An improved method for surface immobilisation of RNA: application to small non-coding RNA-mRNA pairing.
An improved method for surface immobilisation of RNA: application to small non-coding RNA-mRNA pairing.
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DOI：
10.1371/journal.pone.0079142
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Callaghan AJ
中科院分区：
文献类型：
--
作者：
Vincent HA;Phillips JO;Henderson CA;Roberts AJ;Stone CM;Mardle CE;Butt LE;Gowers DM;Pickford AR;Callaghan AJ
随着已知RNA功能的不断扩展，RNA及其分子间相互作用的表征越来越重要。RNA-RNA相互作用是细菌中转录后基因调控机制的核心，细菌小非编码RNA（sRNA）与其mRNA靶标的相互作用是当前许多研究的主题。表面等离子体共振（SPR）技术是一种有吸引力的方法来研究这些相互作用，因为它是高度敏感的，并允许相互作用的测量被实时记录。虽然存在许多方法来标记RNA用于表面固定化，但本文记载的方法简单、快速、有效，并且利用高亲和力链霉亲和素-生物素相互作用。具体来说，我们使用T4 RNA连接酶将生物素化的核苷酸连接到RNA的3′端。虽然这是一种以前公认的方法，但我们已经通过我们的发现优化了该方法，即为了实现高连接效率，需要在RNA的3′端掺入四个或更多个腺嘌呤核苷酸（聚A尾）。我们使用这种方法的背景下，调查小的非编码RNA（sRNA）- mRNA的相互作用，通过应用表面技术，包括定量SPR测定。我们首先专注于验证的方法，使用最近表征的大肠杆菌sRNA-mRNA对，MicA-ompA，特别是证明，无论是RNA的聚-A-尾的添加不影响其随后的结合与合作伙伴分子的相互作用。然后，我们应用这种方法来研究霍乱弧菌Qrr sRNA与伙伴mRNA，hapR和vca 0939的新的相互作用; RNA-RNA配对是重要的介导致病性的毒力。计算的结合参数允许洞察有关sRNA-mRNA相互作用机制。
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.
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