Nucleic Acid Aptamer Selection Against Myostatin as a Therapeutic Treatment for Amyotrophic Disorders

Nucleic Acid Aptamer Selection Against Myostatin as a Therapeutic Treatment for Amyotrophic Disorders

Santiago Diaz

Friday September, 16th 2011

Fall 2011

Pool: N58 RNA

Target: Myostatin

Abstract

The protein myostatin, or GDF8 (growth differentiation factor 8), has been shown to regulate muscle mass growth (myogenesis) by inhibiting myoblast proliferation and differentiation. The theory is that, through a receptor-mediated signal transduction pathway, myostatin stops the cell cycle at G1 phase and prevents myoblasts from undergoing S phase. However, there are people who suffer from muscular dystrophy or other amyotrophic conditions and wish to have greater muscle mass. It is in these scenarios that myostatin inhibition might have beneficial effects.

If the myostatin molecule is somehow disabled or modified so that it can no longer activate the signal transduction pathway, then muscle growth can be promoted. One approach to this task is the use of aptamers, which are RNA oligonucleotides that are capable of binding to a specific target with high affinity and specificity. Aptamers are designed through a careful process of selection, in which the RNA ligands that bind more strongly to the desired target are replicated. Using these aptamers against myostatin might promote muscle development.

Specific aim 1: Aptamer selection against myostatin protein to inhibit myostatin action.

It is hypothesized that the myostatin aptamers will bind to its target protein before these can reach the receptors that activate the signal transduction pathway that negatively regulate proliferation and differentiation. The myostatin-aptamer conjugated protein will have such a foreign shape and form that, when it reaches the receptor, the binding site will not recognize it, rendering it incapable of triggering the signal transduction pathway, and thus, unable to inhibit muscle growth.

Myostatin can be purchased at GenWay at 10ug for $165. Catalog number: 10-663-45269

Introduction and Background

Myostatin, also known as Growth and differentiation factor 8 (GDF-8), is a member of the transforming growth factor beta(TGF-beta) family, and is a protein that is known to negatively regulate muscle tissue growth in humans and other animals(MedicineNet.com). Myostatin is made of two polypeptide chains that account for a total of 375 aminoacids(National Center for Biotechnology Information), and giving it a total molecular weight of 42.75 kDa(Putnam, C. 2006). It is also well known that myostatin usually forms protein complexes with other molecules, such as follistatin, to carry out its function(Amthor, C. 2004).

This intriguing protein was first discovered in mice with a mutation on gene MSTN, the DNA sequence that encodes for myostatin. These myostatin-deficient mice showed muscular hypertrophy and abnormally elevated levels of strength, which led to them naming the strain "mighty mice". Myostatin was thereafter found in humans and other mammals, and it was also accounted for the double-muscled cattle phenomenon found in some breeds of livestock(Wikipedia). In light of these findings, it was concluded that myostatin must inhibit muscle growth.

An extensive number of studies have revealed that myostatin inhibits the proliferation of myoblasts and their subsequent differentiation into myotubes. The action mechanism of this protein is controlled by a receptor-mediated signal transduction pathway, which unleashes a signal cascade that ultimately results in a complete halt of the cell cycle. Myostatin first binds to an activin receptor II-B located on the cell membrane of myoblasts(Wikipedia) to commence its inhibitory pathway. This, as an effect, impairs cyclin dependent kinase, which ultimately causes cell cycle arrest at G1. Consequently, myoblasts can't replicate or differentiate into myotubes, preventing muscle growth this way(Thomas, M. 2001).

Even though myostatin,'s function seems to be unfavorable at first sight, and it may be tempting to eliminate it from our genome, it also plays a crucial role during myogenesis in embryos, by negatively regulating transcription factors necessary for cell identity determination(Amthor, H. 2002). One scenario in which myostatin inhibition may be medically useful, however, is when a person suffers from an amyotrophic disorder, such as Duchenne muscular dystrophy or just simple muscle atrophy. There's plenty of evidence supporting the premise that myostatin blocking can indeed promote skeletal muscle growth and development(Whittemore, L.A. 2003). Although various methods of myostatin blockade have been designed in the past, each one has had different outcomes regarding force output. For example, when using antibodies against myostatin, there is an increase in musculature accompanied by a disproportionate increase in specific force output, making it a not-so-effective method. On the other hand, when using myostatin propeptide to block myostatin activity there is a proportionate increase in muscle mass and specific force output(Amthor, H. 2006). Looking at this variation in outcomes, I desire to try out a new method of myostatin inhibition, using RNA aptamers.

