RNA Aptamer Selection Against Fluorescent Proteins for Signaling Diagnostics

Click here to read the full proposal. Click here to read the final report.

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.


1. Hasegawa, H. et al. (2008) “Improvement of aptamer affinity by dimerization.” Sensors. 8:1090-1098.

2. Nutiu, R. et al. (2005) “Fluorescence-signaling nucleic acid-based sensors.” Landesbioscience.

3. Shcherbo, D. et al. (2007) “Bright far-red fluorescent protein for whole-body imaging.” Nature
Methods. 4:741-746.

4. Stojanovic, M., Kolpashchikov, D. (2004)”Modular aptameric sensors.” Journal of the American Chemical Society. 16(30):9266-9270.

5. Soltenburg, R. et al. (2007) “SELEX – A (r)evolutionary method to generate high-affinity nucleic acid ligands.” Biomedical Engineering. 24:381-403.

6. Hillisch, A. et al. (2001). “Recent advances in FRET: distance determination in protein – DNA complexes.” Current Opinion in Structural Biology. 11(2):201-207.

7. Heim, R. et al. (1995) “Improved green fluorescence.” Nature. 373:663-664.

8. Babendure, J. R. et al. (2003) “Aptamers switch on fluorescence on triphenylmethane dyes.” Journal of the American Chemical Society. 125(48):14716-14717.

9. Famulok, M. (2004) “Chemical biology: green fluorescent RNA.” Nature. 430:976-977.

10. Tsien, R. (1998) “The green fluorescent protein.” Annual Review of Biochemistry. 67:509-544.

11. Pu, Y. et al. (2009) “Aptamers for circulating tumour cells.” Clinical Laboratory International.

12. Payne, H. (2009) “Nobel prize in chemistry: green fluorescent protein.” Dartmouth Undergraduate Journal of Science.

13. Wiednenmann, J. et al. (2009) “Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges.” IUBMB Life. 61(11):1029-1042.


Brad Hall said...


Your writing style is very nice. You set up a situation leading to a problem. However, one of the questions I keep asking about this selection you pointed out but did not directly address. If we can covalently attach a fluorescent protein to an aptamer, why bother with the selection of the pool against the protein? I don't think the purpose is for bridging the GFP with an intracellular target per se. Vlad and Milan suggested that upon binding of GFP to an aptamer there may be a change in fluorescence. Otherwise, two aptamers binding different proteins fluorescent proteins can be covalently attached bringing the proteins together for FRET (fluorescence resonance energy transfer).

Alec Rezigh said...

Brad, I have edited the paper to fix the problem. However, should I focus just on the fluorescence being more intense, or should I continue with its potential biological uses?

Gwen Stovall said...

Cite the original GFP paper in the 1st paper ... sidenote - the 2008 Nobel prize was awarded for the discovery of GFP.

I agree with Brad's comments. Spend some more time considering the applications of such an aptamer & this will help you designing the selection scheme. For instance - the FP are very similar in amino acid sequence & an aptamer against one might bind all the FPs. Will this be OK? Or - will stringent negative selections be required?

Alec Rezigh said...

I have redirected the abstract, hopefully in the right direction. I would appreciate any feedback.

Brad Hall said...

Have Austin read this comment too. Here is information about the target from Milan and Vladislav:

Concentrations are 1mg/ml and the MW is around 27kDa which corresponds to around 37uM. We have mTagBFP (blue), mTagGFP (green) and mTagRFP (red) all with very different external residues. They have polyHis tags at the N-terminal end.

They should be aliquoted and stored at -80C. Here are the refs to the papers:

Merzlyak et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods (2007) vol. 4 (7) pp. 555-7

Shcherbo et al. Bright far-red fluorescent protein for whole-body imaging. Nat Methods (2007) vol. 4 (9) pp. 741-6

Subach et al. Conversion of Red Fluorescent Protein into a Bright Blue Probe. Chemistry & Biology (2008) vol. 15 (10) pp. 1116-1124

Alec Rezigh said...

Thanks, Brad.