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

Abstract

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 K_d 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.