Applications of mass spectrometry to the study of siRNA

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. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:979–998, 2011     

Structure Determination of the Top‐Loop of the Conserved 3′‐Terminal Secondary Structure in the Genome of Flaviviruses

To what extent small differences in RNA sequences (mutations) can have a profound impact on biology remains an intriguing question. This effect can be studied by using untranslated RNA regions as a model. We have studied the influence of mutations on the structure of an RNA hairpin that occurs in the 3′‐untranslated region (UTR) of Flaviviridae, and is known to have a large impact on the vector dependency of flaviviruses. Three related RNA sequences were studied by NMR spectroscopy. The selected sequences represent each one of the three clusters in the flavivirus genes (mosquito‐borne, tick‐borne, and no‐known‐vector viruses). A new strategy was used to obtain chemical shift signatures of carbonyl atoms in unlabeled uridine nucleobases to characterize their involvement in hydrogen bonding. Clear differences occur in the structures and stacking pattern of the three RNA hairpins. The observed differences cannot be predicted based on sequence analysis. A different biology can be correlated with a different RNA tertiary structure. The underlying biological mechanism, however, remains to be studied.     

Quantum‐dots‐based detection of hepatitis C virus (HCV) NS3 using RNA aptamer on chip

BACKGROUND: Over 170 million people, more than 3% of the world's population, suffer from the hepatitis C virus (HCV) infection and the rate of death from liver‐related mortality to HCV has increased. In respect of this, the development of assays for biological imaging should be urgently considered as an essential factor in diagnosis. RESULTS: A novel HCV‐detecting technique using a nanoparticle‐supported aptamer probe was demonstrated. With the aid of nanoparticle quantum dots (QDs) with carboxyl group as an imaging probe, and 5′‐end‐amine‐modified RNA oligonucleotide as a capturing probe, target HCV NS3 was visually detected on chip. The QDs‐based RNA aptamer for HCV NS3 showed high selectivity and specificity against other protein such as BSA. The detection limit of HCV NS3 protein was 5 ng mL^?1 level. CONCLUSION: With a novel strategy for protein–aptamer interaction, the feasibility of applying QDs‐based fluorescent detection technique to HCV viral protein assay for the development of a protein biochip was demonstrated. This scheme of QDs‐mediated imaging with a target‐oriented specific RNA aptamer for the detection of infectious HCV diseases provides an efficient strategy and a promising new platform for monitoring applications. Copyright © 2010 Society of Chemical Industry     

Experience of a pilot-scale hydrogen-producing anaerobic sequencing batch reactor (ASBR) treating food waste

A pilot-scale H₂-producing anaerobic sequencing batch reactor (ASBR) treating food waste was operated. During the operation, the carbon/nitrogen (C/N) ratio was adjusted from 10 to 30 by changing the composition of the food waste. When the C/N ratio was lower than 20, the H₂ yield was maintained at around 0.5 mol H₂/mol hexose_added, accounting for 2.3% of energy conversion efficiency contained in food waste to H₂, but it gradually dropped at higher C/N ratios. The low performance was accompanied by increased production of lactate, propionate, and valerate. In order to recover the performance, alkaline shock (pH 12.5 for 1 day) was imposed on the entire mixed liquor in the fermenter. This alkaline shock method was so effective that the H₂ yield significantly increased to over 0.9 mol H₂/mol hexose_added, and was then stabilized at 0.69 mol H₂/mol hexose_added. In addition, the settling characteristics of H₂-producing ASBR, which was separated into three layers, were investigated. Ribonucleic acid (RNA) as well as volatile suspended solid concentrations of each layer were measured to suggest how to enhance the H₂ production in ASBR operation.