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Expanding the catalytic repertoire of ribozymes to include vitamin synthesis requires efficient labelling of RNA with the substrate of interest, prior to in vitro selection. For this purpose, we rationally designed and synthesized six GMP-conjugates carrying a synthetic pre-thiamine or biotin precursor and investigated their transcription incorporation properties by T7 RNA polymerase. [ABSTRACT FROM AUTHOR]

T4 DNA ligase is capable of ligating 2′OMe-RNA duplexes, HNA, LNA and FANA mixed sequences in the presence of 10% w/v PEG8000 and 3 M betaine. The enzymatic joining of oligonucleotides containing multiple consecutive XNA nucleotides at the ligation site has not been reported before. [ABSTRACT FROM AUTHOR]

Methods for immobilization of DNA on solid supports are in high demand. Herein, we present a generally applicable enzymatic method for the immobilization of DNA without any prior chemical derivatization. This strategy relies on the homopolymerization of the modified triphosphate dImTP by the TdT. The resulting enzymatic his-tag mimic ensures binding of DNA on Ni-NTA agarose. The usefulness of this method is highlighted by the immobilization of functional nucleic acids without impairing their specific activities. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotide (AO) mediated exon skipping has been widely explored as a therapeutic strategy for several diseases, in particular, for rare genetic disorders such as Duchenne muscular dystrophy (DMD). To date, the potential of anhydrohexitol nucleic acid (HNA), cyclohexenyl nucleic acid (CeNA) and altritol nucleic acid (ANA) has not been explored in exon skipping. For the first time, in this study we designed and synthesised HNA, CeNA and ANA-modified 2′-O-methyl (2′-OMe) mixmer AOs on a phosphorothioate (PS) backbone, and evaluated their potential to induce exon 23 skipping in mdx mouse myotubes, as a model system. Our results clearly showed that all three AO candidates modified with HNA, CeNA and ANA could efficiently induce Dmd exon 23 skipping in vitro in parallel to the fully modified 2′-OMePS AO with reduced dual exon 22/23 skipping. In addition, they showed high nuclease resistance and no cytotoxicity compared to the 2′-OMePS AO, demonstrating the applicability of HNA, CeNA and ANA nucleotide-modified AOs in exon skipping. [ABSTRACT FROM AUTHOR]

l-Aspartic acid has recently been found to be a good leaving group during HIV reverse transcriptase catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. This showed that l-Asp is a good mimic for the pyrophosphate moiety of deoxyadenosine triphosphate. The present work explores the thermochemistry and mechanism for hydrolysis of several models for l-aspartic-dAMP using B3LYP/DGDZVP, MP2/6-311++G** and G3MP2 level of theory. The effect of the new compound is gradually investigated: starting from a simple methyl amine leaving group up to the aspartic acid leaving group. The enzymatic environment was mimicked by involving two Mg2+ions and some important active site residues in the reaction. All reactions are compared to the corresponding O-coupled leaving group, which is methanol for methyl amine and malic acid for aspartic acid. With methyl amine as a leaving group a tautomeric associative or tautomeric dissociative mechanism is preferred and the barrier is lower than the comparable mechanism with methanol as a leaving group. The calculations on the aspartic acid in the enzymatic environment show that qualitatively the mechanism is the same as for triphosphate but the barrier for hydrolysis by the associative mechanism is higher for l-aspartic-dAMP than for l-malic-dAMP and pyrophosphate. [ABSTRACT FROM AUTHOR]

The 4′-phosphonomethoxy analogs of inosine and 2′,3′-dideoxyinosine were synthesized and tested for their activity against HCV and HIV, but found to be inactive. During the course of this synthetic investigation, an unexpected oxidative cleavage of the 2′,3′-bond of 2′-deoxyinosine was observed. [ABSTRACT FROM AUTHOR]

The substantial impact of acyclic nucleoside phosphonates (ANPs) on human medicine encourages the synthesis of new ANP analogues with a potentially differentiated antiviral spectrum. Herein, we demonstrate the functionalization of the 2-position of the ( R , S )-3-hydroxy-2-(phosphonomethoxy)propyl side-chain of an inactive ANP with a polar cyano group to generate a thymine analogue with selective inhibition of hepatitis B virus (HBV) replication (SI > 302; EC 50 = 0.33 μM), without significant antiretroviral activity. These findings suggest new strategies to synthesize unique ANPs with a targeted antiviral profile., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Isothiazolo[4,3- b ]pyridines are known to be endowed with potent affinity for cyclin G associated kinase (GAK). In this paper, we expanded the structure-activity relationship study by broadening the structural variety at position 3 of the isothiazolo[4,3- b ]pyridine scaffold. The most potent GAK ligands (displaying Kd values of less than 100 nM) within this series carry an alkoxy group at position 3 of the central scaffold. Unfortunately, these ligands display only modest antiviral activity against the hepatitis C virus.

The pivaloyloxymethyl (PivOM) group is a biolabile 2'-O-ribose protection that is under development in a prodrug-based approach for siRNA applications. Besides an expected cellular uptake, nucleic acid sequences carrying PivOM showed also increased nuclease resistance and, in most cases, an affinity for complementary RNA. The r(CGCU*ACGC)dT:r(GCGUAGCG)dT model duplex containing a single modified residue (U*) was synthesized and its solution structure was determined by NMR. The duplex showed a maintained A-RNA helix. In U*, both 2'-O-acetal ester side chain and ring pucker presented a notable rigid conformation. The PivOM moiety was oriented with the carbonyl group turned outside the minor groove and with trans, -ac and -ac torsion angles around the C2'-O2', O2'-CA and CA-OB1 bonds respectively. Gauche effects and dipolar interactions between the PivOM and the backbone appeared to be the predominant factors influencing the PivOM conformation and the orientation of the two supplementary H acceptors suggested that hydration could also play a role in the duplex stability.

The anti-HIV activity of nucleoside analogues is highly related to their substrate specificity for cellular and viral kinase and, as triphosphate, for HIV-RT. A series of phosphoramidate d4T derivatives have been synthesized and evaluated as substrates for HIV-1 RT, and also tested for their in vitro anti-HIV activity. Compounds 2 and 4 are able to inhibit HIV-1 replication to the same extent as d4T and d4TMP in MT-4 cells as well as in CEM/0 cells and CEM/TK(-) cells. The data suggests that these phosphoramidates are hydrolysed to d4T before exerting their antiviral activity.

A series of sulf(on)ate and phosph(on)ate amino acid phosphoramidate analogues of deoxynucleotides were synthesized as potential substrates for HIV-1 reverse transcriptase. Taurine, L-cysteic acid, 3-phosphono-L-alanine, O-sulfonato-L-serine, and O-phospho-L-serine were investigated as leaving groups in an enzyme catalyzed DNA synthesis protocol. Among these analogues, the phosphonate congener performed best and 3-phosphono-L-alanine can be considered as an excellent mimic of the pyrophosphate (PPi) moiety of deoxyadenosine triphosphate, to be used in enzymatic synthesis of nucleic acids. During a single nucleotide incorporation assay the use of 3-phosphono-L-Ala-dAMP as substrate resulted in 95% conversion to a P + 1 strand in 60 min at 50 μM (a concentration 10 times less than found for L-Asp-dAMP) and with improved incorporation kinetics and less stalling. For the sequences investigated, the efficiency of the incorporation is base dependent and decreases in the order (A ≥ T = G > C). In all cases, the incorporation follows Watson-Crick rules.