Nucleic Acid Aptamers in the Study of Fibroblast Growth Factor 8b

Robert (Robby) Bedenbaugh


Fibroblast Growth Factor 8 (FGF-8) is a member of the growth factor family that plays pivotal roles in a number of developmental processes. FGF-8’s many functions can be attributed to alternative splicing, which generates eight different isoforms in mice(1). One isoform that has become a focus of many studies is Fibroblast Growth Factor 8b (FGF-8b). This isoform is most active during embryogenesis when it helps organize and induce development. For example, FGF-8b is extremely important in early neural development and differentiation of cells (2). Moreover, it has been shown to function in the isthmus where its activation of the Ras-ERK signaling pathway induces midbrain-hindbrain differentiation and the development of the cerebellum (3).

Although a wealth of information is know about FGF-8b there is still a great deal to be discovered. Despite many attempts, an antibody that sufficiently binds FGF-8b has not been developed. Therefore, protein concentrations have been assumed to be proportional to mRNA concentrations determined during RT-PCR studies(4). Not only could this assumption be inaccurate but also since the protein itself cannot be tagged and studied it is possible that the protein is not being produced and secreted despite the presence of FGF-8b mRNA in the cell. Moreover, it is not known whether or not the protein is sequestered within the cell before being secreted or how far it diffuses after secretion.

Aptamers provide an innovative solution to the afore mentioned problems.  If a high specificity, high affinity RNA ligand were developed to bind to FGF-8b it would be a valuable research tool in the study of the protein. This aptamer could be used to tag the protein and indicate which cells it is present in, where it is most concentrated, and even how far it is able to diffuse after being secreted from the cells. This and other information could be used to elucidate how the protein functions in development.

Specific Aim 1: Produce an aptamer via systematic evolution of ligands by exponential enrichment (SELEX) that will bind FGF-8b with high specificity. The SELEX method is a cycle that consists of the basic steps of partitioning the nucleic acid species that bind to the target from those that don’t, eluting these species, and then replicating them. This method starts with a random oligonucleotide library of around 10¹⁴ unique sequences, and after many rounds produces an Aptamer

Specific Aim 2: Employ the Aptamer to help determine which cells FGF-8b is produced in, where it is concentrated, and what roles it plays in development.

The Recombinant Mouse FGF8b that is necessary for the completion of this project will be ordered from R&D systems where its catalog number is 423-F8/CF. The protein is priced at $2000 dollars for .5 milligrams, which makes the cost per round for the target 18 dollars.

1.      Gemel, J. et al. (1996) “Structure and Sequence of Human FGF8.” Genomics 35(1):253-257

2.      Alam, A., Suzuki, H., Tsukahara, T. (2009)”Expression Analysis of FGF8a and FGF8b in Early Stage of P19 Cells During Neural Differentiation.” Cell Biology International. 33(9):1032-7.

3.      Suzuki, A., Harada, H., Nakamura, H. (2012) “Nuclear Translocation of FGF8 and its Implication to Induce Sprouty2.” Growth Development and Differentiation. 54(4):463-73.

4.      Abu-Issa, R. et al. (2002) “FGF8 is Required for Pharyngeal Arch and Cardiovascular Development in the Mouse.” Development. 129(19): 4613-4625.

Proposal
Progress Report 1

Progress Report 2
Final Report