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Thioflavin T fluorescence and NMR spectroscopy suggesting a non-G-quadruplex structure for a sodium binding aptamer embedded in DNAzymes

Recently, a Na+-binding aptamer was reported to be embedded in a few RNA-cleaving DNAzymes, including NaA43, Ce13d, and NaH1. The Na+ aptamer consists of multiple GG stretches, which is a prerequisite for the formation of G-quadruplex (G4) structures. These DNAzymes require Na+ for activity but show no activity in the presence of K+ or other metal ions. Given that DNA can selectively bind K+ by forming a G4 structure, this work aims to answer whether this Na+ aptamer also uses a G4 to bind Na+. Through comparative ThT fluorescence spectrometry studies, while a control G4 DNA exhibited notable fluorescence enhancement up to 5 mM K+ with a Kd of 0.28 ± 0.06 mM, the Ce13d DNAzyme fluorescence was negligibly perturbed with similar concentrations of K+. Opposed to this, Ce13d displayed specific remarkable fluorescence decrease with low millimolar concentrations of Na+. NMR experiments at two different pH values suggest that Ce13d adopts a significantly different conformation or equilibrium of conformations in the presence of Na+ versus K+ and has a more stable structure in the presence of Na+. Additionally, absence of characteristic G4 peaks in one-dimensional 1H NMR suggest that G4 is not responsible for the Na+ binding. This hypothesis is confirmed by the absence of characteristic peaks in the CD spectra of this sequence. Therefore, we concluded that the aptamer must be selective for Na+ and that it binds Na+ using a structural element that does not contain G4.