Nucleic Acid Aptamer selection against myelin basic protein.

Searching for a MS treatment therapeutic by selecting an aptamer against myelin basic protein

Abstract

            Myelin basic protein (MBP), in simple terms, is a main component of the myelin sheath surrounding axons in the nervous system. Myelin, or myelin sheath, is the white fatty substance that forms a sheath about the central nervous system and certain nerve fibers.¹ The myelin sheath is a covering of axon in nerve cells that allows faster conduction of electrical impulses in nerve system. If the sheath is somehow damaged by autoimmune disease or an infection, the speed of electrical propagation slows down dramatically and individuals may experience a difficulty of controlling certain body parts or even a loss of control. In an autoimmune disease such as multiple sclerosis where myelin sheath is attacked by antibodies, victims experience ascending paralysis and cognitive disability.² MBP, a monomer of about 304 amino acids and varying sizes from 14 to 21kD, ³ represents about 30% of the total myelin protein and is an integral structural constituent of the myelin sheath.¹

There has been considerable interest in MBP and its relationship with demyelinating disease. Abnormal amounts of MBP can be detected in the cerebrospinal fluid of individuals with demyelinating disease and in the serum of patients having suffered head injury, leading to a theory that MBP is a response to heal the damaged axon.¹ It was also found that the possible cause of demyelination disease, such as MS, may be the post-translational modification of MBP. In a study where MBPs were isolated from normal and MS human white matter and compared, it was found that MBP from MS patients were missing phosphorylated peptides and increased methylation.^{4, 5.}

 My specific aim (1) is to select an aptamer against MPB and develop a therapeutic that prevents post-translational modification of MBP which seems to a cause of demyelination disease such as MS.

MBP can be purchased from EMD Millipore (Catalog number 13-111). Since the target (BMP) is tagged with biotin, we can use streptavidin beads that form strong covalent bonds. It’s $209.00 per 1 mg. Per round, it will cost $ 8.26 , assuming  each round will use 4 ug. The cost may go down as selecting round advances and conditions become more stringent. The company’s phone number is 978-715-4321.

Gubeom Nam.

Full proposal: https://docs.google.com/open?id=0B2m2NTs-hw5MZnFhQm1ZdGZRajg

Progress report 1: https://docs.google.com/open?id=0B2m2NTs-hw5MTWlkckhQQ0tsUG8

Progress report 2:https://docs.google.com/open?id=0B2m2NTs-hw5MaGswb05qcGJ1ZmM

Final Report https://docs.google.com/file/d/0B2m2NTs-hw5MY0RPY0hlWUJ3LXM/edit

Reference:

      1.     http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/m1891pis.Par.0001.File.tmp/m1891pis.pdf
      2. "Myelin Function." THE MEDICAL NEWS. Creative Commons Attribution-ShareAlike License. Web. 03 May 2012. <http://www.news-medical.net/health/Myelin-Function.aspx

     3.     "Antibody to Myelin Basic Protein by EnCor Biotechnology Inc." EnCor Biotechnology Inc., Manufacturer of Monoclonal and Polyclonal Antibodies for Cell Biological, Protein Chemical and Biomarker Research. Encor Biotechnology Inc. Web. 04 May 2012.

       4.     "Multiple Sclerosis - An Important Role for Post-Translational Modifications of Myelin Basic Protein in Pathogenesis." Www.mcponline.org. Molecular and Cellular Proteomics, 25 June 2003. Web. 21 Sept. 2012. <http://www.mcponline.org/content/2/7/453.long>.

Wood, D. D., MD, M. A. Moscarello, MD, and J. M. Bilbao, MD. "Acute Multiple Sclerosis (marburg Type) Is Associated with Developmentally Immature Myelin Basic Protein." Onlinelibrary.wiley.com. N.p., 8 Oct. 2004. Web. 21 Sept. 2012. <http://onlinelibrary.wiley.com/doi/10.1002/ana.410400106/abstract
5. Wood, D. D., MD, M. A. Moscarello, MD, and J. M. Bilbao, MD. "Acute Multiple Sclerosis (marburg Type) Is Associated with Developmentally Immature Myelin Basic Protein." Onlinelibrary.wiley.com. N.p., 8 Oct. 2004. Web. 21 Sept. 2012. <http://onlinelibrary.wiley.com/doi/10.1002/ana.410400106/abstract>.

