Nucleic Acid Aptamer Selection Against Basic Fibroblast Growth Factor (FGF-2)

Nucleic Acid Aptamer Selection against Basic Fibroblast Growth Factor (FGF-2)

Jessica Nguyen, 18 September, Fall 2012

R50 RNA Pool against Basic Fibroblast Growth Factor

Abstract:

Although science and medicine have developed rapidly over the last decade, some diseases and conditions still evade present diagnostic and therapeutic techniques. For instance, cardiovascular disease, a class of disease that involves vessels of the heart, is the primary cause of death in the industrialized world with approximately 73 million Americans having some form of the disease ⁽¹⁾. Cancer, the uncontrolled growth of abnormal cells, is also considered to be one of the deadliest diseases of the modern world. Although these diseases seem unrelated, scientists have discovered that one protein, basic fibroblast growth factor (FGF-2), plays a vital role in both these conditions and various other conditions of the body. This research proposes that by finding an aptamer (an oligonucleotide with binding properties) against FGF-2 (also known as FGF-b), these diseases and others can be resolved with more efficiency and specificity.

FGF-2 has many effects on the body and is naturally present in basement membranes and in the extracellular matrix of blood vessels ⁽²⁾. It is a single-chain polypeptide growth factor that plays a significant role in the processes of wound healing, inducing angiogenesis or the growth of new blood vessels from existing ones, and supplementing undifferentiated growth in human embryonic stem cells ^{(1), (3), (4),(5)}. Because this protein has such a prominent role in many functions of the human body, an aptamer against FGF-2 could have multiple therapeutic and diagnostic effects in the body and could prove to be very promising. However, despite the many benefits of an aptamer against this protein, the specific aims of this research focus on the role of FGF-2 on cancer progression. 

Specific Aim 1: The first specific aim of this research will be to discover an aptamer that has a high binding affinity for FGF-2.  Seven rounds of selection have already been completed, along with one binding assay of Rounds 2, 4, and 6 (results showed that although positive binding was constant over rounds, negative binders did decrease) and sequencing of the Round 6 pool. More rounds of selection hope to be completed, and a binding assay done with clones found during sequencing will be conducted to test for high affinity binding between the clone and the target.    

Specific Aim 2: The next aim of this research is to modify the aptamer so that it can be used to diagnose and track the progression of cancer in the human body. Modifications that can be used include fluorescence.

Specific Aim 3: The third specific aim of this research will be to modify this aptamer so it can be used for drug delivery. This can be accomplished by collaboration with other labs that have discovered a functioning therapeutic against cancer but have no way of selectively targeting the abnormal cells (Figure 1). 

Figure 1: Specific Aims of Aptamer Research. FGF-2 plays an important role in the angiogenesis and metastasis of cancer. By targeting this protein target for aptamer selection, diagnostic and drug delivery systems can be created that inhibit the progression of cancer.

FGF-2 (17.3kDa) can be purchased from ScienCell Research Laboratories (877.602.8549) for $175 for 50 micrograms (CAT: 104-02). Each round would cost roughly $11.67. Currently, there is some available in the Aptamer -20°C fridge. 

Click here for full proposal. 

Click here for first progress report. 
Click here for second progress report.
Click here for final progress report.

References: 
1. House, Stacey L., et al. "Cardiac-Specific Overexpression of Fibroblast Growth Factor-2 Protects Against Myocardial Dysfunction and Infarction in a Murine Model of Low-Flow Ischemia." 108 (2003).

2. Soulet, Fabienne, et al. "Fibroblast Growth Factor-2 Interactes with Free Ribosomal Protein S19." 289 (2001): 591-596. 6 April 2012.

3. Zhang, Shuang-Xia, et al. "Gekko Sulfated Glycopeptide Inhibits Tumor Angiogenesis by Targeting Basic Fibroblast Growth Factor." (2012).

4. Pereira, Renata C., Aris N. Economides and Ernesto Canalis. "Bone Morphogenetic Proteins Induce Gremlin, a Protein That Limits Their Activity in Osteoblasts." 141.12 (2000).

5. Liu, Yanxia, et al. "A novel chemical-defined medium with bFGF and N2B27 supplements undifferentiated growth in human embryonic stem cells." 346 (2006).

6. Stoltenburg, Regina, Christine Reinemann and Beate Strehiltz. "SELEX - A revolutionary method to generate high-affinity nucliec acid ligands." 24 (2007).

7. Stovall, Gwen. "Protocol - Selection - Filter Based RNA." 2012.

8. Golden, Mace C., et al. "Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers." 81 (2000).

9. McCauley, Thomas G., Nobuko Hamaguchi and Martin Stanton. "Aptamer-based biosensor arrays for detection and quantification of biological macromolecules." 319 (2003).