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1.  An Improved Method for Surface Immobilisation of RNA: Application to Small Non-Coding RNA - mRNA Pairing 
PLoS ONE  2013;8(11):e79142.
Characterisation of RNA and its intermolecular interactions is increasing in importance as the inventory of known RNA functions continues to expand. RNA-RNA interactions are central to post-transcriptional gene regulation mechanisms in bacteria, and the interactions of bacterial small non-coding RNAs (sRNAs) with their mRNA targets are the subject of much current research. The technology of surface plasmon resonance (SPR) is an attractive approach to studying these interactions since it is highly sensitive, and allows interaction measurements to be recorded in real-time. Whilst a number of approaches exist to label RNAs for surface-immobilisation, the method documented here is simple, quick, efficient, and utilises the high-affinity streptavidin-biotin interaction. Specifically, we ligate a biotinylated nucleotide to the 3′ end of RNA using T4 RNA ligase. Although this is a previously recognised approach, we have optimised the method by our discovery that the incorporation of four or more adenine nucleotides at the 3′ end of the RNA (a poly-A-tail) is required in order to achieve high ligation efficiencies. We use this method within the context of investigating small non-coding RNA (sRNA) - mRNA interactions through the application of surface technologies, including quantitative SPR assays. We first focus on validating the method using the recently characterised Escherichia coli sRNA-mRNA pair, MicA-ompA, specifically demonstrating that the addition of the poly-A-tail to either RNA does not affect its subsequent binding interactions with partner molecules. We then apply this method to investigate the novel interactions of a Vibrio cholerae Qrr sRNA with partner mRNAs, hapR and vca0939; RNA-RNA pairings that are important in mediating pathogenic virulence. The calculated binding parameters allow insights to be drawn regarding sRNA-mRNA interaction mechanisms.
doi:10.1371/journal.pone.0079142
PMCID: PMC3828260  PMID: 24244437
2.  PNA HyBeacons for analysis of human mutations related to statin-induced myopathy 
Artificial DNA, PNA & XNA  2011;2(3):79-89.
Aminoalkyl and alkyne-tagged PNA HyBeacons have been synthesized, labeled with fluorescein via conventional amide bond or triazole formation (click chemistry) and used to detect single nucleotide polymorphisms (SNPs) implicated in statin-induced myopathy. The PNA HyBeacons gave much better mismatch/mutant discrimination than conventional DNA HyBeacons but smaller fluorescence changes on melting.
PMCID: PMC3324338  PMID: 22567191
fluorescence melting; genetic analysis; HyBeacon; PNA; SNPs; statin-induced myopathy

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