Spectroscopic, Gel Electrophoretic, and Surface Plasmon Resonance Characterization of G‐Triplex DNA Formation

Abstract

G‐triplex DNA is a recently discovered class of non‐canonical DNA structures related to G‐quadruplex DNA (G4). Originally identified as a folding intermediate in the formation of certain G4, G‐triplex DNA structures can be formed by oligonucleotides corresponding to truncated G4 sequences. Here we present a study of the sequence and environmental effects on G‐triplex formation using spectroscopic, gel electrophoretic, and surface plasmon resonance methods.We examined 16 different variants of the G≥2T1–4G≥2T1–4G≥2 sequence as well as truncated versions of the TBA and human telomeric G4 DNA in both forward and reverse permutations. CD spectroscopy, UV thermal‐difference spectroscopy, CD and UV derived melting temperature measurements, SPR, agarose gel electrophoresis, and native PAGE gels were employed to characterize the topology and thermal stability of these structures in Li+, Na+, K+, Mg2+ and Ca2+ containing buffers. 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 tend 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 noted, with divalent metal ions (Mg2+ and Ca2+) favoring parallel topologies. Using thermal‐difference spectroscopy, we have identified two families of spectra that correlate to G‐triplexes with a parallel topology. Analysis by native PAGE indicates that some sequences may form multiple G‐triplex topologies under certain environmental conditions. Temperature melts show that sequences with longer G‐runs (GGGG > GGG > GG) and shorter T‐loops (TT >TTT > TTTT) form G‐triplex structures that are more thermally stable. Direction of sequence also effects the stability. Environmental conditions such as the presence of different cations have an effect on stability (Ca2+ > K+ > Mg2+ ≥ Na+). Analysis by agarose gel electrophoresis has suggested that some sequences of G‐triplex forming DNA fold into monomeric intramolecular structures, while sequences with long G‐runs form highly oligomeric species. SPR has also been performed in order to probe G‐triplex formation. Immobilized G‐triplex‐forming sequences were probed with short, complementary C‐rich oligonucleotides and the kinetics of hybridization under different buffer conditions provides further insight into the stability and kinetics of G‐triplex formation.The biological relevance of G‐triplex DNA is still not entirely understood. Understanding how sequence effects and environmental conditions affect G‐triplex structure and stability is essential for better studying G‐quadruplex DNA and the biological relevance of G‐triplex DNA.Support or Funding InformationCRPIT HIHR Grant RP160852 and Texas State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.