Nucleic Acid Aptamer Selection against Hcp-6 Surface Protein of Burkholderia pseudomallei for Diagnosis of Melioidosis

Nucleic Acid Aptamer Selection against Hcp-6 Surface Protein of Burkholderia pseudomallei for Diagnosis of Melioidosis
By Dara Goral

Abstract:

Burkholderia pseudomallei is a gram negative bacterium responsible for the disease, melioidosis, which has been the cause of many fatalities in Southeast Asia and Northern Australia[i]. Melioidosis is a septic disease that often causes abscesses in the spleen and liver[ii]. Because of the resilience of B. pseudomallei, many antibiotics have not proven effective in reducing infection and melioidosis is still associated with a high mortality rate of 40%. Most fatalities occur within the first 48 hours of infection and it is difficult to diagnose this disease in that short amount of time[iii].

                  An approach is introduced that seeks to bind short RNA oligonucleotides to the Hcp-6 surface protein of Burkholderia pseudomallei. This surface protein is an excellent marker because it is a major virulence determinant for the melioidosis. In the suggested approach, the use of RNA ligands, called aptamers, prove to be a promising method for binding of Hcp-6 because of successful aptamer selections against many other proteins with various functions. Aptamers are powerful tools in diagnostics and drug delivery due to their high specificity and affinity for protein targets. In finding an aptamer with a high affinity for Hcp-6, it could be a crucial step in the development of a method for diagnosing melioidosis.

Specific Aim 1: Perform SELEX (Systematic Evolution of Ligands by Exponential Enrichment) in order to select an aptamer to bind to the Hcp-6 surface protein of Burkholderia pseudomallei.

A highly specialized aptamer may be developed that binds with great affinity for this surface protein allowing for easy identification of this bacteria in any sample.

Specific Aim 2: Attach a fluorescent tag to the RNA aptamer and experiment use of detection.

The attachment of a fluorescent tag would allow for easy detection of Burkholderia pseudomallei in a given sample. This could expedite the process of diagnosing melioidosis.

Figure 1: The RNA aptamer will bind to the Hcp-6 surface protein of the bacterium. After this specific aim 1 is achieved, a fluorescent tag will be attached to the aptamer for detection.

Previous Research: Two rounds of selection have already been completed against this target.

Purchasing Information: This target has already been obtained from the Katy Brown Lab.

Target Proposal
Progress Report 1
Progress Report 2
Final Manuscript

References:

[i] Brett, P. J., D. DeShazer, and D. E. Woods. (1998) "Burkholderia Thailandensis Sp. Nov., a Burkholderia Pseudomallei-like Species." International Journal Of Systematic And Evolutionary Microbiology 48.1: 317-20.

[ii] Currie, Bart J., Dale A. Fisher, Nicholas M. Anstey, and Susan P. Jacups. (2000) "Melioidosis: Acute and Chronic Disease, Relapse and Re-activation." Transactions of the Royal Society of Tropical Medicine and Hygiene 94.3: 301-04.

[iii] Rajchanuvong, A., W. Chaowagul, Y. Suputtamongkol, M.d. Smith, D.a.b. Dance, and N.j. White. (1995) "A Prospective Comparison of Co-amoxiclav and the Combination of Chloramphenicol, Doxycycline, and Co-trimoxazole for the Oral Maintenance Treatment of Melioidosis." Transactions of the Royal Society of Tropical Medicine and Hygiene 89.5: 546-49.

Nucleic Acid Aptamer Selection Against Basic Fibroblast Growth Factor (FGF-2)

Nucleic Acid Aptamer Selection against Basic Fibroblast Growth Factor (FGF-2)

Jessica Nguyen, 18 September, Fall 2012

R50 RNA Pool against Basic Fibroblast Growth Factor

Abstract:

Although science and medicine have developed rapidly over the last decade, some diseases and conditions still evade present diagnostic and therapeutic techniques. For instance, cardiovascular disease, a class of disease that involves vessels of the heart, is the primary cause of death in the industrialized world with approximately 73 million Americans having some form of the disease ⁽¹⁾. Cancer, the uncontrolled growth of abnormal cells, is also considered to be one of the deadliest diseases of the modern world. Although these diseases seem unrelated, scientists have discovered that one protein, basic fibroblast growth factor (FGF-2), plays a vital role in both these conditions and various other conditions of the body. This research proposes that by finding an aptamer (an oligonucleotide with binding properties) against FGF-2 (also known as FGF-b), these diseases and others can be resolved with more efficiency and specificity.

