Guanines are a quartet's best friend: impact of base substitutions on the kinetics and stability of tetramolecular quadruplexes

Abstract

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well-defined non-guanine base quartets in a number of NMR and X-ray structures, our data suggest that most non-guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G-quartets. Two notable exceptions were found with 8-bromo-guanine (X) and 6-methyl-isoxanthopterin (P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 5' end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/dissociation properties.

Figure 1.

A G-quartet and bases tested here. Top: Chemical formulae of the bases tested here. I = Inosine; 6 = 6-thioguanine; 7 = 7-deazaguanine; 8 = 8-oxoguanine; P = 6MI = 6-methylisoxanthopterin; Q = 3MI = 3-methylisoxanthopterin; M = 6-methyl guanine; X = 8-bromo-guanine. Formula of the regular DNA and RNA bases (A, C, T, U) are not shown. Lower left: Cycling arrangement of four guanine into a G-quartet. Altering the NH₂ group on position 2 will alter the external ring of H-bonds, whereas modifications of the 8-position should leave the H-bond pattern unaffected. Altering the carbonyl group at position 6 not only perturbs the central ring of H-bonds, but may also interfere with cation coordination. Lower right: Scheme of the general folding topology of the TG₄T tetramolecular parallel quadruplex.

Figure 2.

Analysis of the association curves. (A) Representative example of an isothermal renaturation experiment. Formation of a quadruplex with the inosine-containing oligonucleotide TIGGGGT (panel A) (15 µM strand concentration, at 4°C, 0.11 M K⁺). Raw absorbance was recorded simultaneously at two wavelengths (240 nm: blue circles and 295 nm: red inverted triangles). The fitted curves (full lines) are nearly indistinguishable from the experimental data. Fitted k_on values are provided for each curve. (B) Example of a dual wavelength parametric test for TGGIGGT (identical conditions as in Panel A). In this example, absorbance at 240 nm (left Y-scale, blue circles) and absorbance at 295 nm (right Y-scale, red triangles) are plotted versus absorbance at 273 nm. Other examples are provided in Supplementary Data. (C) Relative association constant (k_on) as compared to TG₅T for oligomers in which the first guanine has been replaced by another base (code as in Figure 1). Data obtained in K⁺ (black), Na⁺ (blue) or (red). ‘-’ corresponds to TG₄T. Note that these relative values have been normalized for each cation compared to the unmodified oligonucleotide. k_on values in K⁺ and Na⁺ are respectively 2000 and 10 times higher than in . (D) Association constants in Na⁺: Effect of a single guanine substitution on k_on (corresponding curves in K⁺ or are provided as Supplementary Data). The position of the substitution is indicated on the X-axis: position 1 corresponds to the first guanine (5′ side), position 5 to the last guanine (3′ side). The relative k_on values (±SD) for the formation of the TG₅T variants are indicated on the left Y-axis (k_on for the unmodified TG₅T sequence under the same conditions = 1, corresponding to a horizontal dotted line). Absolute values are shown on the right Y-axis. Experiments were performed in 0.11 M Na⁺ at 3 ± 1°C. Note the semi-log scale: for many mutants, a single substitution may lead a tremendous decrease in k_on. Only a few cases lead to a higher k_on than TG₅T, for example TXGGGGT (blue squares).

Figure 3.

6MI (‘P’) leads to faster association (A) Putative quartet formed by ‘P’ (6MI, or 6-methyl isoxanthopterin). (B) Isothermal quadruplex formation experiments for TGGGGGT (red) TPGGGGT (black), TGPGGGT (blue) and TGGPGGT (green) in 0.11 M Na⁺ at 2.9°C deduced from absorbance measurements at 295 nm. All strand concentrations were identical (10 µM). The fraction of unfolded oligonucleotide is plotted versus time. Note that the fluorescence emission of 6MI is quenched by adjacent purines, preventing us from following kinetics by fluorescence spectroscopy (25). (C) Examples of CD spectra for the quadruplexes containing the base P at 4°C in 0.11 M K⁺. Experimental conditions provided in Supplementary Data. (D) Gel experiments showing that quadruplex formation is faster for TGPGGGT rather than for TGGGGGT (right) in 0.11 M Na⁺ at 4°C. All strand concentrations were identical (80 µM). Size markers (single-stranded oligothymidylate T₂₁, T₉ and T₆) are provided. Time-dependent formation of the tetramolecular quadruplex leads to the apparition of a retarded band. Its mobility is close to the mobility of the reference TG₅T quadruplex.

Figure 4.

Thermal melting experiments. (A) Representative example of a thermal denaturation experiment. Melting of the quadruplex formed with the TIGGGT oligonucleotide may be followed at 240 nm (blue circles; left Y-scale) and 295 nm (red triangles; right Y-scale). The quadruplex was preformed at 4°C at high concentration (300 µM). T_1/2 values determined from the absorbance at 240 and 295 nm are identical (67°C). (B) Comparison of the thermal stability of three different quadruplexes: TGGGXT (open circles), TGGGGT (red diamonds) and TXGGGT (inverted triangles) in 0.11 M Na⁺. Raw absorbance data has been converted to fraction unfolded, using linear baseline assumptions for single strands and quadruplexes. Most melting curves are monophasic; a few samples give a pretransition illustrated here for TXGGGT (black triangles). Temperature gradient: 0.48°C/min. (C and D) Effect of a single guanine substitution on the apparent melting temperature of TG₅T (panel C) and TG₄T (panel D) variants. The position of the substitution is indicated on the X-axis: position 1 corresponds to the first guanine (5′ side), position 5 (or 4 in the case of TG₄T variants) to the last guanine (3′ side). Here, 12 different replacements were tested, each corresponding to a different symbol. The T_1/2 values are provided with a ±1°C accuracy. Experiments were performed in 0.11 M Na⁺ using a temperature gradient of 0.48°C/min. In some cases, no melting of the quadruplex was observed: T_1/2 was arbitrarily fixed at ≥90°C.

Figure 5.

Effect of guanine substitution on the mean number of ions present in the quadruplexes: MS analysis. ESI-MS spectra obtained in gentle condition help in understanding the formation of these tetramolecular structures, not only by providing the strand stoichiometry but also an unambiguous determination of the number of contributing structural cations. The position of each substitution is indicated on the X-axis. The mean number of ammonium ions (N_NH4) present in the complexes is obtained from equation: N_NH4 = [4 × I(G4_{with 4NH4}) + 3 × I(G4_{with 3NH4}) + 2 × I(G4_{with 2NH4}) + 1 × I(G4_{with 1NH4})]/Sum[I(G4all)] where I(G4_n) are the relative intensities of the quadruplex with different number of ammonium ions. Note that the P and Q modifications were not shown here.