ABSTRACT
RNA interference (RNAi) has quickly become a well-established laboratory tool for regulating gene expression and is currently being explored for its therapeutic potential. The design and use of double-stranded RNA oligonucleotides as therapeutics to trigger the RNAi mechanism and a greater effort to understand the RNAi pathway itself is driving the development of analytical techniques that can characterize these oligonucleotides. Electrospray (ESI) and MALDI have been used routinely to analyze oligonucleotides and their ability to provide mass and sequence information has made them ideal for this application. Reviewed here is the work done to date on the use of ESI and MALDI for the study of RNAi oligonucleotides as well as the strategies and issues associated with siRNA analysis by mass spectrometry. While there is not a large body of literature on the specific application of mass spectrometry to RNAi, the work done in this area is a good demonstration of the range of experiments that can be conducted and the value that ESI and MALDI can provide to the RNAi field.
Subject(s)
MicroRNAs/chemistry , RNA, Small Interfering/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Chromatography, High Pressure Liquid/methods , Oligonucleotides/chemistry , Oligonucleotides/therapeutic use , RNA Interference/physiology , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/therapeutic useABSTRACT
A pulsed ultrafiltration cell with a 35 microL binding chamber was evaluated for its ability to screen ligands that formed non-covalent complexes with protein targets. The cell was tested with ligands to the targets of carbonic anhydrase and serum albumin. Non-covalent ligand binding to both of these targets was observed and bound ligands were eluted from the cell in less than five min. The cell was also demonstrated to effectively screen a methanolic fermentation broth extract spiked with a known inhibitor to carbonic anhydrase. In addition to detecting specific binding events, the pulsed ultrafiltration method was investigated for its ability to distinguish non-specific binding events. Using carbonic anhydrase with the zinc-binding site removed, it was found that non-specific complexes observed when using electrospray ionization alone were not detected when using the pulsed ultrafiltration mass spectrometry method.
