Mesyl phosphoramidate antisense oligonucleotides as an alternative to phosphorothioates with improved biochemical and biological properties

Here we describe a DNA analog in which the mesyl (methanesulfonyl) phosphoramidate group is substituted for the natural phosphodiester group at each internucleotidic position. The oligomers show significant advantages over the often-used DNA phosphorothioates in RNA-binding affinity, nuclease stability, and specificity of their antisense action, which involves activation of cellular RNase H enzyme for hybridization-directed RNA cleavage. Biological activity of the oligonucleotide analog was demonstrated with respect to pro-oncogenic miR-21. A 22-nt anti–miR-21 mesyl phosphoramidate oligodeoxynucleotide specifically decreased the miR-21 level in melanoma B16 cells, induced apoptosis, reduced proliferation, and impeded migration of tumor cells, showing superiority over isosequential phosphorothioate oligodeoxynucleotide in the specificity of its biological effect. Lower overall toxicity compared with phosphorothioate and more efficient activation of RNase H are the key advantages of mesyl phosphoramidate oligonucleotides, which may represent a promising group of antisense therapeutic agents.

Oligoribonuclease is a central feature of cyclic diguanylate signaling inPseudomonas aeruginosa

The second messenger cyclic diguanylate (c-di-GMP) controls diverse cellular processes among bacteria. Diguanylate cyclases synthesize c-di-GMP, whereas it is degraded by c-di-GMP–specific phosphodiesterases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of glutamate-alanine-leucine (EAL) domains, which hydrolyze a single phosphodiester group in c-di-GMP to produce 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG). However, to degrade pGpG and prevent its accumulation, bacterial cells require an additional nuclease, the identity of which remains unknown. Here we identify oligoribonuclease (Orn)—a 3ʹ→5ʹ exonuclease highly conserved among Actinobacteria, Beta-, Delta- and Gammaproteobacteria—as the primary enzyme responsible for pGpG degradation inPseudomonas aeruginosacells. We found that aP. aeruginosaΔornmutant had high intracellular c-di-GMP levels, causing this strain to overexpress extracellular polymers and overproduce biofilm. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degrading RNA oligomers comprised of two to five nucleotides (nanoRNAs), including pGpG. Corresponding with this activity, Δorncells possessed highly elevated pGpG levels. We found that pGpG reduced the rate of c-di-GMP degradation in cell lysates and inhibited the activity of EAL-dependent PDEs (PA2133, PvrR, and purified recombinant RocR) fromP. aeruginosa. This pGpG-dependent inhibition was alleviated by the addition of Orn. These data suggest that elevated levels of pGpG exert product inhibition on EAL-dependent PDEs, thereby increasing intracellular c-di-GMP in Δorncells. Thus, we propose that Orn provides homeostatic control of intracellular pGpG under native physiological conditions and that this activity is fundamental to c-di-GMP signal transduction.

A triazole linkage that mimics the DNA phosphodiester group in living systems

We describe the development of a chemical process based on the CuAAC reaction (click chemistry) to ligate DNA strands and produce an unnatural triazole backbone linkage. The chemical reaction is templated by a complementary DNA splint which accelerates the reaction and provides the required specificity. The resultant 1,4-triazole linkage is read through by DNA and RNA polymerases and is biocompatible in bacterial and human cells. This work has implications for the synthesis of chemically modified genes and other large modified DNA and RNA constructs.

