Modified Selection against EGFR Using Known Aptamer E07 to Optimize SELEX Efficiency

Modified Selection against EGFR Using Known Aptamer E07 to Optimize SELEX Efficiency

Marc St. Cyr

               Aptamer research provides a wealth of possibilities for therapeutic, diagnostic, and drug delivery purposes but the methods are constantly evolving and being optimized. It is very difficult to stay updated with the most current and innovative techniques and procedures. However, it is possible to, from time to time, perform selection with positive controls and varying conditions in order to determine the optimum conditions that should be used while performing a Systematic Evolution of Ligands by Exponential Enrichment (SELEX). There are many aspects of the SELEX procedure that can be optimized but the most crucial for these purposes are the time-based and nucleic acid recovery components (Stoltenburg 2007). Reducing the amount of time spent on one round of selection allows for faster results, which lead to expedited aptamer identification. The most effective way to optimize experiments is by comparing the results provided by reagents known to work with current conditions and conditions proposed to be faster, more effective, or otherwise helpful.

The aptamer E07 was successfully selected against epidermal growth factor receptor (EGFR) (Li 2011). Using this aptamer in the SELEX procedure to replace the randomized RNA pool will allow for the optimization of certain aspects of selection. The same reagents provided in the published aptamer paper will be used while the conditions of the selection itself will be varied and analyzed by assaying reactions, incubations, etc. to provide data or comparisons (Avutu 2010).

Specific Aim 1: Provide the FRI Aptamer Lab with optimized selection conditions with the goal of producing more efficient results when using the SELEX method of aptamer selection. This can include a wide and varying range of suggestions from shorter incubation times, new procedures, or preferred versions of already established procedures.

Figure 1. Overall outline of the SELEX method of selection of LNA against a target, including binding, washing, and amplification steps.

References:

1)      Stoltenburg R, Reinemann C, Strehlitz B (2007) SELEX- A (R)evolutionary Method to Generate High-Affinity Nucleic Acid Ligands. Biomedical Engineering 24(4): 381-403.

2)      Li N, Nguyen HH, Byrom M, Ellington AD (2011) Inhibition of Cell Proliferation by an Anti-EGFR Aptamer. PLoS ONE 6(6): e20299. doi:10.1371/journal.pone.0020299

3)      Avutu V (2010) Avidity Effects of MinE07, an anti-EGFR Aptamer, on Binding to A431 Cells <http://repositories.lib.utexas.edu/handle/2152/13407?show=full>

To see full proposal click here.
To see first progress report click here.
To see second progress report click here.
To see final report click here.

Nucleic Acid Aptamer Selection Against Progesterone for use in Preventing and Treating Osteoporosis

Kelsey Gardner

September 1, 3013

           Nucleic Acid Aptamer Selection Against Progesterone for use in Preventing and Treating Osteoporosis

            Osteoporosis is a disease in which the body struggles to create enough new bone tissue to maintain a bone density high enough to support a healthy skeletal system. Type one osteoporosis is most commonly associated with menopausal women, and results in bones become more brittle so that the incidence of fracture increases [1].
          Estrogen and progesterone are both hormones associated with maintaining women’s health, and both drop to record lows during menopause, as depicted in Figure 1, and remain low upon diagnosis of osteoporosis [1]. Hormone therapy along with calcium supplements are considered to be somewhat successful in the prevention of osteoporosis, and are sometimes used to help ease the symptoms of women diagnosed with post-menopausal osteoporosis. Yet, these methods alone are not directly targeted toward the exact progestins associated with early onset osteoporosis. Several antibodies associated with estrogen and progesterone hormone receptors do exist but are not successful because the therapeutic drug does not specifically target the progestin receptors strictly associated with bone growth [2].
             Developing an aptamer for progesterone specifically associated with receptors for bone growth would increase the effectiveness of treating menopausal osteoporosis. Specifically targeting progesterone, so that it can function for drug delivery, would allow for progesterone molecules carrying a drug that increases bone growth to bind at various sites such as the TE85 osteosarcoma receptor , a receptor that plays a large role in promoting regeneration a bone growth [4]. Progesterone is a very small steroid hormone that possesses a high binding affinity inside of the cell membrane, and these characteristics allow for a wide use of aptamers to treat varying degrees and stages of osteoporosis uniquely [1]. Therefore, an anti-progesterone aptamer would be used as a means for highly specified drug delivery to progesterone receptors (via progesterone aptamers) associated with bone growth and result in a prevention of further bone density loss.
Specific Aim: Develop an aptamer for progesterone hormones to prevent or treat the symptoms of menopausal (stage one) osteoporosis.

