A series of nucleobase-modified siRNA duplexes containing ''rare'' nucleosides, 2-thiouridine (s 2 U), pseudouridine (C), and dihydrouridine (D), were evaluated for their thermodynamic stability and gene silencing activity. The duplexes with modified units at terminal positions exhibited similar stability as the nonmodified reference. Introduction of the s 2 U or C units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). In contrary, D unit similarly like wobble base pair led to the less stable duplexes (DTm 3.9 and 6.6°C, respectively). Gene-silencing activity of siRNA duplexes directed toward enhanced green fluorescent protein or beta-site APP cleaving enzyme was tested in a dual fluorescence assay. The duplexes with s 2 U and C units at their 39-ends and with a D unit at their 59-ends (with respect to the guide strands) were the most potent gene expression inhibitors. Duplexes with s 2 U and C units at their 59-ends were by 50% less active than the nonmodified counterpart. Those containing a D unit or wobble base pair in the central domain had the lowest Tm, disturbed the A-type helical structure, and had more than three times lower activity than their nonmodified congener. Activity of siRNA containing the wobble base pair could be rescued by placing the thio-nucleoside at the position 39-adjacent to the mutation site. Thermally stable siRNA molecules containing several s 2 U units in the antisense strand were biologically as potent as their native counterparts. The present results provide a new chemical tool for modulation of siRNA gene-silencing activity.
A series of water-soluble polycationic dendrimers with a phosphoramidothioate backbone (P-dendrimers) was studied in human cell culture. Preliminary studies have shown that P-dendrimers of series 1 and 2, possessing N,N-diethyl-ethylenediamine hydrochloride functions at the surface, show rather moderate cytotoxicity toward HeLa, HEK 293, and HUVEC cells in a standard MTT assay in serum-containing medium, generally lower than lipofectin. The experiments of cellular uptake have shown the necessity for the presence of serum for transfection with P-dendrimers of series 1 and 2. These compounds efficiently delivered fluorescein-labeled oligodeoxyribonucleotide into HeLa cells in serum-containing medium, but they failed to do so in HUVEC cell culture. The dendrimers were found to be successful mediators of transfection of the HeLa cells with a DNA plasmid containing the functional gene of enhanced green fluorescent protein (EGFP).
Four series of phosphorus dendrimers (generations 1 and 4) having various types of amine terminal groups (pyrrolidine, morpholine, methyl piperazine, or phenyl piperazine) are synthesized. After protonation, the fourth generations of three of them are found water-soluble, and used for several biological experiments. First, the cytotoxicity of these polycationic dendrimers towards three cell strains (one healthy: HUVEC, and two cancerous: HEK 293 and HeLa) is assayed and found low. Second, their ability to interact with DNA is tested by electrophoresis: the dendrimer terminated by pyrrolidinium groups is found efficient to form dendriplexes. Finally, the polycationic dendrimers are used as transfection agents to deliver singleand double-stranded DNA into the three above-mentioned cell strains. Here also the dendrimer having pyrrolidinium groups is found the most efficient.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.