William A. Greenberg

Specificity of aminoglycoside antibiotics for the A-site of the decoding region of ribosomal RNA

BACKGROUND Aminoglycoside antibiotics bind to the A-site of the decoding region of 16S RNA in the bacterial ribosome, an interaction that is probably responsible for their activity. A detailed study of the specificity of aminoglycoside binding to A-site RNA would improve our understanding of their mechanism of antibiotic activity. RESULTS We have studied the binding specificity of several aminoglycosides with model RNA sequences derived from the 16S ribosomal A-site using surface plasmon resonance. The 4,5-linked (neomycin) class of aminoglycosides showed specificity for wild-type A-site sequences, but the 4,6-linked class (kanamycins and gentamicins), generally showed poor specificity for the same sequences. Methylation of a cytidine in the target RNA, as found in the Escherichia coli ribosome, had negligible effects on aminoglycoside binding. CONCLUSIONS Although both 4,5- and 4, 6-linked aminoglycosides target the same ribosomal site, they appear to bind and effect antibiotic activity in different manners. The aminoglycosides might recognize different RNA conformations or the interaction might involve different RNA tertiary structures that are not equally sampled in our ribosome-free model. These results imply that models of ribosomal RNA must be carefully designed if the data are expected to accurately reflect biological activity.

Glycan arrays: biological and medical applications

Carbohydrates and their conjugates are involved in various biological events, including viral and bacterial infection, the immune response, differentiation and development, and the progression of tumor cell metastasis. Glycan arrays are a new technology that has enabled the high-sensitivity and rapid analysis carbohydrate–protein interaction and contribute to significant advances in glycomics. Glycan arrays use a minute amount of materials and can be used for high-throughput profiling and quantitative analysis and provide information for the development of carbohydrate-based vaccines and new drug discovery.

Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450BM3 demethylases

Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450BM3) demethylases that provides a highly efficient means to access valuable intermediates, which can be converted to a wide range of substituted monosaccharides and polysaccharides. Demethylases displaying high levels of regioselectivity toward a number of protected monosaccharides were identified using a combination of protein and substrate engineering, suggesting that this approach ultimately could be used in the synthesis of a wide range of substituted mono- and polysaccharides for studies in chemistry, biology, and medicine.

Programmable reactivity-based one-pot oligosaccharide synthesis

A detailed protocol is described for the application of a programmable one-pot oligosaccharide synthesis methodology to the synthesis of fucosyl GM1. This serves as a general example of the application of this method to the synthesis of any desired oligosaccharide. The method relies on a large database of relative reactivities for differentially protected tolyl thioglycoside donor molecules and a computer program to suggest the best order of addition for assembly of the oligosaccharide in optimal yield and with the fewest operations. The product is a protected form of the desired oligosaccharide isolated in 47% yield, which is then deprotected using standard procedures to provide fucosyl GM1 oligosaccharide (1) in 44% yield. The total time for synthesis of 1 from building blocks 3, 4 and 5 is approximately 4 d, whereas synthesis of the same compound by traditional stepwise procedures would take significantly longer. Protocols for the synthesis of thioglycoside building blocks 3 and 4 are also described.

A comparison of H-pin and hairpin polyamide motifs for the recognition of the minor groove of DNA

Solid-phase polyamide synthesis is extended to the H-pin motif using a Boc-protected bispyrrole monomer and bidirectional synthesis. Sequence-specific recognition of a designated seven base pair target site is achieved by the formation of a 10-ring H-pin polyamide⋅DNA complex in the minor groove. DNA binding properties are compared with the corresponding hairpin polyamides (see below).

Evaluation of RNA-binding specificity of aminoglycosides with DNA microarrays

We have developed methods for using DNA array technology to probe the entire transcriptome to determine the RNA-binding specificity of ligands. Two methods were investigated. In the first method, the RNA-binding aminoglycoside antibiotic tobramycin was covalently linked to magnetic beads. The beads were bound to human liver mRNA and washed, and specifically bound RNA was eluted, amplified, and analyzed with DNA array technology. A small number of genes were found to bind specifically to the tobramycin beads. In the second method, the aminoglycoside ligand was added directly to the array hybridization reaction, and the signal was compared with a control experiment in the absence of ligand. The aminoglycosides were found to interfere with a small percentage of all hybridization events. These methods differ from traditional DNA array experiments in that the readout is a direct measure of the interaction between mRNA and a ligand, rather than an indirect measure of effect on expression. We expect that the results will lead to the discovery of new aminoglycoside-binding RNA motifs and may also have relevance toward understanding and overcoming the side effects observed with these antibiotics in the clinic.