RNA Aptamer Selection Against Fluorescent Proteins for Signaling Diagnostics

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Fluorescent proteins (FP’s) function as biological light-switches: switching on and exciting whenever the appropriate wavelength of light is shined upon them. The first fluorescent protein discovered, green fluorescent protein (GFP), was isolated from the jellyfish Aequorea victoria. Through progressive research, modifications have been uncovered that maximize the protein’s potential, increasing fluorescence intensity, photostability, and even shifting the range of excitation. Mutations have also led to different fluorescent colors, such as blue and red (Heim 1995). The distinctive properties of these proteins provide opportunities for noninvasive labeling and in vivo cell tracking. With the development and enhancement of whole-body imaging, fluorescent proteins also allow visualization of changes in target-gene promoter activity, cell inflammation, and tumor trafficking and metastasis (Shcherbo 2007).

However, problems exist. Excitation of the protein, requiring a specific wavelength of light, can induce phototoxic effects in cells, especially at shorter wavelengths. Furthermore, fusing a fluorescent protein to a desired protein target can impair the latter’s function, potentially affecting cellular activity and health (Wiedenmann 2009). However, aptamers could provide an avenue into unimpaired FP interaction and an increase in potential targets.

Aptamers, short nucleic acid oligomers with a complex three-dimensional shape, possess the ability to bind to specific targets, similar in respect to antibodies. However, aptamers are generally more efficient than antibodies as their small size permits them to penetrate into cells and tissues more effectively (Stoltenburg 2007). Although an aptamer pool can be synthesized with a functionalized fluorescent protein, Dr. Milan Stojanovic, a prime researcher in this field, suggests that this diminishes fluorescence intensity. Work performed by Dr. Stojanovic, on the conjugation of the malachite green (MG) and flavin mononucleotide (FMN) aptamers via Watson-Crick base pairs, was particularly intuitive. Upon the binding of FMN to its aptamer, the stability of the entire conjugate was improved, increasing MG binding. This increase in stability led to the transfer of energy from the FMN aptamer to the bound MG, exciting its fluorophore, and thus increasing its fluorescence. Dr. Stanjovic hypothesizes that such signaling, unavailable in functionalized FP-aptamers, could increase the fluorescence intensity, or even alter the color of the fluorescent protein (Stojanovic 2004). The change in color hypothesis is likely derived from GFP’s structure, where a slight change in conformation leads to the many color varieties of FPs commercially available (Tsien 1998). Such a change in color, or increase in fluorescence, could signify tumor progression or inflammation. Through the development of a signaling FP aptamer – target aptamer conjugate, medical diagnostics could be further advanced and perhaps simplified, through a visible change in color.

The fluorescent proteins have been previously provided by Dr. Vladislav Verkhusha of the Albert Einstein College of Medicine in New York. An agreement has been struck with Dr. Brad Hall and Dr. Verkhusha and approximately one to two milligrams of three different fluorescent proteins (all of different colors) have been provided. In exchange for the proteins, rounds of in vitro RNA aptamer selection (SELEX) will be performed.

References

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