Nucleic Acid Aptamer Selection Against Human Transferrin To Enhance Drug Delivery

Ranjeet Kar
September 11, 2012
Nucleic Acid Aptamer Selection against Human Transferrin to Enhance Drug Delivery to Address Anemia
Anemia is a blood disorder suffered by millions of humans who have insufficient normally functioning red blood cells. This can lead to the deprivation of oxygen to vital body tissues. One of the major causes can be a lack of iron, which the body needs to produce enough healthy blood cells. A way to address this would be target a molecule related to iron carrying and transport. One such molecule is human transferrin (often abbreviated to hTF) is a glycoprotein found in cells and blood serum that is responsible for the delivery of iron ions in the body (1). Transferrin, along with its corresponding specific cell surface receptor (TFR), forms a complex that ensures the efficient transport of iron (2). Iron–transferrin binding and delivery occurs through an endocytic cycle controlled by the pH of the environment (1)(3). This event triggers receptor-mediated endocytosis, where the transferrin–TFR complex is pinched inside the cell and combined with an endosome (3).
Transferrin is an attractive target for aptamers because of its importance throughout the body as well as its receptor’s significant presence in the blood–brain barrier (4). It could be used to address anemia. As shown in recent research, an aptamer that competes with transferrin to bind to TFR can be used to enhance drug delivery in cells with the receptor (4). A similar aptamer could bind to transferrin, enhancing its ability to carry siRNAs, which could address many problematic genes in target cells, including those related to anemia (4). Alternatively, an aptamer for transferrin could improve its iron-binding abilities.
Specific Aim 1: Identify an aptamer selection against transferrin using the SELEX method. Five rounds of selection were performed during the summer, followed by a binding assay with a nitrocellulose filter. Although the assay was not conclusive, the data point to the hypothesis that the RNA produced from five rounds did not bind sufficiently (though background binding was minimal), indicating the need for at least three more rounds of selection.
Specific Aim 2: Perform another binding assay after finishing selection to determine whether the generated RNA binds to the target protein. If the assay demonstrates sufficient binding, the next step would be to modify the aptamer so that it could survive the conditions of the human body and perform its intended function: enhance delivery of siRNAs.

Figure 1. The aptamer will bind to transferrin, which will be taken in through endocytosis. This could help enhance drug delivery.

Biotinylated transferrin (CAT # T3915) can be obtained from Sigma-Aldrich (800-325-3010) at a price of $101.00 per 5 mg, which amounts to $0.15 per round (assuming 100 pmol of target is used per round). It is also available in the −80°C freezer in PAI 2.14.

1.    Steere Ashley N. et al. Ionic Residues of Human Serum Transferrin Affect Binding to the Transferrin Receptor and Iron Release. Biochemistry. 2012; 51(2), 686–694
2.    Mason Anne B. et al. How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH. Proc Natl Acad Sci U S A. 2011; 108(32): 13089–13094
3.    Quarles C. et al. Competitive binding of Fe3+, Cr3+, and Ni2+ to transferrin. J Biol Inorg Chem (2011) 16:913–921
4.    Wilner, Samantha E. et al. An RNA Alternative to Human Transferrin: A New Tool for Targeting Human Cells. Molecular Therapy Nucleic Acids (2012) 1(21): 1–12 

Full Proposal
      Progress Report 1
      Progress Report 2
      Final Manuscript


Ankita B. said...

I like that you have used the approach to "enhance drug delivery." My approach for transferrin last semester was to be used as an inhibitor. It was interesting to read another possible use for an aptamer against transferrin.

Gwen Stovall said...

Dear Ranjeet,
Here are a few suggestions:
1. please add your abstract to Blackboard.
2. consider reorg to intro big problem (anemia), then narrow-in on the key player (transferrin) & then offer an aptamer sol'n.
3. abstract may only contain the key details vital to your story. Save extra details for your intro.
4. get rid of TFRs
5. Spseicific aim #1 - identify a transferrin aptamer. And - then go into the progress that you've made.
6. for abstract - remove what you're going to do in the sxn.
7. specific aim #2 - develop application (i.e. deliver siRNA?)
8. repost the figure.
9. budget - include molecular weigth, cost per round, & correct ordering info:
cat. #: T3915
5 mg/$101.00
MW = 76,500
10. consider other #2 ref or just omit
11. revisit the formating of your citations. Please be consistent.
Good work!
Best of luck on your selection!