Figure 1- A decline in progesterone during menopause is a leading 

factor in the onset of osteoporosis in women age 45-60[3].

Vendor: Cayman Chemical

Product catalog number: 9000645

Vendor website: https://www.caymanchem.com/app/template/Product.vm/catalog/9000645

Vendor telephone number: (800) 364-9897

Cost per unit: $72 per 500ug

Cost per round: $0.02 per round

References:

1.  Seifert-Klauss, Vanadin, and Jerilynn C. Prior. (2010) "Progesterone and Bone." National Center for Biotechnology Information. U.S. National Library of Medicine.

2.  A.D.A.M. Editorial. (2012) "Osteoporosis." Osteoporosis-Guideline. U.S. National Library of Medicine.

3.  Lee, John R. "Oestrogen Dominance." (2013) Wellsprings Natural Hormone Health. WellSprings

4. Combs, Donald and Reese, Kimberly. Nonsteroidal Progesterone Receptor Ligands.(1995) Selectivity for Bone Cell Progesterone Receptors, Med Chem, 38, 4880-4884
   
View the full proposal here.
View the final progress report here.

Aptamer E07 Against EGFR for Use as an Anti-Tumor Therapeutic

Zane Alami-Nassif

September 3rd, 2013

Aptamer E07 Against EGFR for Use as an Anti-Tumor Therapeutic

            Epidermal growth factor receptor (EGFR) leads to many cancers when over expressed due to a mutation (Zhang et al., 2007). This is because EGFR is a tyrosine kinase receptor that is responsible for binding to epidermal growth factor and initiating a signal transduction cascade that will lead to DNA synthesis and cell proliferation (Oda et al., 2005). If EGFR is constantly activated then uncontrolled cell division will occur leading to malignant tumors.

            The anti-EFGR aptamer, E07, will compete with epidermal growth factor for binding and will thus inhibit the receptor (Li et al., 2011). In figure 1, the sequence of E07 can be observed with the predicted motif that will bind to EFGR. Currently E07 is not as efficient as alternative drugs such as Cetuximab and monoclonal antibodies until a very high dose is used (Avutu 2010). Increasing the efficiency of E07 is a goal that could be pursued in order to bring E07 into viability for treatment against multiple cancers. The theory that avidity effects could increase the efficiency of E07 was tested with limited results. It was hypothesized that nucleic acid would behave in a similar manner to peptides, which obtain supradditve effects when monomers dimerize or multimerize. (Avutu 2010)

Specific Aim 1: The current goal is to better understand the conditions of selection using E07 with two versions of the FT7 primer and positive controls. Whether or not a concentration of aptamer can be maintained after undergoing selection is one focus.

Specific Aim 2: Another focus is to understand the effects of using different buffers during the binding reaction. Buffers to be tested are HBSS without calcium or magnesium, HBSS with calcium and magnesium, PBS and PBS with magnesium.

Figure 1. Predicted secondary structure of Aptamer E01 and Aptamer E07 (Li et al., 2011)

Link to Proposal

Link to 1st Progress Report
Link to 2nd Progress Report
Link to Final Report

Ordering Information:

Target: Recombinant Human EGFR

Vendor: R&D Systems

Product Catalog Number: 344-ER

Vendor Website: http://www.rndsystems.com/Products/344-er/

Vendor Telephone: (612) 379-2956

Cost: EGFR will be provided by the Altermune lab.

References:

1. Zhang, H., Berezov A., Wang Q., Zhang G., Drebin J., Murali R., Greene M. (2007). ErbB receptors: from oncogenes to targeted cancer therapies. J Clin Invest. 117(8): 2051-2058. 

