R50 RNA pool
Alpha-Synuclein (AS) is a protein that is expressed most abundantly in the brain and other nerve tissues. More is known about the dysfunction of AS than the normal function. (1) This protein is associated with up to 11 different gene mutations that can adversely affect the conformation of the protein and proteins it may interact with.(6) Genetic or environmental factors can cause AS to aggregate and form buildups of the protein in neuronal cell bodies, away from the cell membrane; the aggregations of AS are called Lewy bodies. These structures were originally thought to be the cause of the death of dopaminergic neurons characteristic of Alzheimer’s Disease. (6,11,13) More recent research suggests, however, that the pathogenic form of the protein is the five part oligomer that, when associated with cell membranes, forms a pore-like structure; the going theory is that the Lewy Bodies are the cell’s defense mechanism against the pore-like oligomer.(14)
Specific Aim 1: This research will seek to identify an RNA aptamer with an affinity for the functional monomer of the AS protein, Figure 1 below. Single stranded RNA aptamers will be selected from a pool of random RNA sequences through repeated rounds of selection in a "process of elimination" type assay. An aptamer could functionally inhibit the monomer, preventing it from forming the toxic oligomer. Adding a fluorescent tag to the aptamer would prove useful for helping to further research of how and why the protein misfolds to form the Lewy bodies by allowing for direct observation.(4)
Specific Aim 2: It would be doubly useful to perform simultaneous rounds of selection against samples of the aggregate forms of AS (see Figure 1) including negative selections in order to increase the in vivo affinity and specificity of the respective aptamers. An aptamer that could bind to the aggregated Lewy bodies would be integral in early diagnostics by adding visible tags to the aptamer.(8) Since Lewy body formation is also often associated with decreased proteosome effectiveness, an aptamer that was tagged with ubiquitin could be used in therapeutics to aid the cell in proper destruction of the aggregations. (5, 9,12)
These aptamers could be a stepping stone to better understanding the cause and nature of many Lewy body-associated diseases. AS is available for reorder from rPeptide (678-753-0747) for $300 per 0.5ng, cat # S-1001-1.
Figure 1: Specific Aim 1 proposes selection against the “Native monomer” of alpha-synuclein seen on the far left, Specific Aim 2 selects against the “Fibril” on the far left. The oligomer is a toxic intermediate conformation as the protein goes from monomer to fibril. (1, 14)
Introduction and Background:
Aptamers are derived from synthetically produced random sequences of RNA, ssDNA, or dsDNA. They comprise of a short, variable sequence of nucleic acids (usually about 30-60 bases in length) flanked by forward and reverse primers. A researcher will use the target (a protein, virus, or any molecule of interest) to determine which random sequences out of a pool of 1x1014 synthetic, random sequences exhibit binding affinity, by doing rounds of SELEX in vitro. The SELEX cycle consists of several main steps that are integral in discerning possible aptamers; the target is bound to a filter- or bead-type mechanism and the nucleic acid pool is allowed to interact with the target. The sequences that do not adhere to the target with great affinity during this step are washed away and those that bound are put through rounds of PCR amplification and purification. These newly replicated sequences are used in the subsequent round of selection and the process is then repeated; SELEX represents an inexpensive, process of elimination approach to acquiring highly specific nucleic acids (so named “aptamers”) that will not elicit immune response in vivo. The newly found aptamers can have certain tags added to them (such as a fluorescent tag to show visible signs of binding or a PEG tag to enhance cell uptake, etc) and the SELEX process can be modified to alter the affinity, specificity, and potential application of the aptamers found. (10, 8)
