish that NMM effects photocleavage (420 nm) of c‐MYC more efficiently than the hTel G4 under both Na+ and K+ conditions, and effects photocleavage of c‐MYC G4 even under white light irradiation. Using denaturing polyacrylamide gel electrophoresis (PAGE), we confirmed this selective photocleavage and demonstrate that cleavage occurs at specific guanine residues of the c‐MYC G4. In order to determine if the photocleavage proceeds through the generation of reactive oxygen species like singlet oxygen, we carried out photocleavage reactions in the presence of H2O and D2O, and employed a turn‐on fluorescent singlet oxygen sensor to detect 1O2 generation. We find that NMM acts to destabilize specific G4 topologies, but oligonucleotides annealed in the presence of NMM form alternative topologies that are thermally stabilized. NMM photocleavage is highly selective for G4 DNA versus duplex DNA, and the K+‐stabilized c‐MYC G4 is preferentially cleaved compared to the Na+‐stabilized structure and the hTel G4 in either Na+ or K+. PAGE analysis indicates that NMM photocleavage of c‐MYC G4 occurs preferentially at the 3′ G‐tetrad residues. This photocleavage does not appear to be due to 1O2. These results indicate that NMM may be a useful tool for elucidating the role of promoter G‐quadruplex structures.Support or Funding InformationSupport for this work by the Cancer Prevention and Research Institute of Texas (RP160852) and Texas State University is gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.