1-[2-(Benzyl­amino)-4-pyrid­yl]-2-(4-fluoro­phen­yl)ethane-1,2-dione

The crystal structure of the title compound, C20H15FN2O2, contains two crystallographically independent mol­ecules, which are related by a pseudo-inversion center and linked into dimers via inter­molecular N—H⋯N hydrogen bonds. The 4-fluoro­phenyl ring of mol­ecule A makes dihedral angles of 17.17 (16) and 62.25 (15)°, respectively, with the phenyl and pyridine rings. The 4-fluoro­phenyl ring of mol­ecule B makes dihedral angles of 8.50 (16) and 64.59 (15)°, respectively, with the phenyl and pyridine rings. The dihedral angle between the pyridine ring and the phenyl ring of mol­ecule A [60.97 (15)°] is bigger than in mol­ecule B [59.49 (15)°]. The dihedral angle between the two pyridine rings is 1.37 (14)° and between the two phenyl rings is 3.64 (16)°

3-(4-Fluoro­phen­yl)-6-meth­oxy-2-(4-pyrid­yl)quinoxaline

In the title compound, C20H14FN3O, the quinoxaline system makes dihedral angles of 32.38 (7) and 48.04 (7)° with the 4-fluoro­phenyl and pyridine rings, respectively. The 4-fluoro­phenyl ring makes a dihedral angle of 57.77 (9)° with the pyridine ring. In the crystal, the mol­ecules form dimeric C—H⋯N hydrogen-bonded R 2 2(20) ring motifs lying about crystallographic inversion centers. The dimeric units stack via π–π inter­actions between methoxy­phenyl rings and pyridine–fluoro­phenyl rings with centroid–centroid distances of 3.720 (1) and 3.823 (1) Å, respectively. The respective average perpendicular distances are 3.421 and 3.378 Å, with dihedral angles between the rings of 1.31 (9) and 11.64 (9)°

Stereochemical bias introduced during RNA synthesis modulates the activity of phosphorothioate siRNAs

An established means of improving the pharmacokinetics properties of oligoribonucleotides (ORNs) is to exchange their phosphodiester linkages for phosphorothioates (PSs). However, this strategy has not been pursued for small interfering RNAs (siRNAs), possibly because of sporadic reports that PS siRNAs show reduced inhibitory activity. The PS group is chiral at phosphorous (Rp/Sp centres), and conventional solid-phase synthesis of PS ORNs produces a population of diastereoisomers. Here we show that the choice of the activating agent for the synthesis of a PS ORN influences the Rp/Sp ratio of PS linkages throughout the strand. Furthermore, PS siRNAs composed of ORNs with a higher fraction of Rp centres show greater resistance to nucleases in serum and are more effective inhibitors in cells than their Sp counterparts. The finding that a stereochemically biased population of ORN diastereoisomers can be synthesized and exploited pharmacologically is important because uniform PS modification of siRNAs may provide a useful compromise of their pharmacokinetics and pharmacodynamics properties in RNAi therapeutics.ISSN:2041-172

Site-specific conjugation of drug-like fragments to anti-miRNA oligonucleotides: pros and cons of targeting miRNAs in RISC

MicroRNAs (miRNAs) inhibit gene expression by binding mRNAs in a ribonucleoprotein (RNP) complex, miRISC. Some miRNAs, e.g. miR-122, are targets for oligonucleotide drugs (antimirs) in clinical trials. State of the art in antimir chemistry is the tightly RNA-binding bicyclic nucleic acids derivatives, but as they are not freely available researchers have sought new chemistries. Recently, the field was transformed by work confirming that antimirs inhibit miRNAs in the miRISC. We describe here a parallel synthesis of a miR-122 antimir library in which drug-like fragments were site-specifically introduced to a short 2’-O-methyl-RNA. This affected its silencing activity in cells in a position- and modification-dependent manner. We developed an assay – RNP chemical ligation PCR - and quantified the antimir presence in miRISC and showed it correlated with potency. Together, this approach represents a template for future design of a new class of short minimally-modified antimirs

Properties of short double-stranded RNAs carrying randomized base pairs: Toward better controls for RNAi experiments

Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective down-regulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs. All possible individual sequences were present in the pool with generally an excellent distribution of bases. Melting temperature analyses suggested that pools of randomized guide and passenger strands RNAs with up to eight degenerate positions annealed so that mismatched base-pairing was minimized. Transfections of randomized siRNAs (rnd-siRNAs) into cells led to inhibition of luciferase reporters by a miRNA-like mechanism when the seed regions of rnd-siRNA guide strands were devoid of degenerate positions. Furthermore, the mRNA levels of a select set of genes associated with siRNA off-target effects were measured and indicated that rnd-siRNAs with degenerate positions in the seed likely show typical non-sequence-specific effects, but not miRNA-like off-target effects. In the wake of recent reports showing the preponderance of miRNA-like off-target effects of siRNAs, our findings are of value for the design of a novel class of easily prepared and universally applicable negative siRNA controls.ISSN:1355-8382ISSN:1469-900

Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments

Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective down-regulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs. All possible individual sequences were present in the pool with generally an excellent distribution of bases. Melting temperature analyses suggested that pools of randomized guide and passenger strands RNAs with up to eight degenerate positions annealed so that mismatched base-pairing was minimized. Transfections of randomized siRNAs (rnd-siRNAs) into cells led to inhibition of luciferase reporters by a miRNA-like mechanism when the seed regions of rnd-siRNA guide strands were devoid of degenerate positions. Furthermore, the mRNA levels of a select set of genes associated with siRNA off-target effects were measured and indicated that rnd-siRNAs with degenerate positions in the seed likely show typical non-sequence-specific effects, but not miRNA-like off-target effects. In the wake of recent reports showing the preponderance of miRNA-like off-target effects of siRNAs, our findings are of value for the design of a novel class of easily prepared and universally applicable negative siRNA controls

Carrier-free gene silencing by amphiphilic nucleic acid conjugates in differented intestinal cells

Nucleic acid therapy can be beneficial for the local treatment of gastrointestinal diseases that currently lack appropriate treatments. Indeed, several oligonucleotides (ONs) are currently progressing through clinical trials as potential treatments for inflammatory bowel diseases. However, due to low uptake of carrier-free ONs by mucosal cells, strategies aimed at increasing the potency of orally administered ONs would be highly desirable. In this work, we explored the silencing properties of chemically modified and highly resistant ONs derivatized with hydrophobic alkyl chain on intestinal epithelial cells. We screened a set of lipid-ON conjugates for the silencing of model Bcl-2 mRNA and selected 2′-deoxy-2′-fluoro-arabinonucleic acid modified ON bearing docosanoyl moiety (L-FANA) as the most potent candidate with lowest toxicity. The efficacy of L-FANA conjugate was preserved in simulated intestinal fluids and in the inverted transfection setup. Importantly, L-FANA conjugate was able to downregulate target gene expression at both mRNA and protein levels in a difficult-to-transfect polarized epithelial cell monolayer in the absence of delivery devices and membrane disturbing agents. These findings indicate that lipid-ON conjugates could be promising therapeutics for the treatment of intestinal diseases as well as a valuable tool for the discovery of new therapeutic targets.ISSN:2162-253