2. Oda K., Matsuoka Y., Funahashi A., Kitano H. (2005). A comprehensive pathway map of epidermal growth factor signaling. Mol Syst Biol. 1: 2005.0010. 

3. Li N., Nguyen HH., Byrom M., Ellington AD. (2011) Inhibition of Cell Proliferation by an Anti-EGFR Aptamer. PLoS ONE 6(6): e20299.

4. Avutu, V., (2010) Avidity effects of MinE07, an anti-EFGR aptamer, on binding to A431 cells. 

Using Aptamers against HA33 to Supress the Reaction of Clostridium botulinum against Acetylcholine

Using Aptamers against HA33 to Supress the Reaction of Clostridium botulinum against Acetylcholine

Shashwath Nayak

Abstract:

Clostridium botulinum is a gram-positive bacterium which causes botulism, a very fatal disease that prevents acetylcholine from binding to receptors therefore not removing the muscle’s ability to contract, in people (Shukla 2005). The cells of this bacterium secrete botulinum neurotoxin, (BoNT), which is considered the deadliest toxin up to date and has been deliberated by biodefense researchers as a possible agent for bioterrorism. BoNT, shown in Figure 1, works by binding to the receptor in which acetylcholine is supposed to bind, blocking the binding reaction from occurring. One very important protein in the process of secreting BoNT is HA33. 

BoNT forms a toxin complex (TC) that includes HA33 (Ito et all 2011). HA33 is a protein from Clostridium botulinum that was shown to possibly protect and activate BoNT. HA33 is a neurotoxin- associated protein (NAP) that protects BoNT form acid denaturation in the stomach and gastric tract (Arndt et all 2005). These TCs are actually more toxic than pure BoNT. Without NAPs, like HA33, BoNT are actually susceptible to the acid conditions in the gastric tract. This allows the absorbance of BoNT throughout the body to increase due to the more epithelium tissue being available for the BoNT (Ito et all 2011). This means that HA33 is a problem because it allows BoNT to be even more of a threat than it already is just by allowing it to further penetrate the body. A way that has been discovered to possibly stop this protein from helping BoNT is by the use of aptamers.

Aptamers are oglionucleiotide that can specifically bind to a specific target (Nobuko, Ellington & Stanton, 2001). Aptamers use an in vitro selection process, a process that can be done outside of an organism.

Specific Aim 1: Use aptamers that can bind to HA33 and change its shape in order to prevent the neurotoxin from causing problems in the future.

The aptamer would look for the specific sequence in HA33 by going through a binding selection. The binding selection would take place in a series of rounds in order to narrow a pool of RNA to a specific sequence that will bind to the HA33 protein.

Figure 1: A crystal structure of BoNT in Clostridium botuinum. The arrow in the picture is pointing towards the HA33 present in the toxin complex (Arndt et all 2005)

Target Order Information:
Vendor: Ellington Lab
Vendor Website: https://www.ellingtonlab.org
Central Lab Telephone: 512-471-6445
Office Manager: 512-232-3426
Cost per Unit: $0
Cost per Round: $0

References:

1. Shukla, H. (2005). Clostridium botulinum: a bug with beauty and weapon. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/15839401


2. Arndt, J. W., Gu, J., Jaroszewski, L., Schwarzenbacher, R., Hanson, M. A., Lebeda, F. J., & Stevens, R. C. (2005, 01 01). The structure of the neurotoxin-associated protein ha33/a from clostridium botulinum suggests a reoccurring β-trefoil fold in the progenitor toxin complex. Retrieved from http://www.sciencedirect.com/science/article/pii/S0022283604016092


3. Ito, H., Sagane, Y., Miyata, K., Inui, K., Matsuo, T., Horiuchi, R., Ikeda, T., & Suzuki, T. (2011, 02
01). Ha-33 facilitates transport of the serotype d botulinum toxin across a rat intestinal epithelial cell monolayer. Retrieved from http://onlinelibrary.wiley.com/doi/10.1111/j.1574-695X.2011.00779.x/full

4. Nobuko, H., Ellington, A., & Stanton, M. (2001, June 15).Aptamer beacons for the direct detection of proteins. Retrieved from http://www.sciencedirect.com/science/article/pii/S0003269701951693

5. Yoshimasa , S., Ken, I., Shin-Ichiro, M., Keita, M., Tomonori, S., Koichi, N., & Toshihiro, W. (2012, August 22). Botulinum toxin complex: A delivery vehicle of botulinum neurotoxin traveling digestive tract. Retrieved from http://www.intechopen.com/books/structure-and-function-of-food- engineering/botulinum-toxin-complex-a-delivery-vehicle-of-botulinum-neurotoxin-traveling-digestive-
tract


6. Sugiyama, H. (1980). Clostridium botulinum neurotoxin.Microbiological Reviews, 44(3), 419-448. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373187/



First Proposal
First Progress Report

Identification of Aptamer for Human Telomeric Repeat Binding Factor (TRF1) for the Control of Telomerase Activity

Identification of Aptamer for Human Telomeric Repeat Binding Factor (TRF1) for the Control of Telomerase Activity

Helen Jung

3 September 2013

     

With the rise of nuclear and linear DNA, eukaryotes had to overcome the imperfect DNA replicating mechanism that arises from DNA polymerases’ inability to start their own sequence. The advance of telomeres, non-coding DNA sequences at the end of linear DNA, allows the preservation of genetic information in coding sequences of DNA. These telomeres shorten in length as the specific DNA molecule repeats replication, but excessive shortening of telomere can have devastating consequences, such as HHS to be further described below. In response, yeast and mammals maintain their telomere within a certain size range through telomerase activity (de Lange, 2005). The access to each telomeric end is strictly controlled by a telomere nucleoprotein complex, and they guard telomeres against indiscriminate elongation by telomerase or inappropriate modification by DNA damage repair system. Especially, telomeric repeat binding factor (TRF1), a protein within the telomere nucleoprotein complex, negatively regulates telomere length; its over-expression causes telomere shortening while its under-expression results telomere elongation. (Kishi, etal, 2001).

oyeraal-Hreidarsson Syndrome (HHS) is an X-linked genetic disorder that shows abnormalities in telomere length and its maintenance, including defects in RTEL1 (Le Guen, etal, 2013). This condition impedes development and shows symptoms of bone marrow failure and immunodeficiency. Currently, there is no effective treatment for this condition. The objective of this study is to select an aptamer—an oligonucleotide of three-dimensional orientation that is selected to bind to and inhibit a specific target protein—using repetitive talon bead-based selection method. In a previous study, the overexpression of the protein TRF1 has shown strong telomere shortening and damage, whereas under-expression of the protein has shown telomere elongation (Kishi, etal, 2001). Thus, development of an aptamer against TRF1 can provide an outlook for forced TRF1 inactivation. TRF1 inhibition results promoted telomeric repair activities and offers potential aptamer therapeutics to alleviate the symptoms of HHS.

This study specifically aims to identify aptamer against TRF1 protein target and to develop realistic potential therapeutic purposes of this aptamer against HHS. Currently, there is no effective way of identification or remedy for the symptoms. Potential medical applications for HHS will further be investigated.

His-tagged TRF1 protein (Catalog number: 13044-H07B) can be purchased from Sino Biological Inc. (sinobiological.com; Phone: 86-400-890-9989). Fifty micrograms, or about 990.01 picomoles, of TRF1 protein cost $290. The molecular weight of the target is 50.5Da. Each round of selection will be performed with about 100 picomoles of TRF1 protein and is expected to cost about $30. The selection is performed using talon beads, which are known to and are used in different laboratory (Stovall, 2013) to select against polyhistidine-tagged targets. These beads are already available in the lab with other necessary equipment without any additional cost.