An aptamer is a nucleic acid oligonucleotide made of RNA or DNA that is capable of binding to a target molecule with high affinity and specificity. These aptamers bind in such a way that they adopt a stable secondary structure "that creates a specific binding site for small ligands" (Ellington, A.D. 1990). An aptamer can be applied in many ways; some applications are to deliver small doses of chemicals to a specific target in the body, to bind to a specific protein and keep it from performing its usual function, and to immunoprecipitate desired target proteins from a solution. For this project, it is intended to design an RNA aptamer against myostatin that will bind to it and render it incapable of performing its function. The hypothesis is that the aptamers will bind to myostatin and alter its general three-dimensional shape, which in effect will make it unrecognizable by the Activin receptor II-B and incapable of triggering the signal transduction pathway, promoting myoblast proliferation and thus, muscle growth.

The downside is that myostatin doesn't have any features that may prove useful in binding nucleic acids. It it also burdensome the fact that myostatin has a negative charge at pH 7.2, our intended selection condition(Putnam, C. 2006). As it has been utilized in other studies, it seems that myostatin is stable in PBS buffer(Lee, S.J. 2004). It is also stable in its storage buffer, a pH 4.0 acetate buffer solution (GenWay). However, it would not be possible to use it, as the pH conditions are not appropriate for our study.

It is also noteworthy that, as far as my knowledge concerns, no aptamer against myostatin has ever been designed, hence the intrigue of the outcome. As stated before, there is a lot of variation among the effectiveness of the different myostatin blockade methods. Therefore, we do not know what to expect from this. Another application for this aptamer might be in diagnosing one's levels of myostatin in the body.

Even though there are plenty of myostatin blockers out there, research on this mysterious protein is still going on. Some of the main labs that keep working, trying to decipher myostatin's secrets, are "The Department of Molecular Biology and Genetics at John Hopkins School of Medicine" and "The Department of Veterinary Basic Sciences, Royal Veterinary College in London."

Experimental Design, Methods, Materials

In vitro N58 RNA bead-based selection against myostatin is the method to be used. As the protein has been functionalized with a histidine tag, Nickel-NTA beads will be employed for this selection. Moreover, a negative selection will be performed in an effort to remove any foreign molecules that can potentially bind to the histidine tag of myostatin, and thus, optimize aptamer binding. The buffer to be employed is 10X PBS buffer, as it has been utilized in various in vitro myostatin experiments in the past(Lee, S.J. 2001). It is safe to assume that myostatin is stable at neutral pH, as it is found in the body under neutral pH conditions(pH 7.2). As an aptamer that is able to operate in physiological pH is being sought after, the buffer to be used will have a pH of 7.2. One drawback is that myostatin has an approximate charge of -1.8 at that pH(Putnam, C. 2006). Nevertheless, the charge can be attenuated by adding positive salts to the buffer, in an attempt to bridge similarly charged molecules, such as RNA and myostatin. Taking these conditions into considerations, it has been determined that 10X PBS selection buffer pH 7.2 (1370mM, 100mM Phosphate, 27mM KCL, 50mM MgCl₂) is the ideal buffer for this experiment.

The binding reaction between the protein and RNA will be incubated for 25 minutes at 37̊ C, as the aptamer has to be resistant to physiological conditions for our purposes. A total of 3 washes, 2 volumes each, will be performed in an attempt to remove the unbound RNA pool and RNA pool with very low binding affinity. The starting RNA to protein ratio will be 400:200, as the fewer the proteins, the stronger the selected aptamers. Myostatin can be stored in -20̊ C and remain stable for long periods of time.

The RNA aptamer selection process consists of incubation of a RNA:target reaction in otder to promote RNA binding to the protein. The unbound pool is then washed out whereas the bound specimens are eluated. Because we are working with RNA pools, the bound specimens have to undergo reverse trascription. The produced ssDNA is then amplified via PCR twice; first through a cycle course PCR, in order to determine the ideal number of cycles that will optimize correct amplification; and through a large scale PCR afterward. The amplified dsDNA is transcribed back into RNA, and isolated through polyacrylamide gel electrophoresis and elution. After purifying the RNA, the selection process repeats, using the new RNA as pool this time. As the number of rounds performed increases, the affinity and specificity of the aptamer increases as well. Therefore, multiple rounds of selection have to be performed before the aptamer against myostatin can be considered functional.

Budget

Histidine-tagged human myostatin (Catalog number 10-663-45269) from GenWay will be used for the Aptamer Research Laboratory project. The molecular weight of this conjugated protein is 42,750 g/mol, and our provider is selling 10ug lyophilized pellets at $165 each. The pellet should be resuspended in a 0.1M acetate buffer pH-4 solution of approximately 0.5mg/mL, such as 10ug/20uL, which also equals 11.7uM. If I were to use 200pmol of protein per round, it would take 8.5ug, or 17ul of solution, which is equivalent to paying $140.25/ round. Luckily, the protein is already functionalized with a histidine-tag, so that will save us time and money, as we don’t have to perform the lengthy biotinylation process. Telephone number: (858)458-0866

References

1.Amthor, H.(2002) "The Regulation and Action of Myostatin as a Negative Regulator of Muscle Development during Avian Embryogenesis" Developmental Biology. Volume 251, Issue 2, Pages 241-257.

2.Amthor, H. (2004) " Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis" Developmental Biology. Volume 270, Issue 1, Pages 19-30

3.Amthor, H. (2006) "Lack of myostatin results in excessive muscle growth but impaired force generation" Proceedings of the National Academy of Sciences of the United States of America. Volume 104, Issue 6, Pages 1835-1840

4.Bogdanovich, S. (2005) " Myostatin propeptide-mediated amelioration of dystrophic pathophysiology" The Journal of the Federation of American Societies for Experimental Biology. Volume 19, Issue 6, Pages 543-549

5.Cash, J.N. (2009) " The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding" The EMBO Journal. Volume 28, Pages 2662-2676

6.Ellington, A.E.(1990)"In vitro selection of RNA molecules that bind specific ligands" Nature. Volume 346, Pages 818-822

7.Freshman Research Initiative Aptamer Stream. Complete RNA Bead Selection Protocol. 2010

8.GenWay Biotech Inc. 2011. <http://www.genwaybio.com/>

9.Lee, S.J. (2001) "Regulation of myostatin activity and muscle growth" Proceedings of the National Academy of Sciences of the United States of America. Volume 98, Issue 16, Pages 9306-9311

10.Medicinenet.com. "Definition of Myostatin." June 24th 2006. <http://www.medterms.com/script/main/art.asp?articlekey=33546>

11.National Center for Biotechnology Information. "Myostatin [Homo sapiens]." December 14th, 2006 <http://www.ncbi.nlm.nih.gov/protein/ABI48514.1>

12.Putnam, C. "Protein Calculator v3.3" March 28th, 2006

13.Rios, R. (2001) "Myostatin is an inhibitor of myogenic differentiation" American Journal of Physiology: Cell Physiology. Volume 282. Issue 5. Pages C993-C999.

14.Thomas, M. (2001) "Myostatin, a Negative Regulator of Muscle Growth, Functions by Inhibiting Myoblast Proliferation" The Journal of Biological Chemistry. Volume 275. Pages 40235-40243.

15.Whittemore, L.A. (2003) "Inhibition of myostatin in adult mice increases skeletal muscle mass and strength" Biochemical and Biophysical Research Communications. Volume 300, Issue 4, Pages 965-971

16.Wikipedia. "Myostatin" August 19th, 2011

< http://en.wikipedia.org/wiki/Myostatin>