Impact of a Snap-Back Loop on Stability and Ligand Binding to a Parallel G-Quadruplex

Abstract

Various genomic DNA sequences including a MYC promoter sequence are amenable to the formation of a G-quadruplex featuring a snap-back loop with the incorporation of a 3′-terminal guanine into the quadruplex core. To evaluate relative stabilities and ligand binding in more detail, optical, microcalorimetric, and NMR structural studies were performed on both a minimal mutant sequence Pu22T that exclusively folds into a snap-back loop quadruplex and a parallel MYC quadruplex proposed to be the most relevant fold of the MYC promoter in a cellular environment. Similar thermal stabilities for Pu22T and MYC suggest the coexistence of both quadruplexes when derived from a sequence able to fold into both topologies. Isothermal titration calorimetry indicates a mostly identical enthalpy-driven strong binding of an indoloquinoline ligand but with a reduced number of high-affinity binding sites in Pu22T in line with a novel modified FRET competitive melting assay. Corroborated by fluorescence titrations using 2-aminopurine as a fluorescent probe, NMR chemical shift footprints show binding of the ligand at the Pu22T 5′-outer tetrad with the formation of a binding pocket. On the other hand, steric restrictions due to the snap-back loop severely restrict ligand stacking on the 3′-outer tetrad of Pu22T.

A Thermodynamic Perspective on Potential G‐Quadruplex Structures as Silencer Elements in the MYC Promoter

“…Their symmetric profiles give no obvious indication of superimposed independent meltinge vents. Of note, meltingt emperatures given by the maxima of the DSC curves agree within AE 2 8Cw ith previously reported values from CD meltinge xperiments under the same buffer conditions for MYC-D1,6[1.2.1] and MYC del -D1[1.1.1] [22] as well as from as eparate CD-based T m determination for MYC-D1,6[1.2.1] (data not shown). After proper baseline corrections, calorimetric enthalpies DH o cal of G4 folding were obtainedb yi ntegration of the meltingt ransition.…”

“…Consequently,s uch snap-back loop quadruplexes have been targeted by variousl igands with sometimes promising binding affinities. [13,22,30] To getm ore insighti nto the impact of additional modifications on the stability of the snapback loop structure, we shortened its two-tetrad bridging central propeller loop by deletiono ft wo nucleotides to give MYC del -D1[1.1.1]o ra dded one and two additional 3'-terminal T residues in MYC-D1[1.3.1]T and MYC-D1[1.3.1]TT,r espectively. With a T m of 81 8C, MYC del -D1[1.1.1] shows the highestt hermal stabilityo fa ll quadruplexes, elevated by 6a nd 11 8Cc ompared to MYC-D1,6 and MYC-D1[1.3.1]w ith its longer two-tetrad bridging central propeller loop.…”

Quadruplex–Duplex Junction: A High‐Affinity Binding Site for Indoloquinoline Ligands

“…For Myc3l-dup5 an additional slow-exchanging imino signal of ag uanineb ase within the snap-back loop is observed, in line with its participation in ac apping base triad, as reported previously. [5,37] Thed etection of Watson-Crick imino resonances between 12-14 ppm demonstrate complementary base pairing in the stem-loop structure for the 3'-a nd 5'-flanking sequences. Alli minosi nvolved in Watson-Crick hydrogen bonding of the duplex regionh ave been successfully assigned except for the imino signalo ft he AT base pair adjacent to the T 3 hairpin loop and, in case of Myc-dup3,f or the imino resonance of the GC base pair at the Q-D junction, likely due to its fast exchange with solvent.…”

“…Myc3l-dup5 is based on the quadruplex Myc3l that carriesa na dditional 3'-extension together with ac entral two-nucleotide deletion and was previously shownt o fold into as nap-back loop structure. Because the snap-back loop spans the quadruplex 3'-face and effectively prevents the ligand from bindinga ti ts 3'-tetrad, [5,37] elimination of an additional putative binding site should provide for ab etter resolved thermogram.O nt he other hand, this variant is expected to closely mimic PIQ binding at the Myc-dup5 5'-face with its duplex extension because binding oppositet he snap-back loop should be essentially unperturbed (see below). UV and CD meltingd ata of Myc3l-dup5 are also summarized in Table 1.…”

Structure of a DNA G-Quadruplex Related to Osteoporosis with a G-A Bulge Forming a Pseudo-loop

“…Stacking of A15 on G-quartet III and dynamic residues A9 and G14 can hinder the access and stacking of the potential ligand onto the 3′-G-quartet ( Figure 3 and Figure S7 ). Although steric restrictions can prevent the effective binding of a ligand to one of the G-quartets, they can as well be used as a tool with which we direct the ligand to other high-affinity binding sites on the other side of the G-quadruplex core [ 54 ]. Next to unique loops, targeting the grooves represents one of the most promising binding modes to enhance the selectivity of G-quadruplex ligands [ 55 , 56 , 57 ].…”