References:

https://drive.google.com/file/d/0BwhvTPTt0GX-QnJsa0tJM2xkY2c/edit?usp=sharing

Target Proposal:

  https://docs.google.com/file/d/0BwhvTPTt0GX-ZDVLYnQ4SkRKUG8/edit?usp=sharing

Progress Report:

https://drive.google.com/file/d/0BwhvTPTt0GX-ZDVLYnQ4SkRKUG8/edit?usp=sharing

Second Progress Report

https://drive.google.com/file/d/0BwhvTPTt0GX-QVNVVXk3SGNMUzQ/edit?usp=sharing
Final Manuscript
https://drive.google.com/file/d/0BwhvTPTt0GX-d2IzdjJLS3NBaEE/edit?usp=sharing

 Figure 1. The role of TRF1 in telomerase activity regulation and facilitation during S phase of the cell cycle.

Arianne Alcantara

September 3, 2013

Inhibition of FGF8b Protein Oncogenic Characteristics through Nucleic Acid Aptamer Selection

Prevalent throughout the world and always seemingly one step ahead of society’s most brilliant physicians, cancer has continued to ravage the human population for centuries. As a result, oncology methods to combat the disease seem to shift every year, with experts believing surgery to be the solution in the eighteenth century, to radiation, chemotherapy, and targeted therapy as alternatives in the twenty-first ("Evolution of Cancer Treatments," 2012).  The protein present in humans, Fibroblast Growth Factor 8 (FGF8), can be attributed directly to the third of the aforementioned techniques, targeted therapy, due to its significant role in cell processes such as mitogenesis and angiogenesis. Its isoform FGF8b is medically known as an androgen-induced on dominant in tumorigenesis and hormonal carcinogenesis, with one of the more popular diseases being prostate cancer (Henderson and Feigelson, 1999).

Although FGF8b expression is linked to hormone-related cancers, speculation still remains as to whether it is the cause; furthermore, like all proteins, FGF8 performs various other functions such as those pertaining to embryo development, tissue repair, and cell survival (White and MacArthur, 1995). Consequently, recognizing its role in human development forces the necessary realization that completely eradicating the protein will produce more harmful side effects than good ones. Employing more selective techniques designed to inhibit FGF8 after a certain point instead of abolishing it are sought in this study.

Figure 1 illustrates the affect of an RTK inhibitor if a growth factor like FGF8, binds to a cell. The discovery of an FGF8b aptamer would lead to the prevention of a possibly cancerous cell to divide, and as such remain benign ("Cancer Growth Blockers," 2013).

Specific Aim 1: Employing therapeutic strategies, to determine an aptamer, substance with the capability to bind to a target molecule, through bead-based selection for FGF8 that once bound together, possesses the ability to trigger inhibition or at least antagonization and alter the protein’s affects on an organism.

Specific Aim 2: For cases like prostate cancer, once an aptamer is determined, it can be utilized in blocking the interaction between tumor cells and stromal cells by binding to and preventing the activation of cell surface receptor tyrosine kinases (RTKs) as shown in Figure 1 (Henderson and Feigelson, 1999). This would in turn alter the expected biological response that would typically occur, hopefully leading to decreased oncogenic symptoms, and thus later applied to other types of cancer besides prostate.

The FGF8b protein is made available by Shawn Piasecki through the Keatinge-Clay lab, with other stock sold through mybiosource.com for 0.005mg at $140.00.

References

1.  "Evolution of Cancer Treatments: Targeted Therapy." Evolution of Cancer Treatments: Targeted Therapy. American Cancer Society, 8 June 2012. Web. 02 Sept. 2013. <http://www.cancer.org/cancer/cancerbasics/thehistoryofcancer/the-history-of-cancer-cancer-treatment-targeted-therapy>.
2. Henderson, Brian E., and Heather S. Feigelson. "Hormonal Carcinogenesis." Oxford Journals 21.3 (1999): 427-33. Oxford Journals. Oxford University Press, 1999. Web. 2 Sept. 2013. <3. http://carcin.oxfordjournals.org/content/21/3/427.full>.
3. White, Robert, and Craig MacArthur. "Assignment OfFGF8to Human Chromosome 10q25–q26: Mutations InFGF8May Be Responsible for Some Types of Acrocephalosyndactyly Linked to This Region." Genomics 30.1 (1995): 109-11. Print.
4. "Cancer Growth Blockers." : Cancer Research UK : CancerHelp UK. Cancer Research UK, 15 Apr. 2013. Web. 02 Sept. 2013. <http://www.cancerresearchuk.org/cancer-help/about-cancer/treatment/biological/types/cancer-growth-blockers>.

