ReviewAminoglycoside–RNA interactions
Introduction
The potential of RNA as a new drug target has recently come to the fore 1, 2, 3, with the recognition that RNA molecules can adopt complex three-dimensional structures that, as with proteins, enable the design of specific ligands. Another reason for the present interest comes from the fact that many pathogenic agents, such as retroviruses, encode their genetic information in RNA strands. Aminoglycoside antibiotics (Figure 1) have long been used as very efficient drugs against Gram-positive and Gram-negative bacteria, and against mycobacterial infections [4]. These molecules, however, impair hearing and kidney functions at high doses and resistant strains are appearing at an increasing rate [5]. Thus, the need for new and more specific compounds has emerged. To enable the development of new antibiotics, it is necessary to understand at the molecular level the interactions between aminoglycosides and RNA, often considered as a model system. Interactions between RNAs and aminoglycosides have been reviewed in the past (see 1, 6, 7, 8, 9). We center this review on papers published in the past two years.
Section snippets
Ribosomal RNA
NMR experiments revealed the first atomic insights into complexes between aminoglycosides of the neomycin family (Figure 1a) and a 27 nucleotide stem–loop fragment incorporating the acceptor site (A-site) of 16S rRNA (dissociation constant [Kd] = 0.2 μM) 10, 11••, 12••. It appears that both rings I and II of the aminoglycoside are important for its binding to an A·A mismatch adjacent to a bulging adenine. These two rings (though possessing different side groups) are oriented in the same way
Binding of aminoglycosides to in vitro selected RNA
RNA aptamers binding to aminoglycosides with high affinity (in the nanomolar range) have also been selected by in vitro methods. The structures of the aptamers complexed with small molecules have been extensively reviewed 8, 24, 25, 26. The NMR solution structures of two tobramycin-binding aptamers have been solved 27, 28••, as well as that of the neomycin B binding aptamer [29••]. Interestingly, all aptamers have a similar architectural fold but little sequence homology (Figure 3). In each
Inhibition of catalytic RNAs by aminoglycosides
Catalytic RNAs or ribozymes are RNA molecules that can catalyse chemical reactions, such as the self-cleavage or religation of the phosphor–sugar backbone linkage. They are widely found in nature providing an indispensable biological activity. Thus, in the tRNA-processing enzyme ribonuclease P, the RNA moiety has a catalytic activity, which generates the mature 5′ terminus of an array of tRNA molecules. Self-splicing introns often occur within ribosomal genes, as in Tetrahymena rRNA. The
Small catalytic RNAs
RNA from the human pathogen hepatitis delta virus (HDV) and that from a number of small plant pathogenic viroids and virusoids, such as the Tobacco ringspot virus satellite RNAs, require RNA self-cleavage for the generation of the unit-sized RNA molecules following replication. The ‘hammerhead’ motif was identified on the positive (+) strand of the satellite RNA of the Tobacco ringspot virus RNA, whereas on the negative strand a different structural motif called a ‘hairpin’ was discovered. The
Drug design
Compared to natural sites, the binding affinities provided by in vitro selected aptamers are a factor of at least 1000 higher, most probably because of a reduction in the possibilities of docking to the binding site (despite an induced-fit recognition mechanism). This fact is used to control in vivo gene expression by aminoglycoside-binding aptamers attached to 5′-untranslated regions [56•].
To probe the specificity of neomycin B-RNA interaction, a series of synthetic analogs have been
Progress in techniques
Fluorescently labelled RNA molecules [46] or aminoglycosides 66, 72• can now be used for spectroscopic analyses by polarisation or anisotropy 20, 61, 64, 66, 72• and fluorescence intensity 46, 58, 59; the techniques of lifetime measurements, steady-state and stop-flow kinetics or fluorescence resonance energy transfer (FRET) should come in the future. Fluorescent-dye labelling also allows high-throughput screening of aminoglycoside or RNA libraries [66]. Electron spray ionisation–mass
Conclusions
Aminoglycoside antibiotics interact with a great variety of RNA molecules and any biological function involving RNA is a potential target. Because RNA molecules are highly charged, metal ions participate in RNA three-dimensional folding and provide active centers in catalytic RNA molecules. The interactions between aminoglycosides and RNA are dominated by the number and basicity of amino groups in the aminoglycoside 42•, 76. Experimental and theoretical data show that structural complementarity
Acknowledgements
We thank Dinshaw Patel for sending us the coordinates of the neomycin aptamer. Frank Walter is supported by an European Community Research Network Contract (FMRX-CT97-0154 to Eric Westhof). Quentin Vicens is a Boursier Docteur-Ingénieur CNRS/Rhône-Poulenc-Rorer. Eric Westhof thanks the Institut Universitaire de France for supporting grants.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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