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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. [ABSTRACT FROM AUTHOR]

Binding affinity and selectivity are critical properties of aptamers that must be optimized for any application. The sulforhodamine B binding RNA aptamer (SRB-2) is a somewhat promiscuous aptamer that can bind ligands that vary markedly in shape, size and charge. Here we categorize potential ligands based on their binding mode and structural characteristics required for high affinity and selectivity. Several known and potential ligands of SRB-2 were screened for binding affinity using LSPR, ITC and NMR spectroscopy. The study shows that rhodamine B has the ideal structural and electrostatic properties for selective and high-affinity binding of the SRB-2 aptamer. • Smaller alkyl groups and reduced negative charge result in higher binding affinity. • The single ring and one negative charge are required for binding specificity. • Size and shape of the alkyl amine groups is crucial for binding affinity. • Rhodamine B is the ideal SRB-2 ligand among those tested here. [ABSTRACT FROM AUTHOR]