Alpha-Synuclein (AS) is a protein that is mostly expressed in the dopamine-related nerve cells of the central nervous system. (1) It is 140 amino acids long (6), weighs 14.46kDa, and has a pI of 4.6. There are many theories about what this protein’s purpose in the cell is, the most prominent being that it is of structural function.(6) This protein is of particular interest to us when it is in a mutated state. AS has been found to mutate and form large, fibrillic, insoluble aggregations of the protein that are deposited in the cell body of neurons called Lewy bodies (shown in Figure 2, below). These structures impede normal nerve function in a similar way that sickle-cell anemia creates obstructions in the bloodstream. (1,2,6) There are many genetic mutations in the AS coding region that have been previously associated with the presence of Lewy bodies and Parkinson’s Disease, these mutations are shown in figure 3, below.(9,7,13)
Figure 2: Lewy Bodies in brain cells. Lewy Bodies are comprised of malformed aggregations of the protein AS. (2) Figure 3: the gene that codes for the AS protein is shown here, along with regions of the gene that correspond to proposed functional domains; further research must be done to verify the claims made by this source. (3)
Most prominent in research involving AS, are discussions of the oligomeric form that acts as the toxic intermediate between the monomer and the fibril. An aptamer that binds with affinity to the monomer form could prevent formation of the pore-like oligomer by inhibiting the mutated AS proteins from interacting with each other. (6, 7) By counteracting the formation of the oligomeric pore the effects of Parkinson’s Disease in any remaining dopaminergic cells could be minimized. (14)
There is a modification that could be included in this array of AS-binders that would be useful not only for research, but for diagnostics and therapeutics. If a fluorescent tag was added to the aptamer, it could show specifically in what areas of the neural tissue the AS monomer and Lewy bodies are forming and provide earlier diagnosis of Parkinson’s Disease.(4) For even earlier detection, an aptamer could be developed to detect the presence of the intermediate conformation.(1) This type of fluorescently-tagged aptamer could be just as useful in determining the steps along the pathway from normal conformation to toxic aggregation formation.(4,13) Figure 4, below shows the process and usefulness of developing a florescent tagged aptamer.
Figure 4: this figure describes pictorially how a visibly tagged aptamer could function, although there are some risks involved in the process of probe addition. (4)
The grand finale of this aptamer could occur in conjunction with ubiquitin, a protein that labels a protein, often a defective or old one, for destruction by the cell proteosome. This is a normal part of the “housekeeping” functions that a cell must perform in order to remain efficient.(5,12) When an AS-binding aptamer is found, a ubiquitin-like peptide could be integrated onto the aptamer to help the cell become its own medicine. By delivering the signal necessary for protein destruction to occur to the correct location, the aptamer may be able to induce the proteosome to destroy the toxic form of AS, thus ridding the cell of its Lewy body. Figure 4, below is a diagram that shares in more detail how ubiquitin interacts with the proteosome to induce degradation.
Figure 4: a diagram explaining how ubiquitin is integral in the interaction of the proteosome with the protein that is meant to be destroyed. (5)
These aptamers are exciting in their potential for major downstream effects, however before any of those in vivo applications can be implemented, the basic aptamers that bind to the wild type and mutated forms of the AS protein must be painstakingly recovered from the vast nucleic acid pool. AS-binding aptamers have been found by other teams, so it is possible that this aptamer could really be useful if the proper conditions are met and the rounds of SELEX are properly enacted. (8) Although there is already a DNA aptamer that has been identified, it is always integral to scientific research that results are constantly reproduced and improved upon in order to validate previous findings.
