Structural Differences at Quadruplex‐Duplex Interfaces Enable Ligand‐Induced Topological Transitions

Abstract

Quadruplex‐duplex (QD) junctions, which represent unique structural motifs of both biological and technological significance, have been shown to constitute high‐affinity binding sites for various ligands. A QD hybrid construct based on a human telomeric sequence, which harbors a duplex stem‐loop in place of a short lateral loop, is structurally characterized by NMR. It folds into two major species with a (3+1) hybrid and a chair‐type (2+2) antiparallel quadruplex domain coexisting in a K+ buffer solution. The antiparallel species is stabilized by an unusual capping structure involving a thymine and protonated adenine base AH+ of the lateral loop facing the hairpin duplex to form a T·AH+·G·C quartet with the interfacial G·C base pair at neutral pH. Addition and binding of Phen‐DC3 to the QD hybrid mixture by its partial intercalation at corresponding QD junctions leads to a topological transition with exclusive formation of the (3+1) hybrid fold. In agreement with the available experimental data, such an unprecedented discrimination of QD junctions by a ligand can be rationalized following an induced fit mechanism.

A pH‐Responsive Topological Switch Based on a DNA Quadruplex‐Duplex Hybrid

“…Such an observation strongly suggests that the major structure at pH 8 adopts a hybrid-2 topology, considering recent studies on TelQD at pH 7 which demonstrated a dynamic equilibrium between chair-type and hybrid-2 species. [19] This assumption is corroborated by matching NOE cross-peaks in superimposed NOESY spectral regions of TelQD at pH 8 and of loop-modified TelQD31 (Table 1), previously shown to exclusively fold into the hybrid-2 topology by shortening of its last 3-nt intervening sequence to a 1-nt T loop (Figure 3A). [19] Again, the hybrid-2 structure of TelQD, characterized by a -(llp) loop progression frequently found for human telomeric sequences, features a second lateral loop following the duplex stem-loop.…”

“…[19] This assumption is corroborated by matching NOE cross-peaks in superimposed NOESY spectral regions of TelQD at pH 8 and of loop-modified TelQD31 (Table 1), previously shown to exclusively fold into the hybrid-2 topology by shortening of its last 3-nt intervening sequence to a 1-nt T loop (Figure 3A). [19] Again, the hybrid-2 structure of TelQD, characterized by a -(llp) loop progression frequently found for human telomeric sequences, features a second lateral loop following the duplex stem-loop. However, in contrast to the chair topology, the third intervening sequence forms a TTA propeller loop with no additional interactions of its loop bases with the 3'-tetrad and the interfacial base pair (Figure 3B).…”

“…[21][22][23] Based on these findings, the G-quadruplex is suggested to adopt a chairtype topology with a counter-clockwise lateral loop progression termed-(lll) according to the notation introduced by Webba da Silva. [24] In analogy to the bromo-substituted 32Br TelQD employed in a recent study and shown to fold into a single topology (Table 1), [19] a most downfield-shifted resonance at 14.34 ppm showing a strong NOE contact to A30 H2 can unambiguously be identified through a residue-specific 15 N-labeling to be an imino proton of N1-protonated residue A30 positioned within the third lateral loop (Figure S3). Other fingerprints for adenine N1 protonation come from downfield-shifted A30 amino protons (Figure S3) and from a characteristically upfield-shifted A30 C2 resonance (Figure S2).…”

“…Structure calculations using NMR restraints resulted in a chair-type (2 + 2) antiparallel fold for TelQD, being topologically equivalent to the structure that has recently been determined for modified 32Br TelQD at pH 7 (Table S1, Figure 2). [19] The first Watson-Crick base pair C4•G18 stacks onto the upper G-tetrad, forming a defined QD interface. All three lateral loops progress in a counter-clockwise direction with the duplex stem-loop bridging a wide groove.…”

“…[17,18] In a recent study, a QD hybrid termed TelQD was used as a receptor for ligand binding. [19] TelQD derives from a human telomeric sequence known for its high conformational plasticity [20] but with the first intervening TTA trinucleotide substituted by a putative hairpin duplex-forming domain comprising six Watson-Crick base pairs. At neutral pH, the sequence was demonstrated to fold into two major topologies that could be selected and structurally characterized in detail by introducing specific modifications.…”