Conformational Switching of Human G-Quadruplexed DNA with Ligand Binding under Molecular Crowding Conditions

Abstract

Formation of quadruplexed DNA (qDNA) within telomeric DNA has the potential to effectively inhibit the activity of telomerase, a key enzyme in tumorigenesis. The effects of molecular crowding (MC) within the cell can affect the folding of qDNA and modulate telomerase activity. Furthermore, MC can affect the ability of ligand binding to qDNA in promotion of the folded quadruplex conformation. Thus it is important that quadruplex-ligand binding be evaluated under more physiologically relevant conditions. We have examined the effects of MC on the association of N-methyl mesoporphyrin IX (NMM), a ligand which selectively binds to G-quadruplexed DNA, on the conformation of (TTAGGG)4 (HT4) and (TTAGGG)8 (HT8) human telomeric sequences. We and others have shown that in K+ solutions, HT4 forms a hybrid conformation, whereas the HT8 sequence forms two consecutive hybrid quadruplexes. With addition of crowding mimetics (Ficoll 70; Ficoll 400; and PEG 200 (20 and 40% w/v)) to the HT4 sequence, CD studies show a conformational switch to an antiparallel structure in Ficoll 400 (40% w/v) and a parallel conformation in PEG 200 (40% w/v), with no conformational changes observed in Ficoll 70. For HT8, a similar effect was observed, but in addition an anti-parallel conformation was obtained in Ficoll 70 (40% w/v), which suggests an enhanced sensitivity to dehydration for the HT8 sequence. Additionally, thermal CD melting studies showed stabilization of HT8 conformation in Ficoll compared with HT4. With binding of NMM to HT4 in 20% (w/v) crowding mimetics a shift to the parallel conformation was observed. However, the antiparallel (more dehydrated) conformation for HT8 is promoted in Ficoll 400. We plan more detailed environmental studies of NMM binding to qDNA under crowding conditions using fluorescence spectroscopy.(Supported by NIH 5SC3 GM095437)