Not all G-quadruplexes are created equally: an investigation of the structural polymorphism of the c-Myc G-quadruplex-forming sequence and its interaction with the porphyrin TMPyP4

Abstract

G-quadruplexes, DNA tertiary structures highly localized to functionally important sites within the human genome, have emerged as important new drug targets. The putative G-quadruplex-forming sequence (Pu27) in the NHE-III1 promoter region of the c-Myc gene is of particular interest as stabilization of this G-quadruplex with TMPyP4 has been shown to repress c-Myc transcription. In this study, we examine the Pu27 G-quadruplex-forming sequence and its interaction with TMPyP4. We report that the Pu27 sequence exists as a heterogeneous mixture of monomeric and higher-order G-quadruplex species in vitro and that this mixture can be partially resolved by size exclusion chromatography (SEC) separation. Within this ensemble of configurations, the equilibrium can be altered by modifying the buffer composition, annealing procedure, and dialysis protocol thereby affecting the distribution of G-quadruplex species formed. TMPyP4 was found to bind preferentially to higher-order G-quadruplex species suggesting the possibility of stabilization of the junctions of the c-Myc G-quadruplex multimers by porphyrin end-stacking. We also examined four modified c-Myc sequences that have been previously reported and found a narrower distribution of quadruplex configurations compared to the parent Pu27 sequence. We could not definitively conclude whether these G-quadruplex structures were selected from the original ensemble or if they are new G-quadruplex structures. Since these sequences differ considerably from the wild-type promoter sequence, it is unclear whether their structures have any actual biological relevance. Additional studies are needed to examine how the polymorphic nature of G-quadruplexes affects the interpretation of in vitro data for c-Myc and other G-quadruplexes. The findings reported here demonstrate that experimental conditions contribute significantly to G-quadruplex formation and should be carefully considered, controlled, and reported in detail.

Nucleic acid clamp-mediated recognition and stabilization of the physiologically relevant MYC promoter G-quadruplex

“…A number of equilibrating G4s have been described in the MYC promoter (55), most famously two unique structures with loops of 1, 2 and 1 nucleotide in the 5′-3′ direction, and a few studies highlighting a less frequent 1–6–1 loop isomer (G4 1–5 ) (54). From linear DNA, this 1:2:1 loop isomer structure forms from the second through the fifth run of continuous guanines as demonstrated by both DMS and NMR (G4 2–5 ) (52,53).…”

Hydrodynamic Models of G-Quadruplex Structures

“…For long sequences it is essential to purchase gel purified material to minimize sample heterogeneity. In sample preparation, the annealing protocol used can play an important role in G-quadruplex formation (Le, et al, 2012). Samples at working concentrations of 2 – 10 μM (strand) are annealed by imersion into boiling water contained in a 1 L beaker for 5 minutes, after which the beaker is removed from the heat source and allowed to slow cool to room temperature overnight (12 – 24 hr).…”

“…In contrast, an antiparallel basket quadruplex structure in Na + solution determined by NMR spectroscopy predicted hydrodynamic properties fully consistent with experimentally determined values. Recently we reported a more extensive validation of HYDROPRO predictions for a variety of quadruplex structures obtained by NMR (Le, Buscaglia, Dean, Chaires, & Trent, 2013; Le, Dean, Buscaglia, Chaires, & Trent, 2014; Le et al, 2012; Miller et al, 2011). We have also used this approach to access higher-order quadruplex structures (Petraccone, Garbett, Chaires, & Trent, 2010; Petraccone et al, 2011; Petraccone, Trent, & Chaires, 2008).…”

Metalloporphyrin/G-quadruplexes: From basic properties to practical applications

“…Similarly to native chloroperoxidase, CPO, or cytochrome P450, the hemin/G-quadruplex can also catalyze a two-electron oxidation processes that are characterized by an oxygen atom transfer to the substrate [46]. Examples of these reactions are outlined in Scheme 1, Equations 1-3, where H 2 O 2 stimulates the oxidation of thioanisole, (8), to thioanisole sulfoxide, (9), Equation 1, the epoxidation of styrene, (10), to styrene oxide, (11), (with the phenylacetaldehyde coproduct, (12)), Equation 2, and the oxygen transfer to indole, (13), followed by the C-C coupling of the oxo-indole radical cation, (14), intermediate to indigo, (15), Equation 3.…”

“…For instance, the cationic Mn(III)-tetramethylpyridinium porphyrin, Mn(III)-TMPyP4, (22), bound to the G-quadruplex nanostructure was found to catalyze oxygen transfer reactions to olefins using persulfate, (23), as an oxygen source, to yield epoxides [50], Scheme 3, Equation 12. Specifically, the epoxidation of carbamazepine, (24), to carbamazepine-10,11-oxide, (25), Equation 13, was examined using different Mn(III)-TMPyP4-functiona lized G-quadruplexes as catalysts [50], Fig.…”