Poster abstracts
Poster number 76 submitted by Janan Alfehaid
Impact of Small Molecules on Telomeric Overhang Accessibility
Janan Alfehaid (Department of Physics, Kent State University, Kent, OH 44242, USA), Kazuo Nagasawa (Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo), John J. Portman (Department of Physics, Kent State University, Kent, OH 44242, USA), Hamza Balci (Department of Physics, Kent State University, Kent, OH 44242, USA)
Abstract:
Human chromosomes terminate in 50-300 nucleotide (nt) long single-stranded telomeric overhangs with repeating d(TTAGGG) sequences which can fold into protective tandem G-quadruplex (GQ) structures. Stabilizing GQs with small molecules inhibits telomerase activity, which has inspired considerable efforts in developing them as anti-cancer therapeutics. However, stacking of tandem GQ structures creates a complex and potentially inaccessible structure for small molecule binding. Here, we employ single-molecule fluorescence microscopy and photobleaching step analysis to quantify binding stoichiometry of a fluorescently-labeled oxazole telomestatin derivative (Cy5-7OTD) to telomeric overhangs that can form 1-6 GQs (30-162 nt long), covering a significant portion of the physiologically relevant range. We find that longer overhangs, on average, accommodate a larger number of ligands; however, at consistently lower stoichiometry than the maximum possible. Also, even in constructs that could accommodate 10-12 ligands, 6 or less ligands bind, suggesting structural compaction of stacked GQs. This was further supported by experiments showing enhanced Cy5-7OTD binding when the inter-GQ spacer was increased from 3 to 9 nt, an effect independently confirmed by ensemble fluorescence enhancement experiments utilizing N-methyl mesoporphyrin IX (NMM). These experimental findings were complemented with a transfer matrix model that describes equilibrium binding of small molecules to a partially ordered telomere. Parameters in the model control the GQ folding stabilities in different regions of the overhang, and cooperativity between neighboring GQ structures and between successive bound small molecule ligands. Together, these findings reveal how overhang length regulates ligand accessibility and inform strategies for development of such molecules as therapeutic agents.
References:
Maleki, P. et al. Nucleic Acids Res. (2017)
Iida, K.; Nagasawa, K. Chem. Rec. (2013)
Zahler, A.M. et al. Nature (1991)
Shin-ya, K. et al. J. Am. Chem. Soc. (2001)
Keywords: G-quadruplex, Telomere, Overhang