Click here for full proposal
Click here for Progress Report 1
Click here for Progress Report 2
Click here for Final Progress Report

Selection of an Aptamer Against FGF8b for Breast Cancer and prostate Cancer Treatment

Shuhui Wan

FGF8, the fibroblast growth factor (1), a protein that is inducible by androgen in breast cancer cells, is involved in many hormone related cancers, such as breast cancer and prostate cancer (2,3). More than 67% of breast cancer patients and over 93% of prostate cancer patients showed expression of FGF8 (3). And the breast cancer tissues were show to express much higher level of FGF8 mRNA than the non-malignant tissues (4). One specific isoform, FGF8b, was proved to induce progression of prostate cancer (5).

Because of FGF8b’s significant involvement with tumor growth, it was thought that by inhibiting the activities of this growth protein the tumor growth could also be greatly suppressed. One promising way of doing this would be finding an aptamer, a RNA ligand that binds to specific targets, for FGF8b. The apatamer for FGF8b may bind to it and inhibiting the protein from functioning in promoting tumor growth. In other words, the aptamer could be used for therapeutic purposes to treat breast cancer and prostate cancer in future.

Specific aim #1: find an aptamer that binds specifically to FGF8b.

Specific aim #2: modify the aptamer to be used for therapeutic purposes.

Specific aim #3: use the modified aptamer as a drug for breast and prostate cancer treatment.

Figure 1: structure of FGF8b(1)..

FGF-8b is prepared by Shawn Piasecki and available in lab.


...so the references are not showing up, here's the PDF version file:
https://docs.google.com/file/d/0B38waIP26ToBdDRBX1lLMXItQU0/edit?usp=sharing

progress report 1
https://docs.google.com/file/d/0B38waIP26ToBZUpkX3N1a1pvdzg/edit?usp=sharing

progress report 2
https://drive.google.com/file/d/0B38waIP26ToBQUZWSjFCRU83NUE/edit?usp=sharing

Final report

References:

---

(1) Goetz R, Mohammadi M, Exploring mechanisms of FGF signalling through the lens of structural biology. Nat Rev Mol Cell Biol. 2013 Mar;

(2) Payson RA, Wu J, Liu Y, Chiu IM. The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells. Oncogene. 1996 Jul 4;13(1):47-53.

(3) Mattila MM, Ruohola JK, Valve EM, Tasanen MJ, Seppänen JA, Härkönen PL. FGF-8b increases angiogenic capacity and tumor growth of androgen-regulated S115 breast cancer cells. Oncogene. 2001 May 17;20(22):2791-804.

(4) Tanaka A, Furuya A, Yamasaki M, Hanai N, Kuriki K, Kamiakito T, Kobayashi Y, Yoshida H, Koike M, Fukayama M. “High frequency of fibroblast growth factor (FGF) 8 expression in clinical prostate cancers and breast tissues, immunohistochemically demonstrated by a newly established neutralizing monoclonal antibody against FGF 8.” Cancer Res. 1998 May 15;58(10):2053-6.

(5)Marsh SK, Bansal GS, Zammit C, Barnard R, Coope R, Roberts-Clarke D, Gomm JJ, Coombes RC, Johnston CL. Increased expression of fibroblast growth factor 8 in human breast cancer. Oncogene. 1999 Jan 28;18(4):1053-60.

(6)Valta MP, Tuomela J, Vuorikoski H, Loponen N, Väänänen RM, Pettersson K, Väänänen HK, Härkönen PL. FGF-8b induces growth and rich vascularization in an orthotopic PC-3 model of prostate cancer. J Cell Biochem. 2009 Jul 1;107(4):769-84. doi: 10.1002/jcb.22175.

(7) Gwen Stovall, PowerPoint - “Bead Based RNA Selection, Lecture 09”, slide 21, 2013