FGF-2 has many effects on the body and is naturally present in basement membranes and in the extracellular matrix of blood vessels ⁽²⁾. It is a single-chain polypeptide growth factor that plays a significant role in the processes of wound healing, inducing angiogenesis or the growth of new blood vessels from existing ones, and supplementing undifferentiated growth in human embryonic stem cells ^{(1), (3), (4),(5)}. Because this protein has such a prominent role in many functions of the human body, an aptamer against FGF-2 could have multiple therapeutic and diagnostic effects in the body and could prove to be very promising. However, despite the many benefits of an aptamer against this protein, the specific aims of this research focus on the role of FGF-2 on cancer progression. 

Specific Aim 1: The first specific aim of this research will be to discover an aptamer that has a high binding affinity for FGF-2.  Seven rounds of selection have already been completed, along with one binding assay of Rounds 2, 4, and 6 (results showed that although positive binding was constant over rounds, negative binders did decrease) and sequencing of the Round 6 pool. More rounds of selection hope to be completed, and a binding assay done with clones found during sequencing will be conducted to test for high affinity binding between the clone and the target.    

Specific Aim 2: The next aim of this research is to modify the aptamer so that it can be used to diagnose and track the progression of cancer in the human body. Modifications that can be used include fluorescence.

Specific Aim 3: The third specific aim of this research will be to modify this aptamer so it can be used for drug delivery. This can be accomplished by collaboration with other labs that have discovered a functioning therapeutic against cancer but have no way of selectively targeting the abnormal cells (Figure 1). 

Figure 1: Specific Aims of Aptamer Research. FGF-2 plays an important role in the angiogenesis and metastasis of cancer. By targeting this protein target for aptamer selection, diagnostic and drug delivery systems can be created that inhibit the progression of cancer.

FGF-2 (17.3kDa) can be purchased from ScienCell Research Laboratories (877.602.8549) for $175 for 50 micrograms (CAT: 104-02). Each round would cost roughly $11.67. Currently, there is some available in the Aptamer -20°C fridge. 

Click here for full proposal. 

Click here for first progress report. 
Click here for second progress report.
Click here for final progress report.

References: 
1. House, Stacey L., et al. "Cardiac-Specific Overexpression of Fibroblast Growth Factor-2 Protects Against Myocardial Dysfunction and Infarction in a Murine Model of Low-Flow Ischemia." 108 (2003).

2. Soulet, Fabienne, et al. "Fibroblast Growth Factor-2 Interactes with Free Ribosomal Protein S19." 289 (2001): 591-596. 6 April 2012.

3. Zhang, Shuang-Xia, et al. "Gekko Sulfated Glycopeptide Inhibits Tumor Angiogenesis by Targeting Basic Fibroblast Growth Factor." (2012).

4. Pereira, Renata C., Aris N. Economides and Ernesto Canalis. "Bone Morphogenetic Proteins Induce Gremlin, a Protein That Limits Their Activity in Osteoblasts." 141.12 (2000).

5. Liu, Yanxia, et al. "A novel chemical-defined medium with bFGF and N2B27 supplements undifferentiated growth in human embryonic stem cells." 346 (2006).

6. Stoltenburg, Regina, Christine Reinemann and Beate Strehiltz. "SELEX - A revolutionary method to generate high-affinity nucliec acid ligands." 24 (2007).

7. Stovall, Gwen. "Protocol - Selection - Filter Based RNA." 2012.

8. Golden, Mace C., et al. "Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers." 81 (2000).

9. McCauley, Thomas G., Nobuko Hamaguchi and Martin Stanton. "Aptamer-based biosensor arrays for detection and quantification of biological macromolecules." 319 (2003).

Lab Attendance Hours Spring 2012

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