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Recently, a [Na.sup.+]-binding aptamer was reported to be embedded in a few RNA-cleaving DNAzymes, including NaA43, Ce13d, and NaH1. The [Na.sup.+] aptamer consists of multiple GG stretches, which is a prerequisite for the formation of G-quadruplex (G4) structures. These DNAzymes require [Na.sup.+] for activity but show no activity in the presence of [K.sup.+] or other metal ions. Given that DNA can selectively bind [K.sup.+] by forming a G4 structure, this work aims to answer whether this [Na.sup.+] aptamer also uses a G4 to bind [Na.sup.+]. Through comparative ThT fluorescence spectrometry studies, while a control G4 DNA exhibited notable fluorescence enhancement up to 5 mM [K.sup.+] with a [K.sub.d] of 0.28 [+ or -] 0.06 mM, the Ce13d DNAzyme fluorescence was negligibly perturbed with similar concentrations of [K.sup.+]. Opposed to this, Ce13d displayed specific remarkable fluorescence decrease with low millimolar concentrations of [Na.sup.+]. NMR experiments at two different pH values suggest that Ce13d adopts a significantly different conformation or equilibrium of conformations in the presence of [Na.sup.+] versus [K.sup.+] and has a more stable structure in the presence of [Na.sup.+]. Additionally, absence of characteristic G4 peaks in one-dimensional [.sup.1]HNMR suggest that G4 is not responsible for the [Na.sup.+] 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.sup.+] and that it binds [Na.sup.+] using a structural element that does not contain G4. Key words: aptamers, DNAzymes, sodium, fluorescence, NMR. Recemment, on rapportait qu'un aptamere liant le [Na.sup.+] s'incorporait dans quelques ADNzymes de clivage de l'ARN, dont NaA43, Ce13d et NaH1. L'aptamere a [Na.sup.+] se compose de plusieurs sequences GG, qui sont essentielles a la formation de structures G-quadruplexe (G4). La presence de l'ion [Na.sup.+] est necessaire a l'activite de ces ADNzymes. En effet ces dernieres ne montrent aucune activite en presence de [K.sup.+] ou d'autres ions metalliques. Etant donne que l'ADN peut lier selectivement au [K.sup.+] en formant une structure G4, le but de ces travaux consiste a verifier si cet aptamere a [Na.sup.+] forme aussi une structure G4 pour se lier au [Na.sup.+]. Par des etudes comparatives de spectrometrie de fluorescence a la thioflavine T (ThT), nous avons montre que bien qu'un ADN de structure G4 a titre de temoin presente une amplification notable de la fluorescence a des concentrations allant jusqu'a de 5 mM de [K.sup.+] avec une constante [K.sub.d] de 0,28 [+ or -] 0,06 mM, la perturbation de la fluorescence de l'ADNzyme Ce13d etait negligeable a des concentrations de [K.sup.+] similaires. Au contraire, le Ce13d a affiche une diminution particulierement marquee de la fluorescence aux concentrations millimolaires inferieures de [Na.sup.+]. Les experiences de RMN a deux valeurs de pH differentes semblent indiquer qu'en presence de [Na.sup.+], le Ce13d adopte une conformation ou des conformations a l'equilibre substantiellement differentes par rapport a celles adoptees en presence de [K.sup.+], et que sa structure est plus stable en presence de [Na.sup.+]. De plus, l'absence de pics caracteristiques de la structure G4 en RMN [.sup.1]H 1D laisse supposer que la structure G4 n'est pas responsable de la liaison avec le [Na.sup.+]. Cette hypothese est confirmee par l'absence de pics caracteristiques dans les spectres de dichroisme circulaire de cette sequence. Par consequent, nous avons conclu que l'aptamere doit etre selectif pour l'ion [Na.sup.+] et qu'il se lie au [Na.sup.+] au moyen d'un element structural qui ne comporte pas de structure G4. [Traduit par la Redaction] Mots-cles: aptameres, ADNzymes, sodium, fluorescence, RMN., 1. Introduction Understanding metal binding to DNA is important not only for studying the biological functions of DNA, but also for biosensor development, (1,2) drug development, (3) and nanotechnology. (4) [...]

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]