RNA APTAMER SELECTION AGAINST TRANSFERRIN: “ESCORT APTAMERS” FOR TUMOR TARGETED DELIVERY

Shruti Singh
Target: Transferrin
RNA Pool: N40B



Cancer is the second most leading cause of death in the United Sates according to the Centers for Disease Control and Prevention. Cancer develops from malignant tumor cells, which are abnormal cells that grow uncontrollably. Destroying the tumor cells can inhibit the development of cancer. A protein that is highly expressed in tumor cells, like transferrin, provides a way to delve into this idea and address the issue of cancer.
Transferrin is a protein involved in the transport of ferric iron to all proliferating cells in the body (1). Transferrin binds to receptors on the cell surface and delivers the iron to the cell by endocytosis (2). Tumor cells have higher requirements for iron as they are rapidly proliferating cells, and hence have more transferrin receptors on the cell surface than normal cells (3), making transferrin a great candidate for drug development in cancer research.  Aptamers, which are nucleic acid oligonucleotides having high and specific affinity for molecular targets (4), can be used for this purpose. Anti-transferrin aptamers may be used as delivery agents or “escort aptamers” to deliver radionuclides, toxins, or cytotoxic agents to the tumor cells (3), which can potentially bring about the death of tumor cells. A similar research project was performed by Dr. Levy, which resulted in the discovery of an RNA aptamer with a high affinity for the transferrin receptor. The aptamer bound to the transferrin receptor was internalized by the cells through endocytosis (5). Hence, it could prove useful in applications of targeted drug delivery.
Previously, six rounds of bead-based selection of RNA N50 were carried out against transferrin using the SELEX process. The selection buffer used was 1X PCR Buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, and 5 mM MgCl2). The selection washes were increased over the rounds and ended with a final wash of 3mL(Round 1-Round 4: 3*400ul washes, Round 5: 5*500ul washes, and Round 6: 6*500ul washes). The concentration of transferrin (R1-R3: 400pmol, R4-R5: 200pmol, and R6: 100pmol) and RNA (R1: 200pmol, R2-R3: 400pmol, and R4-R6: 200pmol) was also decreased as the rounds progressed. A bead-based binding assay was performed on Round 2, Round 4, and Round 6 using biotinylated transferrin, streptavidin Dynal beads, and R50 RNA pool. The percentage of RNA bound to transferrin increased from 1.8% in round 2 to 4.6% in round 4, however it decreased to 3.8% in round 6 (Figure 2) . Since the binding assay results were contrary to the expected outcome, the selection was  restarted from Round 1, using a new RNA pool (RNA N40B). This time a more stringent selection will be performed by increasing the number and volume of washes and incorporating negative selections from the beginning.

Specific Aim 1:  Selection of aptamer against transferrin
Transferrin delivers iron to the cells by binding to the transferrin receptors resulting in the intake of the iron-bound transferrin into the cell. Selection of an aptamer which specifically binds to transferrin can allow delivery of toxic materials to tumor cells resulting in their death.

Specific Aim 2:  Modifying the aptamer for delivery of toxins
After finding a specific aptamer which binds to transferrin, it could be used to deliver toxins to tumor cells to cause cell death. A cytotoxic molecule attached  to the transferrin-specific aptamer can be enter a tumor cell through endocytosis by transferrin receptors on the cells.

Figure 1: The transferrin aptamer will deliver the toxic agent into the cancerous cell through endocytosis by transferrin receptor, and result in the cell to die.

Figure 2: Previous Binding Assay Results: 100 pmol of RNA selected from Rounds 2, 4 and 6 was radiolabeled and incubated with 100 pmol bead-immobilized transferrin. The washes and the elution were then passed through a nylon filter which captured the protein and the RNA bound to the protein. This amount of RNA bound was then quantifies by measuring the radioactivity on the filter.


Transferrin-biotin was ordered from Sigma-Aldrich. It was prepared from human holo-transferrin, coupled to biotin and its product number is T3915. The cost for 5mg of transferrin-biotin is $100. The concentration of transferrin provided was 100pmol/ul. Hence, the cost of transferrin (for 400pmol) per round is $0.63.

References:
  1. Gomme, Peter T., Karl B. McCann, and Joseph Bertolini. "Transferrin: Structure, Function and Potential Therapeutic Actions." Drug Discovery Today 10.4 (2005): 267-73.National Center for Biotechnology Information. U.S. National Library of Medicine, 9 Feb. 2005. Web. 04 Apr. 2012. <http://www.ncbi.nlm.nih.gov/pubmed/15708745>.
  2. Hemadi, Miryana, Philippe H. Kahn, Genevieve Miquel, and Jean-Michel E.H. Chahine. "Transferrin's Mechanism of Interaction with Receptor 1." Biochemistry 43.6 (2004): 1736-745. PubMed. Web. 4 Apr. 2012.
  3.  Niitsu, Yoshiro, Yutaka Kohgo, Takuji Nishisato, Hitoshi Kondo, Junji Kato, Yoichi Urushizaki, and Ichiro Urushizaki. "Transferrin Receptors in Human Cancerous Tissues." The Tohoku Journal of Experimental Medicine 153.3 (1987): 239-43.Journal Archive. Tohoku University Medical Press, 31 Aug. 2006. Web. 5 Apr. 2012.
  4.   Hicke, Brian J., and Andrew W. Stephens. "Escort Aptamers: A Delivery Service for Diagnosis and Therapy." Journal of Clinical Investigation 106.8 (2000): 923-28.PubMed Central. U.S. National Library of Medicine, 15 Oct. 2000. Web. 5 Apr. 2012.
  5. Samantha E Wilner, Brian Wengerter, Keith Maier, Maria de Lourdes Borba Magalhães, David Soriano Del Amo, Supriya Pai, Felipe Opazo, Silvio O Rizzoli, Amy Yan, and Matthew Levy. "An RNA Alternative to Human Transferrin: A New Tool for Targeting Human Cells." Molecular Therapy- Nucleic Acids , 15 May 2012. American Society of Gene and Cell Therapy. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390244/>


3 comments:

Gwen Stovall said...

Hi Shruti,
Here are a few suggestions:
1. Capitalize CDC.
2. consider transitional sentence b/t cancer is bad & transferrin intro in 1st paragraph.
3. Include a few more details about what's been performed (binding assay & specific % bound, etc.)
4. include cost per round
5. consider mentioning Dr. Levy's anti-transferrin receptor aptamer applications.
6. Please ck to make sure there's enough biotinylated transferrin in stock. If not, let me know & I'll order more.
7. Add the binding assay results to the abstract.

Nice work!
Gwen

Gwen Stovall said...

what's the molecular weight of your target? 76.5 kDa?

Thanks,
Gwen

Shruti Singh said...

Yes, the molecular weight of transferrin is about 77kDa.