Application Notes

The Decondensation factor 31 specifically binds to ssRNA but not to ssDNA or dsDNA

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2 Fig. 1 Measurement of Df31 binding to nucleic acids. MST values were normalized to fraction bound. 3 independent measurements where performed to obtain the depicted binding curve. The K d of the Df31-ssRNA interaction was 24.01 ± 3.15 µM. The calculated K d from the measurements of the Df31 binding to ssRNA was 24.01 ± 3.15 µM. As control we also measured the interaction of BSA and GST with our target ssRNA. The experiment was performed as described for the Df31 protein. No interaction could be detected. Conclusion The study provides an example that MicroScale Thermophoresis is capable of measuring and detecting specific interactions between nucleic acids and their interacting proteins. Straightforward control experiments proof the specificity of the interaction. Experiments are easily setup and affinities can be determined in a timely manner. Material and Methods Assay conditions For the experiment random FAM or Cy5-labeled ssRNA, ssDNA and dsDNA molecules of same sequence and length were used at the concentration of 50 nM. Unlabeled Df31 was added in 1:1 dilutions beginning at 680 µM. Samples were prepared in a buffer containing 20 mM Tris-HCl pH 7.4, 1.5 mM MgCl 2 , 0.5 mM EGTA, 200 mM KCl, 10 % Glycerol and 0.05 (v/v) % NP-40. For the measurement the samples were filled into standard capillaries. Instrumentation The measurements were performed on a NanoTemper Monolith NT.115 instrument. The measurement was performed in standard capillaries at 30 % LED and 30 % MST power with Laser-On time 30 sec and Laser-Off time 5 sec. References Luger, K., and Richmond, T. J. (1998). The histone tails of the nucleosome. Curr Opin Genet Dev 8, 140-146. Schubert, T., Pusch, M. C., Diermeier, S., Benes, V., Kremmer, E., Imhof, A., and Langst, G. (2012). Df31 Protein and snoRNAs Maintain Accessible Higher-Order Structures of Chromatin. Mol Cell. van-Holde, K. E. (1989). Chromatin. Springer Verlag, 497. Wolffe, A. P. (1997). Histones, nucleosomes and the roles of chromatin structure in transcriptional control. Biochem Soc Trans 25, 354-358. © 2013 NanoTemper Technologies GmbH

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