Hybrid G-Quadruplex and i-Motif Formation in G/C-Rich Repeat DNA: Beyond the Competitive Paradigm

Technical Summary: Hybrid G-Quadruplex and i-Motif Formation in G/C-Rich Repeat DNA

Problem Statement
G-quadruplexes (G4s) and i-motifs (iMs) are traditionally viewed as mutually exclusive, non-canonical DNA structures formed on complementary guanine-rich (G-rich) and cytosine-rich (C-rich) strands, respectively. G4s are stabilized by Hoogsteen hydrogen bonding and monovalent cations, while i-motifs rely on hemiprotonated C•C⁺ base pairs under acidic conditions. The prevailing paradigm suggests that the formation of one structure precludes the other due to topological and structural constraints. However, this view may be overly simplistic for intrinsically G/C-rich sequences, particularly tandem repeats like C₃G₂ and G₃C₂. While sequence-based predictors (e.g., G4Hunter) indicate these motifs lack sufficient intrinsic propensity to form stable G4s or iMs, and thermodynamic models favor canonical Watson–Crick duplexes, their conformational behavior under varying physicochemical conditions remains poorly understood. This study addresses the gap in understanding how G/C-rich tandem repeats navigate the equilibrium between duplex DNA and alternative higher-order structures, specifically investigating whether G4s and iMs can coexist or structurally couple rather than compete.

Methodology
The researchers employed a systematically designed series of short DNA oligonucleotides containing tandem G₂ and C₃ tracts (e.g., C₃G₂, G₂C₃, G₃C₂, and their variants with terminal flanking bases or thymine substitutions). The study utilized a multi-faceted biophysical approach to characterize structural transitions:

• Circular Dichroism (CD) Spectroscopy: Used to monitor structural topology (parallel vs. antiparallel) and stability across a temperature range (0–100°C) and varying pH levels (5.0 and 7.4).
• Thermodynamic Analysis: Melting curves were analyzed to determine melting temperatures ($T_m$), enthalpy ($\Delta H$), Gibbs free energy ($\Delta G$), and cooperativity (Full Width at Half Maximum, FWHM).
• Ion Titration: Experiments were conducted in the presence of varying concentrations of K⁺ and Na⁺ ions (2.5–100 mM) to assess cation dependence.
• Electrophoretic Mobility: Non-denaturing and denaturing PAGE were used to evaluate the compactness and folding state of the oligonucleotides.
• Temperature Difference Spectroscopy (TDS): Employed to confirm specific G4 spectral signatures.

Key Results

1. pH-Dependent G4 Formation in "Non-Propensity" Sequences: Contrary to sequence predictions, the C₃G₂ and T[C₃G₂]A sequences formed stable, pH-responsive G-quadruplex structures under acidic conditions (pH 5.0) in the presence of K⁺ ions. These structures exhibited characteristic antiparallel CD signatures (positive band at ~295 nm, negative band at ~240/273 nm) and TDS profiles consistent with folded G4s. This formation was absent at pH 7.4 and was strictly dependent on the presence of four intact GG tracts.
2. Structural Coupling of G4 and i-Motif Features: The study demonstrates that G4s and i-motifs are not strictly antagonistic. In the C₃G₂ system, protonation-dependent C•C⁺ interactions appear to stabilize the G4 topology. The authors propose a model where C•C⁺ pairs form within the loops or at the terminal regions of a two-tetrad antiparallel G4 core, effectively "capping" the structure. This coupling reduces conformational heterogeneity and stabilizes otherwise marginally stable G4 architectures.
3. Sequence Orientation and Stability: While C₃G₂ and G₂C₃ share similar base compositions, their structural behaviors differ slightly due to orientation. G₂C₃ showed a unique ability to form i-motif-like signatures under ion-free acidic conditions, whereas C₃G₂ did not. The G₃C₂ variant (inverted G:C ratio) formed a highly stable, three-tetrad antiparallel G4 reinforced by lateral C•C⁺ interactions, exhibiting significantly higher thermodynamic stability ($\Delta G \approx -30$ kJ/mol at 25°C) than the C₃G₂ variant.
4. Cation Specificity and Cooperativity: The formation of these hybrid structures was highly dependent on K⁺ ions; Na⁺ did not support G4 formation, likely due to insufficient stabilization of the G-tetrad core. Thermodynamic analysis revealed that C-containing sequences (C₃G₂, T[C₃G₂]A) displayed broader melting transitions (FWHM ~24–26°C) compared to the highly cooperative, narrow transitions of the thymine-only control (T₃G₂, FWHM ~17°C). This suggests a multistep unfolding process involving the coexistence of distinct interaction domains (G-tetrad core and C•C⁺ stabilized regions).
5. Electrophoretic Behavior: Despite the expectation that C•C⁺ protonation would reduce negative charge and decrease mobility, the folded C₃G₂ and T[C₃G₂]A structures exhibited increased electrophoretic mobility compared to their denatured forms. This indicates that the reduction in hydrodynamic volume due to the formation of highly compact structures outweighs the charge effects.

Significance and Claims
The paper argues that the relationship between G4s and i-motifs should be re-evaluated from a purely competitive paradigm to one of structural and functional coupling. The authors claim that:

• Coexistence is Possible: In G/C-rich genomic regions, G4 and i-motif features can coexist within the same sequence context, acting cooperatively rather than antagonistically.
• Protonation as a Stabilizer: Cytosine protonation (C•C⁺) is not merely a driver for i-motif formation but can directly influence G4 topology and stability, expanding the known mechanisms of pH-responsive DNA architecture.
• Biological Relevance: These findings suggest that tandem C₃G₂ and G₃C₂ repeats, which are widespread in regulatory regions of the human genome, may function as finely tunable conformational switches. Their ability to respond to subtle physicochemical fluctuations, such as local pH changes in hypoxic or tumor-associated tissues, could have implications for gene regulation, epigenetic control (via cytosine methylation susceptibility), and genome stability.
• Beyond Competition: The study provides experimental evidence that "hybrid" G4/i-motif states represent a distinct class of non-canonical DNA structures, challenging the assumption that G-rich and C-rich strands must fold into mutually exclusive structures.

The authors maintain a modest tone, presenting their structural models (e.g., the specific C•C⁺ pairing schemes) as tentative interpretations consistent with experimental data rather than definitive atomic-resolution structures. They emphasize that these behaviors expand the current understanding of structural plasticity in G/C-rich regions without overstating specific disease mechanisms beyond the known association of such repeats with regulatory processes.