Novel synthesis and RNA-binding properties of aminoglycoside dimers conjugated via a naphthalene diimide-based intercalator.

The synthesis and RNA-binding properties of naphthalene-based diimide conjugated bis-aminoglycoside antibiotics are reported. Compared to the monomeric aminoglycoside, the conjugated ligands were observed to attain up to 35-fold enhancement in binding affinity towards a novel RNA construct that contained two 16S rRNA A-sites.

Binding of dimeric aminoglycosides to the HIV-1 rev responsive element (RRE) RNA construct.

Through a series of elegant fluorescence measurements, particularly through stopped-flow kinetic measurements, it was recently demonstrated that aminoglycoside antibiotics are able to bind to the HIV-1 Rev responsive element (RRE) RNA construct in more than a 1:1 stoichiometry (Lacourciere, K. A.; Stivers, J. T.; Marino, J. P. Biocheminstry 2000, 39, 5630). Here, we present the binding study results of dimeric neomycin ligands through fluorescence anisotropy studies, to the HIV-1 RRE RNA construct. The dimeric neomycin molecules are observed to be able to bind the HIV-1 RRE RNA construct approximately 17-fold higher when compared to the monomeric neomycin, lending evidence that there are indeed two or more neomycin binding sites within the HIV-1 RRE construct.

Binding of a cyclic BIV beta-Tat peptide with its TAR RNA construct.

The ability of RNA structures to adopt diverse yet complex tertiary structures has resulted in numerous fascinating RNA-protein recognition events. It was recently reported that a close relative of the HIV Rev peptide, namely a 17 residue Tat peptide from bovine immuno-deficiency virus (BIV), is able to bind to the 28 nucleotide BIV TAR RNA construct. Here we report that by simply converting the 17 residue beta-ribbon peptide structure to a 19 residue cyclopeptide, the binding affinity (Kd) of the resulting cyclopeptide to the TAR RNA target, observed by fluorescence binding study, was enhanced approximately 5-fold.

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

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 stem-loop 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.

Enhanced binding of aminoglycoside dimers to a "dimerized" A-site 16S rRNA construct.

In this work, we investigated the binding of a series of dimeric aminoglycoside molecules to (i) a 27 nt A-site 16S rRNA construct, and (ii) an artificially grafted 46 nt 'dimerized' A-site 16S rRNA construct. It was observed that the dissociation constants of dimeric aminoglycosides to the dimerized A-site 16S rRNA construct can achieve up to approximately 19-fold enhancement compared to the monomeric aminoglycoside molecules.

Xenognosin sensing in virulence: is there a phenol receptor in Agrobacterium tumefaciens?

BACKGROUND: The mechanisms of signal perception and transmission in the 'two-component' autokinase transmitters/response regulators are poorly understood, especially considering the vast number of such systems now known. Virulence induction from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens represents one of the best understood systems with regard to the chemistry of the activating signal, and yet the existing data does not support a receptor-mediated perception event for the xenognostic phenols. RESULTS: Here we provide the first conclusive evidence that a specific receptor must be involved in xenognostic phenol perception, detail structural requirements of the xenognosins necessary for perception by this receptor, and develop a genetic strategy that demonstrates critical components of the phenol recognition system are not encoded on the Ti plasmid. CONCLUSIONS: Although the basic elements of the two-component system required for phenol-mediated induction of virulence gene expression are encoded on the Ti plasmid, they are dependent on the chromosomal background for even the very first stage of signal perception. This discovery suggests a curious evolutionary history, and also provides functional insight into the mechanisms of two-component signal detection and transmission in general.

Aminoglycoside antibiotics are able to specifically bind the 5'-untranslated region of thymidylate synthase messenger RNA.

The translational initiation codon for thymidylate synthase (TS) mRNA is located in a unique stem-loop structure which contains an internal cytosine-cytosine (CC) bubble. This stem-loop structure is thought to be important in the regulation of TS translation, which is itself an important target for anticancer drugs, such as 5-fluorouracil. Internal bubble or bulge structures are candidate receptors for the aminoglycoside antibiotics. It is shown here that aminoglycosides bind in a specific and saturable fashion with dissociation constants of approximately 1 microM to a TS mRNA site 1 construct and that the binding site for the aminoglycosides is located in the CC bubble region. In fact, the CC bubble, when grafted into other stem-loop structures, confers aminoglycoside binding on them. These studies reveal an additional binding domain for aminoglycosides and also suggest how novel anti-cancer drugs might be designed that affect TS mRNA translation rather than enzyme function.