A label-free method for determining the 5'-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quantitative and qualitative information on the 5' cap was determined from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA preparations was determined to be between 88 and 98 % depending on the modification type and length of the mRNA. mRNA of 2.2K and 9K nucleotides in length and containing the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping. Graphical abstract mRNA 5' end analysis with RNAse H cleavage and capture probe.
Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed ''Dicer-substrate siRNA'' (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as duration of silencing in vivo. Duplexes from both structural classes tolerated 29-OMe chemical modifications well with respect to target silencing, although some modified dsiRNAs demonstrated reduced activity. On the other hand, dsiRNAs were more immunostimulatory as compared with the shorter siRNAs, both in vitro and in vivo. Because the dsiRNA structure does not confer any appreciable benefits in vitro or in vivo while demonstrating specific liabilities, further studies are required to support their applications in RNAi therapeutics.
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