Coenzymes II: Metallic Coenzymes

The original coenzymes were small organic molecules that activated enzymes and participated directly in catalyzing enzymatic reactions. Most of them were derived from vitamins and were known as biologically “activated” forms of vitamins such as niacin, riboflavin, thiamine, and pyridoxal. Heme was in a separate category, perhaps because of its widespread biological role as an oxygen carrier, and because it was not a vitamin, it was not widely regarded as a coenzyme. However, heme was clearly an enzymatic prosthetic group in enzymes such as peroxidases and catalase, and it was known to participate in catalysis. Today, heme takes its place among the coenzymes. Other, more recently discovered metallic cofactors round out this chapter on metallocoenzymes. Most of the detailed mechanisms of metallocoenzyme-dependent reactions are not known. Hypothetical mechanisms can often be written, and some of them are supported by a few experiments. Emerging principles are emphasized here for several of the more extensively studied metallocoenzymes. In other cases, the detailed mechanisms that we include in figures and schemes must be regarded as conjectural. We do not regard them as fanciful, but they have not been proved and are referred to as “a mechanism for” in recognition that other possible mechanisms have not been excluded. Space does not permit all conceivable mechanisms to be aired, and we hope that those shown here will stimulate discussion and experimentation. Vitamin B12 coenzymes may be regarded as transitional from traditional coenzymes, in that the parent cyanocobalamin is a true vitamin, and its biologically activated forms adenosylcobalamin and methylcobalamin, with their covalent cobalt-carbon bonds, are organometallic compounds. For these reasons, we begin by discussing the vitamin B12 coenzymes. The structure in fig. 4-1 is that of adenosylcobalamin, the first B12 coenzyme to be discovered. The molecule consists of the tetradentate corrin ring, cobalt in its 3+ oxidation state held within the corrin ring, the lower axial dimethylbenzimidazole α-ribotide ligand linked by a phosphodiester group to the corrin, and the 5'-deoxyadenosyl moiety covalently bonded to cobalt. The corrin ring is structurally and biosynthetically related to heme, but it differs in a number of respects, including that it is more highly reduced and incorporates extensive stereochemistry.

Stroke in an Early Adolescent with Systemic Lupus Erythematosus and Coexistent Antiphospholipid Antibodies

Thromboembolic events in systemic lupus erythematosus (SLE) are an occasional but significant occurrence. Cerebrovascular disease in adults with SLE has been well described.1-4 Antiphospholipid antibodies, such as lupus anticoagulant, an immunoglobulin directed against the platelet phospholipid component of the prothrombin activator complex, and the false-positive serologic tests for syphilis, which detect reaginic antibodies that react with the purified beef cardiolipin substrate of these assays, have been reported for a long time in association with these events.5-9 More recently, development of an assay for anticardiolipin antibodies, immunoglobulins directed against the phosphodiester group of negatively charged phospholipids, has led to wider exploration of this subset of antiphospholipid antibodies and their relation to thrombosis.10,11

Alkylation of deoxyribonucleic acid by carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3′-5′)thymidine

1. The ethyl phosphotriester of thymidylyl(3′-5′)thymidine, dTp(Et)dT, was identified as a product from reaction of DNA with N-ethyl-N-nitrosourea, by procedures parallel to those reported previously for the methyl homologue produced by N-methyl-N-nitrosourea. 2. Enzymic degradation to yield alkyl phosphotriesters from DNA alkylated by these carcinogens and by dimethyl sulphate and ethyl methanesulphonate was studied quantitatively, and the relative yields of the triesters dTp(Alk)dT were determined. The relative reactivity of the phosphodiester group dTpdT to each of the four carcinogens was thus obtained, and compared with that of DNA overall, or with that of the N-7 atom of guanine in DNA. Relative reactivity of the phosphodiester group was lowest towards dimethyl sulphate, the least electrophilic of the reagents used, and was highest towards N-ethyl-N-nitrosourea, the most electrophilic reagent. 3. The nature of the alkyl group transferred also influenced reactivity of the phosphodiester site, since this site was relatively more reactive towards ethylation than would be predicted simply from the known Swain-Scott s values of the alkylating agents. It was therefore suggested that the steric accessibility of the weakly nucleophilic phosphodiester group on the outside of the DNA macromolecule favours its reaction with ethylating, as opposed to methylating, reagents. 4. Taking a value of the Swain-Scott nucleophilicity (n) of 2.5 for an average DNA nucleotide unit [Walles & Ehrenberg (1969) Acta Chem. Scand. 23, 1080-1084], a value of n of about 1 for the phosphodiester group was deduced, and this value was found to be 2-3 units less than that for the N-7 atom of guanine in DNA. 5. The reactivity of DNA overall was markedly high towards the alkylnitrosoureas, despite their relatively low s values. This was ascribed to an electrostatic factor that favoured reaction of the negatively charged polymer with alkyldiazonium cation intermediates.