RRM
2006
Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Fluorescence based techniques have proven very useful in studying the structure and function of proteins and nucleic acids in general. However, purifying fluorophore labeled proteins can be particularly challenging if the protein denatures easily and the labeling reaction is not 100% efficient. In order to overcome this challenge, we are developing an efficient method to separate fluorophore labeled proteins from unlabeled proteins using an RNA aptamer based affinity column specific to the fluorophore.
SRB 2 is a 54 nucleotide long RNA aptamer that has been previously evolved in vitro to bind sulforhodamine. [Wilson C., et al. (1998), Folding & Design 3:423] Previous work suggests that the aptamer selectively binds the fluorophore by recognizing both the planar aromatic ring system and the negatively charged sulfonate group.
Here, we show proof of concept for the use of SRB 2 in purifying tetramethylrhodamine (TAMRA) labeled proteins. Our fluorescence anisotropy measurements show that SRB 2 also binds TAMRA tightly and selectively (KD = 18 ± 8 nM) and this binding requires Mg2+ ions (K1/2 = 260 ± 100 nM). We have exploited this property to effectively release TAMRA from the aptamer by chelating Mg2+ ions in solution using only 5 mM EDTA. Finally, we have successfully labeled the 3’ end of SRB 2 aptamer with biotin and immobilized it on streptavidin-coated beads to purify rhodamine labeled proteins.
This method is of particular interest because it allows protein purification in very mild conditions. Furthermore, it can be easily expanded to purify proteins labeled with any fluorophore by simply changing the RNA aptamer.
Keywords: RNA Aptamer, Anisotropy, Tetramethylrhodamine
