Parallel reaction pathways accelerate folding of a guanine quadruplex

Abstract

G-quadruplexes (G4s) are four-stranded, guanine-rich nucleic acid structures that can influence a variety of biological processes such as the transcription and translation of genes and DNA replication. In many cases, a single G4-forming nucleic acid sequence can adopt multiple different folded conformations that interconvert on biologically relevant timescales, entropically stabilizing the folded state. The coexistence of different folded conformations also suggests that there are multiple pathways leading from the unfolded to the folded state ensembles, potentially modulating the folding rate and biological activity. We have developed an experimental method for quantifying the contributions of individual pathways to the folding of conformationally heterogeneous G4s that is based on mutagenesis, thermal hysteresis kinetic experiments and global analysis, and validated our results using photocaged kinetic NMR experiments. We studied the regulatory Pu22 G4 from the c-myc oncogene promoter, which adopts at least four distinct folded isomers. We found that the presence of four parallel pathways leads to a 2.5-fold acceleration in folding; that is, the effective folding rate from the unfolded to folded ensembles is 2.5 times as large as the rate constant for the fastest individual pathway. Since many G4 sequences can adopt many more than four isomers, folding accelerations of more than an order of magnitude are possible via this mechanism.

Figure 1.

G4 structures and sequences were investigated in this study. (A) GR isomers with exchanging dG residues colored red and blue. (B) G-tetrad structure. (C) The WT c-myc Pu22 and trapped mutant sequences were investigated in this study. dG to dI mutations to the WT sequence are shown in pink. Full corresponds to the fully trapped sequences that contain dual mutations, whereas half indicates the half-trapped sequences with a single mutation that are capable of undergoing two-state GR exchange. Numbers indicate the direction that the G-tract is locked by mutation; for example, the 33 sequence has both exchanging G-tracts locked in the 3′ direction. The exchanging G-tract for the half-trapped sequences is denoted by X.

Figure 2.

Structural characterization of the c-myc Pu22 G4 and trapped mutants by CD. The WT CD spectrum is shown overlaid with those for the fully trapped (A) and half-trapped (B) mutant structures.

Figure 3.

G4 structural analysis by proton NMR. Imino proton spectra for the fully trapped (A), half-trapped (B) and WT structures (C). Imino peaks for the 12 major resonances in each spectrum are numbered. Minor state peaks due to incomplete trapping of the 35 and 33 mutants are indicated with # symbols. The resonances for minor state GR isomers in the WT and X5 and X3 half-trapped mutants are labeled with underlined numbers. Numbers with asterisks indicate regions where the major and minor state GR isomer resonances are overlapped. In panels (B) and (C), the equilibrium population-weighted average spectra of the corresponding fully trapped mutants are shown as colored dashed lines, calculated from the extracted TH global fitting parameters (see ‘Materials and Methods’ section for details). The 5X, 3X, X5, X3 weighted average spectra are given by the weighted sums of the 55 + 53, 35 + 33, 55 + 35 and 53 + 33, respectively. The WT weighted average spectra are the weighted sums of the 5X + 3X, X3 + X5 and 33 + 53 + 35 + 33 spectra.

Figure 4.

Global fits of a parallel pathways model to TH data for the WT and trapped-mutant G4s. TH datasets (295 nm) for the fully trapped (A), half-trapped (B) and WT (C) G4s. Fit residuals are shown in the subpanel below each dataset. Light to dark blue and orange to red indicate slowest to fastest annealing and melting scan rates, respectively. Experimental data are shown as colored circles, while optimized global fit data are colored lines.

Scheme 1.

Folding mechanisms for the WT c-myc Pu22 ensemble and trapped mutants. (A) The fully trapped G4s adopt a single folded (F) conformation from the unfolded state (U). (B) The half-trapped G4s fold into two GR isomers (A and B) from the unfolded state, which can then slowly equilibrate by direct interconversion (indicated by small arrows) (23). (C) The WT c-myc Pu22 ensemble primarily folds by directly adopting the four GR isomers from U in parallel, with slow GR exchange between isomers.

Figure 5.

Theoretical prediction of folding acceleration in the c-myc Pu22 WT ensemble by TH and experimental validation by isothermal NMR folding experiments. (A) Short- and long-timescale isothermal population distributions predicted by TH are shown in the top and bottom plots, respectively. U (green) denotes the unfolded state, and the WT trace is the sum of the profiles for the four GR isomers (colors matching Figures 2 and 3). Isothermal simulations were performed according to Scheme 1C using the optimized parameters from Table 1 without direct GR isomer interconversion (37 °C and k_ex = 0 min⁻¹, see Supplementary Methods for details). (B and C) Imino proton region of the 1D ¹H spectra of 3x-NPE-caged and native (after irradiation) c-myc Pu22 oligonucleotides: WT (B) and 33 fully trapped mutant (C) at 37 °C. For the 3x-caged oligonucleotides, the absence of imino proton signals indicates an unfolded state with no stable Hoogsteen base-pair interactions. (D) Photolysis reaction for O6-(R)-NPE-caged dG with laser light at 355 nm. Folding is initiated after the caging group is released from the dG residue. (E and F) Normalized kinetic NMR data for the light-induced folding of photocaged c-myc Pu22 WT and 33 fully trapped G4s using imino proton signal integration limits of 11.2–11.3 and 10–12.5 ppm, respectively. Data (colored points) were fit to a monoexponential function (colored lines) with fit residuals shown underneath each panel. Extracted rate constants are indicated.