Monovalent Ion Dependence of Neomycin B Binding to an RNA Aptamer Characterized by Spectroscopic Methods

Abstract

Understanding the interactions of small molecules like antibiotics with RNA is a prerequisite for the development of novel drugs. In this study we address structural and thermodynamic features of such interactions by using a simple model system: the binding of the highly charged antibiotic neomycin B to a short hairpin RNA molecule. Nucleotide A16, which acts as a flap over the neomycin B binding pocket, was substituted by the fluorescent adenine analogue 2-aminopurine (2-AP). Steady-state and time-resolved fluorescence measurements were complemented by UV-melting and circular dichroism studies. The binding of neomycin B at three sites was found to have a strong inverse correlation with Na(+) concentration. For the highest-affinity site, both fluorescence and UV absorption experiments were consistent with a model assuming at least three neomycin NH(3) (+) groups participating in addition to hydrogen bonds in electrostatic interactions with the RNA. The variation of fluorescence intensity and lifetime upon neomycin B binding indicated unstacking of 2-AP16 from neighbouring bases as it flipped over the binding pocket. RNA conformational changes upon binding of the antibiotic were confirmed by circular dichroism. The two weaker binding sites were characterized as unspecific binding to the aptamer, while the high-affinity binding event was shown to be highly specific even at high ionic concentration. In addition, 2-AP was confirmed to be a noninvasive fluorescent probe; it serves as a sensitive spectroscopic tool to investigate details of the interactions between small molecules and RNA.

An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures

“…1). Applying polyelectrolyte theory by the following linear relationship: logK a = logK 0 − mψlog [I], where K a is the measured association constant, K 0 is the limiting equilibrium constant, m is the number of ion pairs formed between drug and host RNA, and ψ is the thermodynamic counter-ion binding parameter for the host RNA (Stampfl et al 2007), resulted in the prediction of up to two ionic contacts involved in the binding of ribostamycin by NEO1A and up to three ionic contacts in the paromomycin-NEO1A interaction. For neomycin-B binding the nonlinear relation suggests that the aptamer shifts in its interaction mode with [I] of the buffer.…”

“…RNA aptamers that specifically bound to neomycin-B over paromomycin were identified by in vitro selection (Wallis et al 1995) and this aptamer-ligand interaction has been studied intensively (Wallis and Schroeder 1997;Cowan et al 2000;de-los-Santos-Alvarez et al 2007Stampfl et al 2007). With the intent to use NEO1A in synthetic biology applications that would require that the aptamer be functional in the cell cytoplasm, we examined the interaction of NEO1A with its ligands in Buffer A that emulates the free ion concentrations of the mammalian cell cytoplasm.…”

“…The complex is stabilized by electrostatic and Hbonding interactions (Wallis and Schroeder 1997;Cowan et al 2000;de-los-Santos-Alvarez et al 2007Stampfl et al 2007). Our NMR results (performed in a similar buffer as the original study) are consistent with the reported structure of the neomycin-B-NEO1A complex.…”

“…The focus of the current study is a 23-nt RNA aptamer selected against neomycin-B (NEO1A), which is the smallest RNA motif reported to have high affinity and specificity for neomycin-B over paromomycin (Wallis et al 1995;Wallis and Schroeder 1997;Cowan et al 2000;de-los-SantosAlvarez et al 2007de-los-SantosAlvarez et al , 2009Stampfl et al 2007). An NMR-derived structure is available for the NEO1A in complex with neomycin-B (Jiang et al 1999) but there is no equivalent structure for the apo-aptamer.…”

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“…4,5 In their most recent work, Herrmann and co-workers relied on an RNA structure that was generated by another molecular biology tool, namely a SELEX experiment (Systematic Evolu tion of Ligands by EXponential Enrichment). 6 This RNA aptamer binds the aminoglycoside antibiotic neomycin B (1) that contains six amino and seven hydroxyl groups (Figure 1). Andreas Bastian, the PhD student working on the aptameric protective group project, mentions: "After searching the literature for suitable aptamers for our new approach, I immediately realized that this was the right one.…”