An Improved Method for Surface Immobilisation of RNA: Application to Small Non-Coding RNA - mRNA Pairing

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.

Insights into transcription termination of Hfq-binding sRNAs of Escherichia coli and characterization of readthrough products

“…Another approach for studying RNA-RNA and RNA-protein interactions is the use of biotin-labeled RNAs along with streptavidin beads. Indeed, this approach has proved to be useful to study the sRNA-mRNA interaction (Vincent et al 2013). In this study, as we have outlined in results, we used the biotin-streptavidin system to study the interaction between sRNAs and Hfq.…”

Definition and identification of small RNA sponges: Focus on miRNA sequestration

“…After stringent washes, elute the associated RNAs and analyze them as per RAP method [97]. In vitro capture of miRNA target can also been performed by pulling down a cell lysate through the single-strand biotinylated miRNA directly coupled with the streptavidin support (beads or surface) [104,105]. The purified RNAs can be identified using several methods including RNA-Seq.…”

Reprogramming Gene Expression by Targeting RNA-Based Interactions: A Novel Pipeline Utilizing RNA Array Technology

“…We elected to use functional-RNA arrays 23 as the basis for the RNA−RNA Qrr sRNA−hapR mRNA binding interactions. Although other interaction methodologies could be employed, e.g., surface plasmon resonance (SPR) 36 or electrophoretic mobility shift assays (EMSAs), an advantage of using RNA array technology is its high-throughput capacity. This capacity can be used to perform multiple experimental repeats in parallel on a single RNA array, for a small number of related interactions, as we have done here for the four Qrr sRNA−hapR mRNA interactions.…”