Abstract
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.
Highlights
RNA is a multifaceted molecule with an ever-expanding repertoire of intra, inter-molecular and ligand-binding functions
The interactions of bacterial trans-acting small non-coding RNAs with their mRNA targets are the subject of much current research. sRNAs, which range from 50–200 nucleotides, have become increasingly recognised as a novel and ubiquitous class of gene expression regulator [6]
We have described a simple, quick and efficient strategy to biotinylate the 39 end of biologically relevant RNAs, ranging in length from 61 to 592 nucleotides, in order to facilitate their immobilisation to a sensor surface
Summary
RNA is a multifaceted molecule with an ever-expanding repertoire of intra-, inter-molecular and ligand-binding functions These include acting as the messenger for gene expression (mRNA), regulating gene expression (e.g. riboswitches, non-coding RNAs, siRNAs), catalysing biological processes (e.g. self-splicing ribozymes, the ribosome, telomerase) and providing mechanisms for viral infection [1], [2], [3], [4], [5]. SRNAs, which range from 50–200 nucleotides, have become increasingly recognised as a novel and ubiquitous class of gene expression regulator [6] They function through pairing with their mRNA targets via short regions of imperfect complementarity [7], in some cases promoted by the RNA chaperone protein, Hfq [8]. Determining the affinity of the interactions between such sRNAs and their mRNA targets is important for furthering our molecular-level understanding of this important post-transcriptional gene regulation mechanism
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