Targeting the G‐Triplex Intermediate in G‐Quadruplex DNA Folding for Potential Chemoprevention Applications

The mechanisms underlying genetic instabilities that lead to events such cancer‐associated chromosomal deletions, translocations, and rearrangements have yet to be fully clarified, but it has recently been shown that DNA sequences with the capacity to adopt alternatively structured DNA ( i.e. non‐B DNA) often co‐localize with hotspots of genetic instability. We hypothesize that the genetic instability associated with non‐B DNA‐forming sequences is a function of the stability of the non‐B DNA structures that these sequences form, and that ligands that destabilize these structures will decrease the DNA damage and cancer‐associated genomic instability associated with these sequences. Our approach to identify non‐B DNA destabilizing ligands exploits recent findings that specific non‐B DNA structures (G4‐DNA and H‐DNA) fold via long‐lived ( i.e. seconds) intermediates. We propose that ligands that target these intermediates will effectively destabilize these non‐B DNA structures by inhibiting their folding. We used CD spectroscopy, UV thermal‐difference spectroscopy, and Tm measurements to characterize the topology and thermal stability of the folding intermediates (G‐triplexes) for a variety of truncated G4‐DNA sequences. We also carried out a virtual screen in order to identify ligands that bind to a structurally well‐characterized G‐triplex, the truncated thrombin‐binding aptamer (TBA) G4 DNA. G‐triplex formation is a general phenomenon for a wide range of truncated G4 DNA sequences. We examined 16 different variants of the G ≥2 T 1–4 G ≥2 T 1–4 G ≥2 sequence as well as truncated versions of the TBA and human telomeric G4 DNA in both forward and reverse permutations. All of these sequences can adopt G‐triplex structures. However, we note that the number of nucleotides in the loop and sequence direction affect G‐triplex topology. Sequences with longer G‐tracks tends to form parallel G‐triplex, as do sequences with fewer loop residues. Permutation of the direction of the truncated G4 DNA sequence also affect G‐triplex folding topology. Environmental effects on topology were also noted, with divalent metal ions (Mg 2+ and Ca 2+ ) favoring parallel topologies. Virtual screening against the antiparallel truncated TBA G‐triplex reveals a number of viable ligand binding sites that are predicted to interfere with folding to G4 DNA. G‐triplex folding intermediates of G4 DNA structures are promising targets for small molecule G4 DNA destabilizing ligands. The effect of the destabilization of G4 DNA on the genetic instability associated with these sequences in human cells will provide crucial evidence for the role of structural stability in genetic instability and potential lead chemopreventive agents. Support or Funding Information We gratefully acknowledge funding from CPRIT HIHR Grant RP160852 Space‐fill model of top hit from virtual screening for TBA13 G‐triplex ligandsG‐triplex structures are intermediates in the folding of G4‐DNA, and targeting these structures with small molecule ligands may result in the destabilization of the corresponding G4‐DNA.Anti‐parallel G‐triplex CD spectrumParallel G‐triplex CD spectrumSequence and salt effects on G‐triplex CD spectrumDirectional effects on G‐triplex CD spectrumEvidence for multiple G‐triplex conformationsTable of highest scoring hits for screening of G‐triplex ligandsZinc ID Binding Energy (kcal/mol)19325434 −7.5 9828011 −7.1 6885996 −7.0 97463340 −7.0 17836980 −7.0 42783800 −7.0 4004793 −7.0 371296 −6.9 57612466 −6.9 70631631 −6.9This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .