Nucleic Acid Aptamer Selection Against Tie-2 to Limit Tumor Growth

Amika Alibhai 

Link to Proposal

Link to Progress Report 1 

Link to Progress Report 2 

Link to Final Manuscript

Nucleic Acid Aptamer Selection Against Tie-2 to Limit Tumor Growth

A tumor is a population of cancer cells, or cells that stop dividing in an orderly manner and start rapidly growing, dividing, and gaining mutations.  However, due to a lack of nourishment, lone tumors cannot grow past one or two cubic millimeters (1).  In order to grow larger, tumors require nutrients, which are usually delivered though blood vessels.
Tie-2, a receptor tyrosine kinase, is an endothelial cell surface receptor that binds to angiopoietins and mediates cell signaling through phosphorylation and binding and activation of enzymes (2).  An angiopoietin is a protein growth factor required for angiogenesis, the formation of blood vessels.  Angiopoietins function by binding receptor tyrosine kinases, such as Tie-2, which is mandatory for normal embryonic vascular development and tumor angiogenesis (3).  While angiogenesis is vital in the healing of wounds, it also serves in the growth and transition of tumors from dormant to malignant, as well as contributing to metastasis.  Tie-2, by mediating the effects of angiopoietins, is an integral receptor in the pathway of blood vessel formation (4).
As tumors are nourished and led through metastasis, the process becomes more agonizing for cancer patients and doctors alike.  To manage this there are general antibody treatments available, but another method of therapy is possible.  Such a possibility comes in the form of an aptamer, which is a short sequence of nucleic acids that binds with great specificity and strength to a particular target.  Isolated in vitro from vast libraries of sequences, aptamers can fold into tight binding pockets for nearly any molecule (5).  Aptamers are expected to be able to bind Tie-2; an aptamer that blocks the binding site of angiopoietins would reduce signaling vital to the growth of tumors by angiogenesis.  In this respect, an aptamer would be beneficial in cancer treatment and therapy by limiting the progression of tumors in early stages.
Specific Aim 1:  RNA aptamer selection against Tie-2
RNA aptamers are selected through the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process (6).  Tie-2 proteins are functionalized with 6X Histidine tags, lending to use of Nickel-NTA magnetic beads in the process of selection.
Specific Aim 2:  Develop an aptamer therapeutic to limit tumor growth
Upon the binding of the angiopoietins, Tie-2 provides the signaling essential to angiogenesis.  Tie-2 also mediates the actions of angiopoietins in their prevention of apoptosis, further aiding the growth of blood vessels (7).  An aptamer selected against Tie-2 may inhibit the receptor, thus suppressing the development and spreading of tumors by depriving the pathway of the required signaling.  

Figure 1.  The progression from Tie-2 to tumor growth.
A comparison is shown between an angiopoietin and an aptamer in binding to Tie-2.

Recombinant Human Tie-2 with a 6-His tag is available online from R&D Systems (  The catalog number is 313-TI-100 and the price is $340 for 100 micrograms.  The protein is 165 kDa and is to be resuspended at 100 ug/mL; this will provide six rounds of selection at approximately $56 per round, if using 100 pmol per round.


1. McDougall, S.R, Anderson, A.R.A., Chaplain, M.A.J., (2006) “Mathematical modelling of dynamic adaptive tumour-induced angiogenesis: Clinical implications and therapeutic targeting strategies.” Journal of Theoretical Biology. 241.

2. Cox, M., Nelson, D.R. (2008). Lehninger: Principles of Biochemistry (fifth ed.). W H Freeman & Co.

3. National Library of Medicine – Medical Subject Headings, (2011).  “Receptor, Tie-2. 

4. Fagiani, E., Christofori, G.,  (2012).  “Angiopoietins in Angiogenesis.”  Cancer Lett.  Ahead of print.

5. Famlouk, M., Hartig, J.S., Mayer, G., (2007). “Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy.”  Chem Rev. 107:3715-43.

6. Ellington, A.D. Szostak, J.W., (1990.) “In vitro selection of RNA molecules that bind specific ligands.” Nature. 346:818-822.

7. Milner, C.S., Hansen, T.M., Singh, H., Brindle, N.P., (2009).  “Roles of the receptor tyrosine kinases Tie1 and Tie2 in mediating the effects of angipoietin-1 on endothelial permeability and apoptosis.” Microvasc Res. 77:187-91.

1 comment:

Gwen Stovall said...

Dear Amika,
Here are a few thoughts:
1. ck your cost/rd - I got ~$56/rd if using 100pmol per a round.
2. ck the molecular weight - the R&D systems site says it is 165 kDa. The 100 kDa is for the monomer, but your protein is a homodimer (emphasis on the dimer) & runs at 165 kDa on a SDS-PAGE.
3. How do you want to do the selection? Bead-based or filter-based? If you opt to use beads, then work with Alice to use the his Dynabeads and read-up on these beads.
4. for your own edification, please review (i.e. do the math) the amount of Tie-2 remaining in the -80. (you told me there are R&D aliquots - 7 aliquots - 100 uM - 5.1 uL/tube - 50 pmol/tube [sic] - 10/6/2008).
5. consider reorg to introduce a prob & offer an aptamer sol'n.
6. specific aim 1 - 1-3 sentences on how you'll achieve this.
7. consider doing a toggle selection!
8. develop specific aim #2 - develop aptamer therapeutic against cancer & unchecked vascular growth
9. consider figure improvements - I like cartoons
10. add "ahead of print" to citatoin #4

Good abstract & good luck with the selection!