Oanh Tran's HIF-1a Aptamer Project

HIF-1A Aptamer Selection

Introduction & Background

Medical News Today states that Cancer is the 2nd leading cause of death in the United State (Medical News Today). Malignant Cancer is also predicted to affect around 1.7 million people and 40% of men and women (National Cancer Institute). A research by Reinhard Bos showed that presence of HIF-1a in quantities has a relationship to the rise in detecting lymph nodes in breast cancer (Reinhard Bos). HIF-1a, or Hypoxia Inducible Factor- 1 alpha, binds onto DNA (figure 3). HIF-1a then encourages transcription of proteins that eventually lead to formation of new vessels in which redirect a fresh source of supplies to the cells. This gives cells, more importantly, cancer cells, fresh oxygen resources to continue growing and spreading to other parts of the body.

HIf-1a weights 32.8 kDa and is present in the cytoplasm as well as the nucleus in human cells. Its isoelectric point is 5.17 and the pH for this target varies within the condition of human pH. It is commonly found in heart and kidneys tissues as well as cancer tissues. HIF-1a structure is a Helix-loop-helix. This target is from Genway Biotech in which it extracted and manufactured for research purposes. HIF-1a amino acid sequence is shown in table 1.

HIF-1a is commonly degraded by Oxygen (Figure 1). In cancer, cells are multiplying at an immense rate and therefore used up all the oxygen for cellular respiration: This cause hypoxia, lack of Oxygen, in that specific portion of the body. Because there is little to no oxygen, HIF-1a, present in large quantities, triggers a formation of new vessels otherwise known as angiogenesis. In doing so, this vessels give rise to a fresh supplies of Oxygen for the malignant cancer cells to continuously progress and extend to other parts of the body.

Therefore, a possible solution to this problem that is focused on by this research is aptamer. Once, aptamer bind and surround HIF-1a transcription factor, it prevents HIF-1a from attaching itself on to the DNA. Accordingly, this stops transcriptions for proteins that are involved in angiogenesis. Aptamer is an Onglio-nucleotides RNA or ssDNA strand. When folds into its natural conformation, aptamer can bind with targeted proteins for inhibition, detection, and/or connecting 2 different target together. For HIF-1a, aptamer is use for competitive inhibition application. One of the many reasons why aptamer is a better approach than normal antibodies that is produced by different labs is that it is stable at extreme’s pH or temperature change. Antibodies, which are proteins, will denature if the condition of the lab does not match its natural environment. Where as antibody proteins must be modified or extracted, RNA can be formed from RNA polymerase and reverse transcript into DNA. If this research yields favorable result, aptamer could also be use for detection of cancer, or any hypoxia condition of cells in the body. This application could be use via attaching detection molecules to aptamer that bind to HIF-1a with a great affinity. Thus, under radar’s readings, one could locate the location of cancer tissues before angiogenesis and its progression to other parts of the body.

This target proved a little challenging due to its negative nature. Since RNA is also negative, in terms of charges, it is complicated to bind both of these together because of its repealing properties. Thus, using by PBs buffer for this target during filter-based selection with 50 mM of MgCl2, Mg 2+ then can acts like a disulfide bridge and connects the RNA, a negative charged molecules, together with the HIF-1a target, another negative charged molecules. The pool for this selection is N71 with a RNA base pair of 112 and the DNA base pair of 132. The process for HIF-1a target is In Vitro selection with filter-based selection and undergo 3 washes because HIF-1a does not contains any tags. The round selection for HIF-1a is featured in figure 4. This target is also incubated at 37C, human’s body temperature, because it is mostly found in human cells.

Currently, the project is still at the beginning of round 3. The major set back that this research has experience is failed cycle course PCR results depicted from agarose gels. In round 1, I did another ccPCR recently up to thirty cycles and it worked because this time with the usage of the right Taq DNA polymerase. The test I am currently preparing is side-by-side comparison between the results from round 2 and round 3 selections to see what changed from the R2 to R3. Therefore, in the next few weeks, N71 pool will be more specific into multiple copies of RNA aptamer that hopefully have a high binding affinity of HIF-1a. This aptamer is then use for competitive inhibition and/or detection to stop cancer progression.

Click here for the Final Report

Citations

Bos, Reinhard Bos. "Cancer." Levels of Hypoxia-Inducible Factor-1 Independently Predict Prognosis in Patients with Lymph Node Negative Breast Carcinoma 97.6 (2003): 1573-581. Print.

Ellington, A. D., and J. W. Szostak. "Trends in Aptamer Selection Methods and Applications." Analyst. Royal Society of Chemistry, 9th June 2015. Web. 01 Sept. 2015.

"HIF-1a (Human)." PhosphoSitePlus. Cell Signaling Technology Inc., n.d. Web. 29 Sept. 2015.

KA, Mace. "U.S. National Library of Medicine." National Center for Biotechnology Information. PubMed.gov, Sept.-Oct. 2007. Web. 01 Sept. 2015.

P, Vaupel, and Mayer A. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 26 June 2007. Web. 29 Sept. 2015.

Pescarmona, Gianpiero. "HIFTranscription Factors." HIF RSS. Flipper E Nuvola, 6 Sept. 2008. Web. 28 Sept. 2015.

"The Top 10 Leading Causes of Death in the US." Medical News Today. MediLexicon International, 30 Sept. 2014. Web. 28 Sept. 2015.

Xia, Xiaobo, Madeleine E. Lemieux, Wei Li, Jason S. Carroll, Myles Brown, X. Shirley Liu, and Andrew L. Kung. "Integrative Analysis of HIF Binding and Transactivation Reveals Its Role in Maintaining Histone Methylation Homeostasis." Proceedings of the National Academy of Sciences of the United States of America. National Academy of Sciences, 17 Mar. 2009. Web. 29 Sept. 2015.

Xiao, Haijuan, Zhenyang Gu, Guoxing Wang, and Tongfeng Zhao. "The Possible Mechanisms Underlying the Impairment of HIF-1α Pathway Signaling in Hyperglycemia and the Beneficial Effects of Certain Therapies." International Journal of Medical Sciences. Ivyspring International Publisher, 22 Aug. 2013. Web. 01 Sept. 2015.

Zimna, Agnieszka, and Maciej Kurpisz. "Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological Angiogenesis: Applications and Therapies." BioMed Research International. Hindawi Publishing Corporation, 4 June 2015. Web. 02 Sept. 2015.