Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

Abstract

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C(+) basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C(+) hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.

i-Clamp phenoxazine for the fine tuning of DNA i-motif stability

“…Figure 6c summarizes pH sensitivities of i -clamp absorbance and fluorescence in T 1 Z. The observed quenching of i -clamp fluorescence and the bathochromic shift of its absorption band under acidic conditions (Figure 6c ) are in line with the reported data for related dC analogues ( 46 ). The transition pH value calculated based on i -clamp absorbance and fluorescence changes (pH i = 6.7±0.1, middle panel in Figure 6c ) agrees with our estimates based on the T 1 Z CD data (6.6±0.1,middle panel in Supplementary Figure S11 ).…”

“…A phenoxazine nucleotide per se (i.e. without the clamp-specific aminoalkyl/guanidinoalkyl tether) has been reported to cause insignificant destabilization when introduced into the iM core ( 46 ). In the case of G-clamp and guanidino-G-clamp, one could expect stabilization due to additional H-bonding between the protonated clamp tether and the oxo-group of the opposing cytosine/clamp (Figure 2B ).…”

DNA i-Motifs With Guanidino-i-Clamp Residues: The Counterplay Between Kinetics and Thermodynamics and Implications for the Design of pH Sensors

“…The kinetics of IM formation have previously been studied by a number of methods, including SPR, FRET-based techniques and NMR spectroscopy [ [38] , [39] , [40] , [41] , [42] , [43] ] – all these methods are only applicable to relatively slow processes. Recently, stopped flow-based analyses of rapid IM folding/unfolding (typically induced by 8 → 5/5 → 8 pH jumps) have been reported [ 44 , 45 ]. In these studies, the pH conditions were selected to ensure complete (pseudo-irreversible) formation/disruption of the structures, which simplifies the interpretation of the kinetic curves and apparent rate constants.…”

“…Previous studies of IM kinetics (reported for the C20T mutant of the NHEIII c-MYC promoter fragment) revealed two-step folding: rapid formation of the intermediate and its subsequent slow rearrangement [ 45 ]. For our inter- and intramolecular IMs, no clear signs of intermediates were observed.…”

Effects of 5-Hydroxymethylcytosine Epigenetic Modification on the Stability and Molecular Recognition of VEGF i-Motif and G-Quadruplex Structures

“…Recordings were made over the wavelength range 225–350 nm. For thermal denaturation, increasing temperatures of 20–100°C with a ramp rate of 1°C per minute with a 1 minute hold per degree were used [ 35 , 36 ]. Thermal melt values were determined by nonlinear regression fitting on KaleidaGraph software (Synergy Software, Reading, PA) of the spectral data to determine the midpoint of the transition ( T M ) and Δ H for the folded to unfolded transition.…”