RNA aptamers that specifically bind to a 16S ribosomal RNA decoding region construct

Jeffrey B.-H. Tok 0 1 Junhyeong Cho 0 1 Robert R. Rando 0 1 0 Present address: Jeffrey B.-H. Tok, Indiana University-Purdue University at Fort Wayne, Department of Chemistry , 2101 E. Coliseum Boulevard, Fort Wayne, IN 46803, USA 1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School , 45 Shattuck Street, Boston, MA 02115, USA RNA-RNA recognition is a critical process in controlling many key biological events, such as translation and ribozyme functions. The recognition process governing RNA-RNA interactions can involve complementary Watson-Crick (WC) base pair binding, or can involve binding through tertiary structural interaction. Hence, it is of interest to determine which of the RNA-RNA binding events might emerge through an in vitro selection process. The A-site of the 16S rRNA decoding region was chosen as the target, both because it possesses several different RNA structural motifs, and because it is the rRNA site where codon/anticodon recognition occurs requiring recognition of both mRNA and tRNA. It is shown here that a single family of RNA molecules can be readily selected from two different sizes of RNA library. The tightest binding aptamer to the A-site 16S rRNA construct, 109.2-3, has its consensus sequences confined to a stem-loop region, which contains three nucleotides complementary to three of the four nucleotides in the stemloop region of the A-site 16S rRNA. Point mutations on each of the three nucleotides on the stem-loop of the aptamer abolish its binding capacity. These studies suggest that the RNA aptamer 109.2-3 interacts with the simple 27 nt A-site decoding region of 16S rRNA through their respective stem-loops. The most probable mode of interaction is through complementary WC base pairing, commonly referred to as a loop-loop 'kissing' motif. High affinity binding to the other structural motifs in the decoding region were not observed. - Ever since in vitro selection was introduced in the early nineties (13), the power of this combinatorial approach has been utilized to generate aptamers that bind targets ranging from organic molecules to proteins, and even DNA (4,5). There also have been recent reports on the selection of RNA motifs that bind RNA tetraloops (6) and on the selection of an RNA substrate for the P RNA of Bacillus subtilis (7). Studies on RNARNA binding and recognition are not at the stage where it is possible to predict the nature of the RNARNA binding interactions that would prevail in a particular instance. Therefore, the fundamental question of understanding the principles that govern intermolecular RNARNA recognition still remains in the exploratory stage. RNARNA interactions are known to control many key biological events, such as translation and ribozyme function. Most of the RNARNA recognition processes occur through complementary WatsonCrick (WC) base pair binding. Specifically, looploop interactions generally occur from the single stranded region of the RNA, e.g. stemloop, internal bulge, followed by formation of an extended intermolecular helix. These types of interactions have been observed in the dimerization of the HIV-1 genome (8,9), the formation of ribonucleoprotein particles for transport and localization in Drosophila (10), and in the self-splicing of subgroups in Tetrahymena thermophilia ribozymes (11). Alternatively, recent reports demonstrate that the catalytic P RNA ribozyme is able to recognize its substrate (RNA) through its threedimensional structural or tertiary interactions (12,13). Hence, it is of interest to investigate which of the RNARNA binding events will emerge through an in vitro selection protocol. The decoding region of prokaryotic 16S rRNA is a region of RNA which is thought to interact with mRNA and tRNA (1416). It has long been known that the interaction of the codon and anticodon occurs on the A-site of the decoding region of prokaryotic 16S rRNA, which is itself part of the 30S ribosomal subunit (17,18). This interaction is disrupted by aminoglycoside antibiotics, which cause misreading of the mRNA, as well as inhibition of translocation (18). The aminoglycoside antibiotics are thought to primarily bind to 16S rRNA (19,20). While the entire 16S rRNA is too large for molecular dissection, recent experiments have shown that the 16S rRNA can be treated in a modular fashion (21). The binding-site of the aminoglycoside antibiotics in 16S rRNA appears to be confined to a discrete region (decoding region) as revealed by a series of mutational and chemical protection experiments (20,21). A simple decoding analog of 49 nt has been shown to be able to bind to aminoglycosides, tRNA and mRNA (21). The decoding region contains the A and P sites for tRNA binding, mRNA binding, as well as an important aminoglycoside binding region (21). The aminoglycoside binding region has been studied in some detail. An NMR spectroscopic study on the interaction between the aminoglycosides paramomycin and gentamycin with the internal bulged A-site of a 27-nt 16S rRNA construct has been reported (2224). A more extensive 16S rRNA construct has been shown to stoichiometrically bind aminoglycosides known to interact with the A-site, while not binding to antibiotics known not to bind to the A-site (25,26). In contrast to the relatively advanced state of knowledge concerning A-site decoding regionaminoglycoside interactions, little is known about the nature of the interactions between tRNA and mRNA with the A-site. Thus the work described should allow for a further understanding of the RNARNA recognition process. In addition, it could also reveal novel molecules able to prevent the interaction of aminoglycosides with the 27-nt A-site decoding region construct of 16S rRNA first described by Fourmy et al. (22). We have recently reported the development of a sensitive and quantitative binding fluorescence method that allows the efficient and accurate measurements of the dissociation constant (Kd) for aminoglycosideRNA interactions (2528). The method entails the use of fluorescent dye tagged aminoglycosides to measure binding affinities to RNA molecules selected to bind to particular aminoglycosides. Here, we report a modification of this technique that enabled us to study RNARNA interactions efficiently and quantitatively. In this instance, the attachment of a fluorescein fluorophore to the target RNA, instead of to the aminoglycoside, allows us to monitor and measure the Kd of RNA species through fluorescence anisotropy measurements. In addition, previously published aminoglycoside binding measurements were performed on a substantially more complex decoding region construct (157 nt) than the one under investigation here (25). Therefore, it was of interest to determine the specificity and affinity of aminoglycoside binding to the simplified 27-nt 16S rRNA construct which comprises only the A-site. MATERIALS AND METHODS Neomycin B sulfate (>90%), and paramomycin sulfate were purchased f (...truncated)