The solution structure of Dead End bound to AU-rich RNA reveals an unusual mode of tandem RRM-RNA recognition required for mRNA regulation.

Dead End (DND1) is an RNA-binding protein essential for germline development through its role in post-transcriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1's tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA.

One- and Two-Dimensional High-Resolution NMR from Flat Surfaces.

Determining atomic-level characteristics of molecules on two-dimensional surfaces is one of the fundamental challenges in chemistry. High-resolution nuclear magnetic resonance (NMR) could deliver rich structural information, but its application to two-dimensional materials has been prevented by intrinsically low sensitivity. Here we obtain high-resolution one- and two-dimensional P NMR spectra from as little as 160 picomoles of oligonucleotide functionalities deposited onto silicate glass and sapphire wafers.

Towards Improved Oligonucleotide Therapeutics Through Faster Target Binding Kinetics.

When used as inhibitors of gene expression in vivo, oligonucleotides require modification of their structures to boost their binding affinity for complementary target RNAs. To date, hundreds of modifications have been designed and tested but few have proven to be useful. Among those investigated are mono- and polyamino-groups.

Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain.

The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood.

Chemical synthesis of long RNAs with terminal 5'-phosphate groups.

Long structured RNAs are useful biochemical and biological tools. They are usually prepared enzymatically, but this precludes their site-specific modification with functional groups for chemical biology studies. One solution is to perform solid-phase synthesis of multiple RNAs loaded with 5'-terminal phosphate groups, so that RNAs can be concatenated using template ligation reactions.

Site-Specific Labeling of MicroRNA Precursors: A Structure-Activity Relationship Study.

Functionalized oligoribonucleotides are essential tools in RNA chemical biology. Various synthetic routes have been developed over recent years to conjugate functional groups to oligoribonucleotides. However, the presence of the functional group on the oligoribonucleotide backbone can lead to partial or total loss of biological function.

A Small-Molecule Inhibitor of Lin28.

New discoveries in RNA biology underscore a need for chemical tools to clarify their roles in pathophysiological mechanisms. In certain cancers, synthesis of the let-7 microRNA tumor suppressor is blocked by an RNA binding protein (RBP) Lin28, which docks onto a conserved sequence in let-7 precursor RNA molecules and prevents their maturation. Thus, the Lin28/let-7 interaction might be an attractive drug target, if not for the well-known difficulty in targeting RNA-protein interactions with drugs.

Site-Specific Difunctionalization of Structured RNAs Yields Probes for microRNA Maturation.

Modified oligonucleotides bearing multiple functional labels are valuable tools in RNA biology. Efficient synthetic access to singly modified short DNAs and RNAs has been developed in the past years and paved the way to a first generation of oligonucleotide tools. Here, we describe an efficient procedure for the site-specific hetero bis-labeling of long RNAs.

Site-specific conjugation of drug-like fragments to an antimiR scaffold as a strategy to target miRNAs inside RISC.

We synthesized a miR-122 antimiR library in which drug-like fragments were site-specifically introduced to short 2'-O-methyl-RNAs. At some sites selected fragments elevated cellular antimiR activity to that of an unmodified 23mer antimiR, whereas at others the same fragments abolished activity. The potency of the antimiRs correlated with uptake into miRISC.

β-D-2'-C-Methyl-2,6-diaminopurine Ribonucleoside Phosphoramidates are Potent and Selective Inhibitors of Hepatitis C Virus (HCV) and Are Bioconverted Intracellularly to Bioactive 2,6-Diaminopurine and Guanosine 5'-Triphosphate Forms.

The conversion of selected β-D-2,6-diaminopurine nucleosides (DAPNs) to their phosphoramidate prodrug (PD) substantially blocks the conversion to the G-analog allowing for the generation of two bioactive nucleoside triphosphates (NTPs) in human hepatocytes. A variety of 2'-C-methyl DAPN-PDs were prepared and evaluated for inhibition of HCV viral replication in Huh-7 cells, cytotoxicity in various cell lines, and cellular pharmacology in both Huh-7 and primary human liver cells. The DAPN-PDs were pan-genotypic, effective against various HCV resistant mutants, and resistant variants could not be selected.

Short loop-targeting oligoribonucleotides antagonize Lin28 and enable pre-let-7 processing and suppression of cell growth in let-7-deficient cancer cells.

MicroRNAs (miRNAs) originate from stem-loop-containing precursors (pre-miRNAs, pri-miRNAs) and mature by means of the Drosha and Dicer endonucleases and their associated factors. The let-7 miRNAs have prominent roles in developmental differentiation and in regulating cell proliferation. In cancer, the tumor suppressor function of let-7 is abrogated by overexpression of Lin28, one of several RNA-binding proteins that regulate let-7 biogenesis by interacting with conserved motifs in let-7 precursors close to the Dicer cleavage site.

