Carbohydrates Binding to RNA

In addition to binding to sialic acid residues of the carbohydrate side chains of cellular proteins that the virus exploits as receptors, hemagglutinin has a second function in the infection of host cells. Viruses, bound to the plasma membrane via their membrane receptors, are taken into the cells by endocytosis. Proton pumps in the membrane of endocytic vesicles that now contain the bound viruses cause an accumulation of protons and a consequent lowering of the pH inside the vesicles. The acidic pH (below pH 6) allows hemagglutinin to fulfill its second role, namely, to act as a membrane fusogen by inducing the fusion of the viral envelope membrane with the membrane of the endosome. This expels the viral RNA into the cytoplasm, where it can begin to replicate. 

This biosensor consists of two surface-tethered components. The biorecognition element can be an antibody (Section 19-5), DNA, RNA, or carbohydrate with specific affinity for analyte. A structural analog of the analyte is bound to a flexible arm adjacent to the recognition element. In the absence of analyte, the tethered analog binds to the recognition element. 

The carbohydrate moiety of RNA is D-ribose with the / -D-ribofurano-side ring. The 2 - and 3 -OH groups are cis to each other and easily form the cyclic phosphate intermediate. Although the 2 -deoxynucleotides bind to the enzyme, they only serve as inhibitors. The 2 -OH group is mandatory for the catalytic activity of pancreatic RNase. 

The synthesis of the library proceeded smoothly as planned, and only two purifications were necessary. The four intermediates 8.20 were chromatographed and the final, basic library individuals were purified by ion-exchange chromatography, both steps being amenable to automation for synthesis of a larger library. The library LI validated the chemical route and confirmed the structural hypothesis of 1,3-hy-droxyamine-containing carbohydrate scaffolds as RNA binders. The compounds were tested and showed RNA-binding activity, even if the desired sequence specificity was not observed (54). 

One of many other types of carbohydrate/noncarbohydrate interactions that lead to bioactivity involves the aminoglycoside class of antibiotics, of which neamine 44 is a simple example. They bind to a specific part of the decoding region of prokaryotic ribosomal RNA to induce their activity, and this has led to the synthesis of analogs designed to have stronger binding [50]. 

RT is the major protein of the Ricinus communis seed. It is composed of two polypeptide chains, RTA and RTB, of approx the same molecular mass (30-32 kDa) linked to each other with a disulfide bond (Fig. 2). RTA is an N glycosidase, which removes a specific adenine residue from the 28S ribosomal RNA, thereby inhibiting protein synthesis. The RTB chain is a galactose-specific lectin that allows the RT to bind to the cell-surface glycoproteins and glycolipids on virtually all mammalian cells. Both chains also contain carbohydrate moieties, which are responsible, at least in part, for their interaction with the carbobydrate-binding lectins of liver cells. The procedure used in our laboratory for isolation and purification of RT and its RTA chain is as follows (Fig. 6). 

Many questions still remain, such as whether extracellular Ap modulates intracellular Ap, or the mechanism by which Ap accumulation leads to synaptic dysfunction. Other factors, such as oxidative stress, which is extensive in AD, may aid the early accumulation of Ap (Butterfield et al., 2(X)2b). AP peptides stimulate oxidative stress by direct and indirect mechanisms. AP-induced oxidative stress may result from an imbalance between reactive oxygen species (ROS) and reactive nitrogen species (RNS), which could react with a number of cellular macromolecular targets including proteins, lipids, carbohydrates, DNA, and RNA. An early marker for oxidative stress is the formation of protein carbonyls, 4-hydroxy-2-tra 5-nonenal (4-HNE) and 3-nitrotyrosme (3-NT), a marker for the nitration of proteins (Butterfield, 2002). Ap peptide can bind to mitochondrial proteins to generate free radicals, or it can promote oxidative stress via neuroinflanunation. Ap peptides stimulate microglial cells to release a neurotoxin, quinoUnic acid, which may also play a role in neurotoxicity (Guillemin et al., 2003). 

Different reporter groups have been attached to C5 of uracil, e.g., a number of fluorophores including naphthalene, pyrene, fluor-ene, benzofuran ° and the biaryl derivative (36) that also has an NMR label ( F). The structure of an RNA-DNA hybrid has been examined by EPR using a spin label attached to C5 of du/ A terpyridine moiety has been attached to C5 to chelate metal ions/ Different carbohydrates have been attached via a linker to C5 of dU and the resultant DNA was hybridised to form a three-way junction with the carbohydrate displayed on each arm to probe binding to lectin/ Two nitroimidazole derivatives attached to C5 via a methylene bridge have been described where they form interstrand crosslinks under hypoxic conditions/ The acyclic pyrimidine analogue (37) has been incorporated into the thrombin-binding aptamer where it was found to increase the efficacy of the aptamer/ ... 

Most eukaryotic mRNA molecules have up to 250 adenine bases at their 3 end. These poly (A) tails can be used in the affinity chromatographic purification of mRNA from a total cellular RNA extract. Under high salt conditions, poly (A) will hybridize to oligo-dT-cellulose or poly(U)-sepharose. These materials are polymers of 10 to 20 deoxythymidine or uridine nucleotides covalently bound to a carbohydrate support. They bind mRNA containing poly (A) tails as short as 20 residues. rRNA and tRNA do not possess poly (A) sequences and will not bind. After washing the mRNA can be eluted with a low salt buffer. 

---