Experimental Design, Methods, and Materials:
Filter-based SELEX was deemed appropriate for selecting against AS based on the fact that the AS was suspected of being resuspended in TRIS, rendering biotinylation ineffective; the AS available at the start of the project was from an unknown vendor (though available for reorder from a number of vendors). Although the nitrocellulose filters generally require a protein greater than 15 kDa in weight, the filters usually have a negative charge. (10) AS has a pI of 4.6, meaning that in pH environments near or below this value, the protein will accrue more positive charges on its surface. The more positive the protein, the more likely it is to be bound to the negatively charged protein. The selection buffer used was MES (MW 195.2) at pH 5.6 in order to induce more positive charge on the AS protein, allowing the 14.4kDa AS protein to be better retained on the filter than if the filter-selection was performed at physiological pH. The working concentration of salts is crucial to the proper folding of proteins and nucleic acids; the working concentrations of NaCl and MgCl2 were 150mM and 5mM, respectively. Other papers suggested the importance of Ca2+ in determining the folding of AS, this variable was disregarded for simplicity. (6)
Filter SELEX is useful because the nitrocellulose has pores that are too small to allow the passage of RNA-protein complex yet large enough to allow the passage of unbound RNA, allowing the separation of bound and unbound species. The flow-through and eluants obtained after each of the two binding reactions was ethanol precipitated in order to isolate the RNA. The elutions contained the RNA most interesting to the project, this was the RNA that showed affinity to AS. The precipitated RNA was resuspended in 10uL of diH2O and then reverse transcribed so that ssDNA was now present. The ssDNA derived from the eluted filter underwent a large scale PCR so that the effective pool size was increased. This new dsDNA was transcribed in order to convert the DNA back to ssRNA. The RNA was then isolated from the transcription reaction using a denaturing dye in conjunction with a PAGE gel. The RNA was extracted from the gel using the crash soak method and subsequently ethanol precipitated and quantified. This made the RNA available for use as the starting pool for the next round of selection. (10)
Two rounds of selection were performed using the method described above and the arbitrarily selected R50 RNA pool. For the first, an R50 RNA: AS protein ratio of 400pmol:300pmol was used. For the second the R50 RNA: AS protein ratio was 150pmol:120pmol. Both binding reactions used the MES selection buffer and were allowed to incubate for 30 minutes; the first round incubated at 37C and the second at 25C.
The monomer was the easiest to procure and was the first (and thus far, only) AS protein formation to be selected against. In order to increase the binding specificity of this aptamer, negative selections will need to be performed in order to eliminate background RNA sequences that exhibit affinity for the nitrocellulose filter. (10) Also, the potential AS monomer aptamers will need to be screened against other conformations of AS in order to increase specificity in this regard.
At the start of the project, the AS was available in the lab but was from an unknown vendor. AS is available for reorder from rPeptide; 0.5ng of AS costs $300 and would allow for 86.5 rounds of selection at $3.47 apiece in addition to any costs added by any new equipment, etc. The catalog number is S-1001-1 and can be ordered by calling 678-753-0747.
Results and Discussion:
The first observable results for Round 1 came with the cycle course gel. Samples were taken from the reverse transcription reactions of the first and third flow-through (W1 and W3) as well as the elution. During the 72C extension step of PCR, 5uL aliquots of each of the samples were removed from the PCR reaction mix. This step allows the determination of the optimal number of cycles of PCR in relation to pool concentration after selection has occurred. By observing the image of Gel 1, below, it was determined that 11 cycles of PCR would provide a strong amplification of the eluted pool without over amplification. The bands of the gel may appear distorted, this is due to imperfections/lumps in the gel as it solidified, a human error; the streak running through the bottom half of the gel is a scratch on the imaging bed.
Gel 1: Round 1 cycle course gel. This shows that 11 cycles of PCR is sufficient for amplifying R1 RNA.
The next observable data came from the PAGE gel in which the binding species of RNA were separated from the transcription reaction components. The shadow band of RNA observed in the PAGE, rendered in Gel 2, below, was quite small, though dark. The RNA band was cut out of the gel and eluted through 3 washes using the crash-soak method and an ethanol precipitation.
Gel 2: Round 1 PAGE gel. RNA band was small, yet of condensed appearance.
The Nanodrop spectroscopy revealed that the concentration of RNA indeed was low; the round one RNA yield was 5.3uM, which is not enough to start round 2. I performed three more washes of crash-soak and an ethanol precipitation in order to attempt to extract more RNA out of the gel to add to my final sample of RNA. This increased the concentration of round one RNA to 6uM. This was still an extremely low yield. A check on the large scale PCR reaction was run in order to verify that template was indeed present during the transcription reaction. Gel 3, below, is an exhibition of the lsPCR check.
Gel 3: the large-scale PCR check exhibits a well-defined band; there was template provided for transcription. (Ladder is not shown because it was too dilute.)