Azetidines and spiro azetidines as novel P2 units in hepatitis C virus NS3 protease inhibitors.

Herein, we report the synthesis and structure-activity relationship studies of new analogs of boceprevir 1 and telaprevir 2. Introduction of azetidine and spiroazetidines as a P2 substituent that replaced the pyrrolidine moiety of 1 and 2 led to the discovery of a potent hepatitis C protease inhibitor 37c (EC50=0.8 μM).

Synthesis and antiviral evaluation of bis(POM) prodrugs of (E)-[4'-phosphono-but-2'-en-1'-yl]purine nucleosides.

Seventeen hitherto unknown bis(POM) prodrugs of novel (E)-[4'-phosphono-but-2'-en-1'-yl]purine nucleosides were prepared in a straight approach and at good yields. Those compounds were synthesized by the reaction of purine nucleobases directly with the phosphonate synthon 3 bearing POM biolabile groups under Mitsunobu conditions. All obtained compounds were evaluated for their antiviral activities against a large number of DNA and RNA viruses including herpes simplex viruses 1 and 2, varicella zoster virus, Feline herpes virus, human cytomegalovirus, HIV-1 and HIV-2.

Synthesis of 5'-methylene-phosphonate furanonucleoside prodrugs: application to D-2'-deoxy-2'-α-fluoro-2'-β-C-methyl nucleosides.

A new and facile synthetic pathway to metabolically stable 5'-methylene-bis(pivaloyloxymethyl)(POM)phosphonate furanonucleoside prodrugs is reported. The key step involves a Horner-Wadsworth-Emmons reaction of a tetra(pivaloyloxymethyl) bisphosphonate salt with appropriately protected 5'-aldehydic nucleosides. This efficient approach was applied for the synthesis HCV related 2'-deoxy-2'-α-fluoro-2'-β-C-methyl nucleosides.

Balancing antiviral potency and host toxicity: identifying a nucleotide inhibitor with an optimal kinetic phenotype for HIV-1 reverse transcriptase.

Two novel thymidine analogs, 3'-fluoro-3'-deoxythymidine (FLT) and 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (Ed4T), have been investigated as nucleoside reverse transcriptase inhibitors (NRTIs) for treatment of HIV infection. Ed4T seems very promising in phase II clinical trials, whereas toxicity halted FLT development during this phase. To understand these different molecular mechanisms of toxicity, pre-steady-state kinetic studies were used to examine the interactions of FLT and Ed4T with wild-type (WT) human mitochondrial DNA polymerase γ (pol γ), which is often associated with NRTI toxicity, as well as the viral target protein, WT HIV-1 reverse transcriptase (RT).

Substrate mimicry: HIV-1 reverse transcriptase recognizes 6-modified-3'-azido-2',3'-dideoxyguanosine-5'-triphosphates as adenosine analogs.

β-D-3'-Azido-2',3'-dideoxyguanosine (3'-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3'-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3'-azido-ddG in primary cells. To gain insight into their structure-activity-resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT).

Microwave-assisted syntheses of nucleosides and their precursors.

In recent decades, nucleosides analogs have been the cornerstone in the treatment of various diseases, such as AIDS, herpes and hepatitis. More than 40 modified nucleosides are officially approved by the US FDA and represent the major compound class for inhibition of viral replication. By comparison with traditional conditions, microwave irradiation offers a powerful tool that can increase yields and decrease reaction time, with simple manipulation and an environmentally friendly way.

Novel antiviral C5-substituted pyrimidine acyclic nucleoside phosphonates selected as human thymidylate kinase substrates.

Acyclic nucleoside phosphonates (ANPs) are at the cornerstone of DNA virus and retrovirus therapies. They reach their target, the viral DNA polymerase, after two phosphorylation steps catalyzed by cellular kinases. New pyrimidine ANPs have been synthesized with unsaturated acyclic side chains (prop-2-enyl-, but-2-enyl-, pent-2-enyl-) and different substituents at the C5 position of the uracil nucleobase.

Preparation of ribavirin analogues by copper- and ruthenium-catalyzed azide-alkyne 1,3-dipolar cycloaddition.

In this study, we described the synthesis of 1,4- and 1,5-disubstituted-1,2,3-triazolo-nucleosides from various alkynes with 1'-azido-2',3',5'-tri--acetylribose using either copper-catalyzed azide-alkyne cycloaddition (CuAAC) or ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC), respectively. Optimized RuAAC conditions were realized with the commercially available [Cp*RuCl(PPh)] under microwave heating, which allows a significant acceleration of the reaction times (from 6 h to 5 min). This reaction can work under water-containing system.