Kinetic resolution of bimolecular hybridization versus intramolecular folding in nucleic acids by surface plasmon resonance: application to G-quadruplex/duplex competition in human c-myc promoter

Abstract

The human oncogene c-myc is regulated by G-quadruplex formation within the nuclease hypersensitive element (NHE III(I)) in the c-myc promoter, making the quadruplex a strong anti-cancer target. With respect to this, the competing equilibrium between intramolecular quadruplex folding and bimolecular duplex formation is poorly understood and very few techniques have addressed this problem. We present a method for simultaneously determining the kinetic constants for G-quadruplex folding/unfolding and hybridization in the presence of the complementary strand from a single reaction using an optical biosensor based on surface plasmon resonance (SPR). Using this technique, we demonstrate for the first time that quadruplex formation in the c-myc promoter is favored at low strand concentrations. Our results indicate favorable quadruplex folding (equilibrium folding constant K(F) of 2.09 calculated from the kinetic parameters: folding rate constant, k(f) = 1.65 x 10(-2) s(-1) and unfolding rate constant, k(u) = 7.90 x 10(-3) s(-1)) in 150 mM K+. The hybridization rate constants detected concurrently gave a bimolecular association constant, k(a) = 1.37 x 10(5) M(-1) s(-1) and dissociation constant, k(d) = 4.94 x 10(-5) s(-1). Interestingly, in the presence of Na+ we observed that G-quadruplex folding was unfavorable (K(F) = 0.54). Implication of our results on the c-myc transcription activation model is discussed in light of aberrant c-myc expression observed on destabilization of the G-quadruplex.

Figure 1

Hybridization of C1 and G1 monitored by CD. An aliquot of 1.28 μM G1 (solid line) was treated with 1.28 μM (gray line), 2.56 μM (gray dashed line) or 3.84 μM (solid dashed line) C1 in 10 mM HEPES, 150 mM NaCl and 3 mM MgCl₂, pH 7.4. Spectra were recorded at 25°C, 2 h after each addition.

Figure 2

Sensorgrams analyzed with simple versus QC hybridization models. Sensorgrams were obtained by hybridization with immobilized G1B (a–c) or C1B (d) using 16, 32, 64, 128, 256, 512 or 1024 nM of the respective complementary strand in the mobile phase. Fitted curves (red) were obtained by fitting the sensorgrams (black) with either the QC model using Equation 8 (a and b) or simple hybridization model using Equation 2 (c and d) as in Material and Methods. Sensorgrams were obtained at 25°C and pH 7.4 in either 150 mM KCl (a) or NaCl (b–d). Hybridization of C1 and G1 shows no triplex formation (e). Reactions had 10 nM of 5′ end labeled and 0.5 μM unlabeled C1 (lane 1) with either, 0.5 μM G1 (lane 2) and 5 U DNase I (lane 3) or 1.5 μM G1 (lane 4) and 5 U DNase I (lane 5). Samples were incubated at 4°C for 18 h before 15 min DNase I treatment (lanes 3 and 5). G1 shows multiple folded conformations (f). An aliquot of 10 nM of 5′ end labeled and 0.5 μM unlabeled G1 (lane 1), C1 (lane 2) and 31mer control dT31 (lane 3) were incubated for 4 h at 25°C before separation. Both (e) and (f) were incubated in 10 mM HEPES, 150 mM NaCl, 3mM MgCl₂ and pH 7.4. Bands were separated in a 20% non-denaturing PAGE in 0.5× TBE buffer (pH 8.0) at 4°C for 6 h at 90 V and visualized using autoradiography on phosphorimager (Fujifilm FLA 2000).

Figure 3

Resolution of two modes of duplex formation on sensor surface using QC hybridization model. Folding/unfolding equilibrium of quadruplex molecules on sensor surface present pre-equilibrated unfolded molecules as well as molecules unfolding during injection, for hybridization. QC hybridization model has been used to separate these components. Sensorgrams obtained with immobilized G1B and 16 (a), 32 (b), 64 (c), 128 (d), 256 (e) and 1024 (f) nM C1 are shown in black. Simulated curves for hybridization with pre-equilibrated unfolded G1B (D_e) and unfolding G1B during injection (D_t) were generated using Equations 6 and 7, respectively, and are marked in the figures. The theoretically derived hybridization curve is shown in red and is the sum of D_e and D_t in all cases. The dependence of the unfolding rate constant (k_u) and k_obs for hybridization on strand concentration using the kinetically extracted parameters obtained form QC model is shown in (g). Relationship between D_e and D_t, as percentage contribution towards total duplex formation as a function of strand concentration is shown in (h). All experiments were done in 150 mM NaCl as described in Figure 2.