2'-O-Methyloligoribonucleotides have been synthesised on solid phase from base protected 5'-O-dimethoxytrityl-2'-O-methylribonucleoside-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) using 5-(4-nitrophenyl)-1H-tetrazole as activator. Coupling yields greater than 99% were achieved, as judged by trityl cation release. The preparation of a modified 2'-deoxycytidine building block bearing an N4-(5-trifluoroacetylaminopentyl) spacer is also described. The latter compound enabled the chemical synthesis of 2'-O-methyloligoribonucleotide probes carrying several 5'- terminal biotinylation sites (in general four modified residues were used), which can be conveniently 32P end-labelled enzymatically using polynucleotide kinase. Used in conjunction with streptavidin-containing derivatives, such biotinylated probes have important applications in biochemical purification and electron microscopy of RNA-protein complexes. The 2'-O-methyloligoribonucleotides are completely resistant to degradation by either RNA or DNA specific nucleases. In contrast, nucleases with dual RNA/DNA specificity show a complete spectrum of cleavage rates.

alpha,gamma-Diketo acids (DKA) were discovered from screening as selective and reversible inhibitors of hepatitis C virus NS5b RNA-dependent RNA polymerase. The diketo acid moiety proved essential for activity, while substitution on the gamma position was necessary for selectivity and potency. Optimization led to the identification of a DKA inhibitor of NS5b polymerase with IC(50) = 45 nM, one of the most potent HCV NS5b polymerase inhibitors reported.

ABSTRACT2'-O-Methyl oligoribonucleotides have recently been introduced as antisense probes for studying RNA processing and for affinity purification of RNA-protein complexes. To identify RNA analogues with improved properties for antisense analysis, 2'--alkyl oligoribonucleotides were synthesized in which the alkyl moiety was either the threecarbon linear allyl group or the five-carbon branched 3,3-dimethylallyl group. Both these analogues were found to be completely resistant to degradation by either DNA-or RNAspecific nucleases. Use of biotinylated derivatives of the probes to affinity-select ribonucleoprotein particles from crude HeLa cell nuclear extracts showed that the presence of the bulky

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

The syntheses of the four novel, base protected 5'-(S-triphenylmethyl)mercapto-2',5'-dideoxyribonucleoside-3 '-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) are described. These compounds have been used to prepare 5'-(S-triphenylmethyl) mercapto-oligodeoxyribonucleotides, which are readily purified by reversed phase h.p.l.c., owing to the highly lipophilic trityl group. After cleavage of the S-trityl group by silver or mercuric ions, the free thiol moiety can be coupled to a wide variety of reagents, generating very useful probes. Fluorescent labelled 5'-mercapto-oligodeoxyribonucleotides are being used for automated DNA sequencing without radioactivity, and heavy metal labelled 5'-mercapto-oligonucleotides will be used in X-ray crystallography.

The synthesis of base protected 5'-O-dimethoxytrityl-2'-O-[1-(2- fluorophenyl)-4-methoxypiperidin-4-yl]-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) is described, using phenoxyacetyl protection for the exocyclic amino groups of guanosine and adenosine and acetyl protection of the amino group of cytidine. High yield assembly of these building blocks into oligoribonucleotides on aminopropyl controlled pore glass was achieved using 5-(4-nitrophenyl)-1H-tetrazole as activator. Mixed sequences containing selected 2'-O-methylation were also synthesised and their significance for the study of RNA biochemistry is discussed.

In vivo electroporation of plasmid DNA (DNA-EP) is an efficient and safe method for vaccines resulting in increased DNA uptake, enhanced protein expression and increased immune responses to the target antigen in a variety of species. To further enhance the efficacy of DNA-EP, we have evaluated the toll-like receptor7 (TLR7) agonist-2, 9, substituted 8-hydroxyadenosine derivative or SM360320-as an adjuvant to vaccines against HER2/neu and CEA in BALB-neuT and CEA transgenic mice (CEA.Tg), respectively. SM360320 induced in vivo secretion of interferon a (IFNa) and exerted a significant antitumor effect in CEA.Tg mice challenged with a syngenic tumor cell line expressing CEA and an additive effect with a CEA vaccine. Additionally, combination of SM360320 with plasmid encoding the extracellular and transmembrane domain of ratHER2/neu affected the spontaneous tumor progression in BALB-neuT mice treated in an advanced disease setting. The antitumor effect in mice treated with DNA-EP and SM360320 was associated with an anti-CEA and anti-p185 neu antibody isotype switch from IgG1 to IgG2a. These data demonstrate that SM360320 exerts significant antitumor effects and can act in association with DNA-EP for CEA-positive colon cancer and HER2-positive mammary carcinoma. These observations therefore emphasize the potential of SM360320 as immunological adjuvant for therapeutic DNA vaccines.