Abstract
A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose beads. The beads are washed to remove non-specific complexes and then the RNA Mango containing complexes are eluted by the addition of free biotin to the beads. Since the eluted complexes are native and fluorescent, a second purification step such as size exclusion chromatography can easily be added and the purified complexes tracked by monitoring fluorescence. The high purity native complexes resulting from this two-step purification strategy can be then used for further biochemical characterization.
Keywords: RNA Mango, TO1-Desthiobiotin, RNP complex purification, Fluorophore, Native purification, Non-coding RNAs, RNA-protein complexes
Background
Current RNA tags suffer from limitations such as poor KD, large size, potential biological interference or lack of intrinsic fluorescence (Panchapakesan et al., 2015). RNA Mango is small, can be simply integrated into stem-loop structures, in particular, GNRA tetraloops, is biologically tolerated and above all has a high affinity for its thiazole orange-based (TO1) ligand, TO1-Desthiobiotin (TO1-Dtb). This allows Mango tagged complexes to be easily bound and washed on streptavidin beads (summarized in Figure 1). The Mango:TO1-Desthiobiotin complex is highly fluorescent and can be eluted from streptavidin beads by the addition of biotin. The fluorescence of Mango tagged native complexes allows additional purification steps to be used to obtain highly purified native complexes. Figure 1. Purification of Mango tagged RNA-protein complexes out of native extract. RNA constructs are designed (Procedure A, Figure 2) so that the Mango tag is located in a biologically compatible location. After bacterial expression of this construct, a native extract containing the Mango tagged RNA (Mango tag shown in red highlight), is prepared (Procedure B). Mango tagged RNA and RNA complexes are then bound to streptavidin beads (Black circle containing S) that have been derivatized with TO1-Desthiobiotin (Dtb, Procedure C). The thiazole orange moiety of TO1-Dbt is shown in purple and becomes highly fluorescent once bound by Mango (bright red highlight). Bound complexes are then extensively washed in native conditions (Procedure D). After washing fluorescent Mango tagged complexes can be eluted in native conditions by addition of biotin (Procedure E). Downstream analysis including further purification steps can then be simply implemented (Procedures F and G).
Materials and Reagents
Equipment
Procedure
Data analysis
For more data analysis such as denaturing gel (Step F2), SEC trace of the purified RNP complex (Step G1), the readers may refer to (Dolgosheina et al., 2014; Panchapakesan et al., 2017).
Notes
Recipes
Note: Buffers (prepare as 10x sterile filtered stocks, store at -20 °C or lower).
Acknowledgments
PJU acknowledges an NSERC Discovery operating grant. The authors declare no conflicts of interest or competing interests.
References
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Hi Laura, Solubility of Biotin is very low in water. You can make 100 mM stock or so by titrating with NaOH. Just add biotin in water and Keep on adding NaOH, say from a 2 M stock, till biotin dissolves. You can then use this stock to dilute into elution buffer of your choice. My email is shankershyams.panchapakesan@yale.edu. If you have any questions, you can contact me directly in that email. Cheers,Shyam
Sorry, read that as 200 mM biotin stock