ABSTRACT
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.
Subject(s)
Chromatography, Liquid/methods , Mass Spectrometry/methods , Molecular Probe Techniques , RNA Caps/chemistry , RNA, Messenger/chemistry , Ribonuclease H/chemistry , Sequence Analysis, RNA/methods , Molecular Probes/chemistry , Molecular Probes/genetics , RNA Caps/genetics , RNA, Messenger/genetics , Reproducibility of Results , Ribonuclease H/genetics , Sensitivity and Specificity , Staining and LabelingABSTRACT
Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle mediated delivery. The most clinically advanced siRNA-containing nanoparticles are polymer-coated supramolecular assemblies of siRNA and lipids (lipid nanoparticles or LNPs), which protect the siRNA from nucleases, modulate pharmacokinetics of the siRNA, and enable selective delivery of siRNA to target cells. Understanding the mechanisms of assembly and delivery of such systems is complicated by the complexity of the dynamic supramolecular assembly as well as by its subsequent interactions with the biological milieu. We have developed an ex vivo method that provides insight into how LNPs behave when contacted with biological fluids. Pulsed gradient spin echo (PGSE) NMR was used to directly measure the kinetics of poly(ethylene) glycol (PEG) shedding from siRNA encapsulated LNPs in rat serum. The method represents a molecularly specific, real-time, quantitative, and label-free way to monitor the behavior of a nanoparticle surface coating. We believe that this method has broad implications in gaining mechanistic insights into how nanoparticle-based drug delivery vehicles behave in biofluids and is versatile enough to be applied to a diversity of systems.
Subject(s)
Blood Chemical Analysis/methods , Lipids/chemistry , Magnetic Resonance Spectroscopy , Nanoparticles/chemistry , Polyethylene Glycols/analysis , RNA, Small Interfering/chemistry , Animals , Liposomes/chemistry , Male , RatsABSTRACT
A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the determination of clotrimazole in bulk drug and tablet dosage form. The stationary phase used was precoated silica gel 60F(254). The mobile phase used was a mixture of cyclohexane:toluene:methanol:triethyleamine (8:2:0.5:0.2 v/v/v/v). The detection of spot was carried out at 262 nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 200 to 1000 ng/spot for clotrimazole. The limit of detection and the limit of quantification for clotrimazole were found to be 50 ng/spot and 200 ng/spot, respectively. The proposed method can be successfully used to determine the drug content of bulk drug and marketed formulation of tablet.