Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye.

Purpose: The major challenges of developing an RNAi therapeutic include efficient delivery to and entry into the cell type of interest. Conventional ("naked" and chemically stabilized) small interfering RNAs (siRNAs) have been used in the eye in the past but they demonstrated limited clinical efficacy. Here we investigated a recently developed class of small, hydrophobic, asymmetric RNAi compounds.

Mechanism of the sulfurisation of phosphines and phosphites using 3-amino-1,2,4-dithiazole-5-thione (xanthane hydride).

Contrary to a previous report, the sulfurisation of phosphorus(III) derivatives by 3-amino-1,2,4-dithiazole-5-thione (xanthane hydride) does not yield carbon disulfide and cyanamide as the additional reaction products. The reaction of xanthane hydride with triphenyl phosphine or trimethyl phosphite yields triphenyl phosphine sulfide or trimethyl thiophosphate, respectively, and thiocarbamoyl isothiocyanate which has been trapped with nucleophiles. The reaction pathway involves initial nucleophilic attack of the phosphorus at sulfur next to the thiocarbonyl group of xanthane hydride followed by decomposition of the phosphonium intermediate formed to products.

Synthesis of phosphorothioamidites derived from 3'-thio-3'-deoxythymidine and 3'-thio-2',3'-dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3'-S-phosphorothiolate linkage.

The synthesis of N4-benzoyl-5'-O-dimethoxytrityl-2',3'-dideoxy-3'-thiocytidine and its phosphorothioamidite is described for the first time, together with a shortened procedure for the preparation of 5'-O-dimethoxytrityl-3'-deoxy-3'-thiothymidine and its corresponding phosphorothioamidite. The first fully automated coupling procedure for the incorporation of a phosphorothioamidite into a synthetic oligodeoxynucleotide has been developed, which conveniently uses routine activators and reagents. Coupling yields using this protocol were in the range of 85-90% and good yields of singularly modified oligonucleotides were obtained.

NMR and UV studies of 3'-S-phosphorothiolate modified DNA in a DNA : RNA hybrid dodecamer duplex; implications for antisense drug design.

High-resolution NMR spectroscopy has been used to establish the conformational consequences of the introduction of a single 3[prime or minute]-S-phosphorothiolate link in the DNA strand of a DNA : RNA hybrid. These systems are of interest as potential antisense therapeutic agents. Previous studies on similarly modified dinucleotides have shown that the conformation of the sugar to which the sulfur is attached shifts to the north (C(3[prime or minute])-endo/C(2[prime or minute])-exo).

Probing the effect of a 3'-S-phosphorothiolate link on the conformation of a DNA:RNA hybrid; implications for antisense drug design.

The effects of a single 3'-S-phosphorothiolate link in the DNA strand of a DNA:RNA dodecamer duplex is described; the sulfur induces a conformational shift in the (attached) sugar pucker, as shown by 1H NMR studies, and increases the thermal stability of the duplex compared to the non-modified system.