RNA Aptamer Selection Against Fluorescent Proteins for Signaling Diagnostics

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Fluorescent proteins (FP’s), naturally found in such sea species as Aequorea victoria, serve to tag and label the various cellular elements present in the biological research field (Shcherbo 2007). As fluorescence implies, excitation with a specific wavelength of light causes the protein to emit a specific color. The most notable of the FP’s is green fluorescent protein (GFP), originating from the previously mentioned Aequorea victoria (Tsien 1998). From GFP, additional proteins have been derived that emit different colors and possess increased fluorescence and photostability (Heim 1995).

While this unique property of noninvasive labeling can be applied to such avenues as the visualization of tumor progression and the death of tissue, the following problems arise (Shcherbo 2007): fusing an FP protein to a protein of interest can impair the function of the target protein, potentially adversely affecting cellular function (Wiedenmann 2009); the lack of specificity between FP’s and the target protein limits the necessary protein-protein interaction. However, small oligonucleotides known as aptamers could allow for reduced or eliminated cytotoxicity as well as more specific and increased protein binding (Stoltenburg 2007).

These short nucleic acid sequences, successfully selected against protein targets in such fields as therapeutics and drug delivery, warrant hope for fluorescent imaging with their high binding affinity and antibody-like specificity (Stoltenburg 2007). While the selection process of an aptamer against FP’s could seemingly be avoided through direct covalent attachment of FP’s to target aptamer’s, there is reason to believe that non-covalent linkages (i.e. aptamer:protein interactions) could yield amplified FP fluorescence versus covalent linkages. Additionally, the research of Dr. Milan Stojanovic on modular aptameric sensors presents the possibility of aptamer:aptamer modules bolstering aptamer:target stability.

Using malachite green (MG), an organic dye, bound to a previously selected MG aptamer (MGA), the conjugate was attached to a flavin mononucleotide (FMN): FMN aptamer (FMNA) conjugate (Stojanovic 2004). Through experimental methods it was determined that the presence of FMN, as opposed to large concentrations of MG alone, resulted in a significantly reduced Kd for the aptameric sensor (from >750 to 30 μM) and a 30 to 50 fold increase in the fluorescence of MG (Stojanovic 2004). These findings demonstrate a detector, FMNA, channeling released energy from the stabilization of the aptameric complex, through the Watson-Crick bases communication module. From here, the energy traveled to an awaiting reporter, MGA, and manifested itself as augmented fluorescence of MG (Stojanovic 2004). In application to FP’s, Dr. Stojanovic hypothesizes that this signaling pathway could result in the altered emission color for each FP, a conclusion stemming from the observation that only a slight change in the conformation of GFP results in the FP color varieties (Tsien 1998). Consequently, if a highly malignant and progressive tumor was examined preliminarily with GFP, its observation 6 months later could yield a bluish color, a color of higher energy on the visible light spectrum, as opposed to the anticipated green.

Through the dealings of Dr. Brad Hall of the University of Texas at Austin and Dr. Vladislav Verkhusha of Albert Einstein College of Medicine, approximately one to two milligrams of three different fluorescent proteins, mTagBFP (blue), mTagGFP (green), and TagRFP(red), will be provided by the latter. In return for the proteins, rounds of in vitro RNA aptamer selection (SELEX) will be performed by Dr. Brad Hall’s students.


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Brad Hall said...

Good ideas Austin. As I commented to Alec, you try to think about why binding an aptamer non-covalently for signaling may be more beneficial than covalent interactions. If it is, then great! If it isn't, then the selection aims may be less useful. And a different purpose may be necessary.

Gwen Stovall said...

Take a look at the sequences of these targets. I believe they're really similar. I bet that if you find an aptamer against one of the FP that the aptamer will bind all of the FPs ... unless, of course, you do some really stringent negative selections against the unwanted FPs.

I agree with Brad's comments. It isn't really clear to me when a covalent attachment to GFP wouldn't work. Has your collaborator had problems?

And - could an aptamer alter the optical or fluorescent properties of a FP? That could be interesting. Has anyone seen this before?