Computational protocol: The 5′-tail of antisense RNAII of pMV158 plays a critical role in binding to the target mRNA and in translation inhibition of repB

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Protocol publication

[…] RNA structures were predicted by using the Mfold (3.2) program (Zuker, ). [...] Previous to the binding reaction, the antisense and target RNAs were incubated separately at 65°C for 10 min and then at 37°C for 10 min, in order to obtain their native conformation. The binding mixtures (10 μl) contained 0.2 nM of any of the 5′-end-labeled antisense RNA variants and various concentrations (0–400 nM) of the unlabeled target RNA (mRNA80) in TMN buffer (100 mM NaCl, 20 mM Tris-OAc, 10 mM Mg(OAc)2. Binding was allowed for 30 min at 37°C, and terminated by adding an equal volume of loading buffer (glycerol 30%, tRNA 4%, xylene cyanol 0.4%, and bromophenol blue 0.4% in TMN buffer) and placing the mixture on ice. The samples were applied to non-denaturing 10% PAA gels. Gels were run at 300 V for 2 h.Radioactive bands were visualized and quantified from fixed and dried gels using the storage phosphor technology, with the aid of a FLA-3000 (FUJIFILM) imaging system and the Quantity One software (Bio-Rad). The mRNA concentration required for half-maximal binding under the described experimental conditions (Kd) was obtained with the Sigmaplot 9.0 software from data point fitting to the equation:where y is the fraction of complexed antisense RNA, Bmax is the amplitude of the reaction, and x is the concentration of the target RNA. […]

Pipeline specifications

Software tools Mfold, SigmaPlot
Applications Miscellaneous, RNA structure analysis