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The complex folding energy landscape of DNA G-quadruplexes leads to numerous conformations for this functionally important class of noncanonical DNA structures. A new layer of conformational heterogeneity comes from sequences with different numbers of G-nucleotides in each of the DNA G-strands that form the four-stranded G-quartet core. The mechanisms by which G-quadruplexes transition from one folded conformation to another are currently unknown. To address this question, we studied two different G-quadruplexes, selecting a single conformation by blocking hydrogen bonding with photolabile protection groups. Upon irradiation, the block can be released and the kinetics of re-equilibration to the native conformational equilibrium can be determined by time-resolved NMR. We compared the NMR-derived refolding kinetics with data derived from thermal hysteresis folding kinetic experiments and found excellent agreement. The outlined methodological approach allows separation of K+-induced G-quadruplex formation and subsequent refolding and provides key insight into rate-limiting steps of G-quadruplex conformational dynamics.

We present a rapid and efficient method to generate a family of platinum supramolecular square complexes, including previously inaccessible targets, through the use of ball milling mechanochemistry. This one-pot, two-step process occurs in minutes and enables the synthesis of the squares [Pt4(en)4(N∩N)4][CF3SO3]8(en= ethylenediamine, N∩N = 4,4′-bipyridine derivatives) from commercially available precursor K2PtCl4in good to excellent yields. In contrast, solution-based assembly requires heating the reagents for weeks and gives lower yields. Mechanistic investigations into this remarkable rate acceleration revealed that solution-based assembly (refluxing for days) results in the formation of large oligomeric side-products that are difficult to break down into the desired squares. On the other hand, ball milling in the solid state is rapid and appears to involve smaller intermediates. We examined the binding of the new supramolecular squares to guanine quadruplexes, including oncogene and telomere-associated DNA and RNA sequences. Sub-micromolar binding affinities were obtained by fluorescence displacement assays (FID) and isothermal titration calorimetry (ITC), with binding preference to telomere RNA (TERRA) sequences. ITC showed a 1:1 binding stoichiometry of the metallosquare to TERRA, while the stoichiometry was more complex for telomeric quadruplex DNA and a double-stranded DNA control.