With a defined band in the PAGE and a successful lsPCR, the only potential causes of the low RNA yield were failed RNA polymerase for the transcription or RNAse contamination. However, in spite of all this, there was still RNA present and round two could be started using the low concentration round one RNA pool using extreme caution and conversely low concentrations of AS protein. The RNA: protein ratio was lowered to 150pmol:120pmol; this amount of RNA constituted of the entire round one yield, so it was a risky pursuit. The first confirmation of whether or not the second round of selection was successful was with the second cycle course gel, gel 4, below. This gel indicates that the low concentration of RNA did not impede the usual binding process and indicates that 9 cycles of PCR are sufficient in order to amplify the second round bound species.
Gel 4: Round 2 cycle course gel indicates success in spite of low round 1 yield. 9 rounds show optimal amplification. (Only eluted binding species run through PCR in order to conserve materials.)
Overall Problems Encountered:
The most obstructive challenge faced with this project was trying to find an effective way to immobilize the AS protein. Thinking at first that the protein was too small, and therefore ineligible, for filter-based SELEX, biotinylation was attempted. However, the buffer that the protein was resuspended in was a mystery as no information was left by the previous AS user as to the details of this protein’s storage. Because of the possibility that the protein was suspended in TRIS, all of the “biotinylated” AS had to be discarded; TRIS compound reacts with biotinylate in the same way that the amino acid lysine does, this could have prevented a majority of the biotinylate from ever reacting with the AS. Without biotinylation, the only other option was filter-based SELEX, which seemed risky as AS was small enough to potentially pass right through the filter pores. This problem was resolved by buffering the selection reaction at a pH of 5.6, near the pI of AS (4.6) in order to make the protein “stick” to the filter for electrochemical reasons as opposed to mechanical reasons. The process of elimination in deciding which method of SELEX to use took several weeks to overcome yet paid off when one and a half successful rounds of selection were easily procured.
Conclusion and Future Work:
The focus of this semester-long project was to identify (or begin to identify) and aptamer that showed affinity for the AS protein that is assigned responsibility for the onset of Parkinson ’s disease. The AS monomer aptamer could have the potential to be a diagnostic and research tool when tagged with florescent compounds and could also be a therapeutic if the binding of the aptamer inhibited the association of one AS protein monomer with another. The one and a half rounds of selection performed in working towards this goal are outlined in Table 1, below.
Table 1: specifics of each round for R50 RNA selection against AS
Future work could include continuing to hone in on the RNA aptamer for the AS monomer and adding the tags to what is found to adapt the aptamer’s function. Once the AS monomer aptamer is found and studied, it would be a useful therapeutic tool to isolate an AS aptamer that binds to the Lewy Body aggregate or the oligomer formation. The AS LB aptamer could be tagged with ubiquitin peptides to aid in the natural destruction of the LB or of the pore-like oligomer.
Though I am not entirely sure of what I will be doing in the next five or ten years, being a part of the FRI has opened up an option for my future by showing me what it is like to perform biological research and the different types of research that are out there. Regardless of whether or not I end up researching aptamer s in the future or if I even end up doing research at all, my time here in the Aptamer Stream was very enriching to my college experience. I am thrilled that I have been placed in the Undergraduate Rotation Program, because now, after two semesters of performing research alongside my schoolwork, I can’t imagine myself not saying “I have to go to lab”; working in the lab has truly become a major part of my academic experience here at UT
I have taken away so much from this process even though my selection is still only in the first few phases. Not only have I developed a greater understanding for Parkinson’s Disease and aptamers, but I have also learned about the triumphs and tribulations of research and how to problem solve through unforeseen issues with a project. I have also discovered things about myself, my preferences as a scholar, and what I would like to consider for a career path. Through designing my experiment and outlining the parameters for the SELEX reactions, I now understand the creativity that scientific research requires. By analyzing solutions to frustrating issues with getting my project started, I was challenged to think outside the box. Through my preliminary research about Parkinson’s Disease and alpha-Synuclein, I experienced the excitement and gratification that comes from knowing that the work I will be doing both now and in the future could have a real impact on people who may know nothing about science but so desperately need its innovations. Before becoming involved in the FRI and beginning my individual research project I was unsure of my capability and zeal for research, but now that I have become a part of the research community at my university I am realizing what an exciting, relevant, difficult, and flexible field biological research can be.
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