Abstract
Recently, we reported an inhibitory effect of guanine substitutions on the conformational switch from antiparallel to parallel quadruplexes (G4) induced by dehydrating agents. As a possible cause, we proposed a difference in the sensitivity of parallel and antiparallel quadruplexes to the guanine substitutions in the resulting thermodynamic stability. Reports on the influence of guanine substitutions on the biophysical properties of intramolecular parallel quadruplexes are rare. Moreover, such reports are often complicated by the multimerisation tendencies of parallel quadruplexes. To address this incomplete knowledge, we employed circular dichroism spectroscopy (CD), both as stopped-flow-assisted fast kinetics measurements and end-point measurements, accompanied by thermodynamic analyses, based on UV absorption melting profiles, and electrophoretic methods. We showed that parallel quadruplexes are significantly more sensitive towards guanine substitutions than antiparallel ones. Furthermore, guanine-substituted variants, which in principle might correspond to native genomic sequences, distinctly differ in their biophysical properties, indicating that the four guanines in each tetrad of parallel quadruplexes are not equal. In addition, we were able to distinguish by CD an intramolecular G4 from intermolecular ones resulting from multimerisation mediated by terminal tetrad association, but not from intermolecular G4s formed due to inter-strand Hoogsteen hydrogen bond formation. In conclusion, our study indicates significant variability in parallel quadruplex structures, otherwise disregarded without detailed experimental analysis.
Highlights
Guanine quadruplexes are secondary structures of nucleic acids, composed of several stacked square-shaped tetrads, each formed from four Hoogsteen hydrogen-bonded guanines [1]
Q migrates on native polyacrylamide gel electrophoresis (PAGE) as several smeared bands (Figure 1B) ranging from ~20 bp up to ~35 bp, i.e., even the fastest band is significantly slower than the bimolecular species
Any addition of terminal overhangs leads to an increase in PAGE migration speed, but the migration speed cannot be attributed to the molecularity of the complex
Summary
Guanine quadruplexes are secondary structures of nucleic acids, composed of several stacked square-shaped tetrads, each formed from four Hoogsteen hydrogen-bonded guanines [1] This structure is stabilized by a cation, especially potassium, located in the central space between each pair of tetrads, and is strengthened by crowding conditions [2,3]. The effect of mutations in PQS in genomic context might be reduced by replacing either only the affected guanine by another guanine located outside the quadruplex-forming motif [16,17], or by replacing the whole affected G-tract with another [18,19,20] For promoter quadruplexes such G-tract redundancy is quite common [21]. We were interested how the lesions in guanines, simulated here by G to T substitutions, affect the properties of the parallel intramolecular quadruplexes and to which extent the guanines within each tetrad differ in terms of the effect of their substitution on the quadruplex properties
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