Hey Brad or anyone who knows,
Is it necessary to turn in a written copy of your abstract or does the online submission suffice?
Thanks,
Austin
RNA Aptamer Selection Against HCP1 for the Therapeutic Attenuation of Melioidosis
Complete proposal available here.
Melioidosis, a highly virulent disease caused by infection of the soil-borne bacterium Burkholderia pseudomallei, is currently and voraciously expanding its geographical dominion from the general propinquity of southeast Asia and northern Australia1 to adjacent regions of the world due to the glaring absence of efficacious therapies, namely antibiotics. Hcp1, a protein involved in the secretion system (Type VI) of the organism as either a translocator or effector macromolecule2, is therein implicated in the intrinsic perniciousness of B. pseudomallei.3 Hcp1 then, represents a feasible and promising target for aptamer selection, as inhibition of the bacterial secretion mechanisms which undergird the persistence of the infection would have the potential to devastate entire colonies and extinguish the pathogen altogether.
The specific aims of this proposal involve the isolation of a specific, anti-Hcp1 binding aptamer sequence for therapeutic utility, and, as more of an extant potentiality, the application of this aptamer to a population of B. pseudomallei in vitro for the determination of the specific function of HCP1 and additionally to afford greater insight into the functional nature of Type VI bacterial secretion systems (T6SS).
Figure 1: Explication of basic methodological progression from initial selection rounds to latent applications of derived anti-Hcp1 aptamer.
In effect, the goal of this proposal is to select an aptamer that binds with high affinity to HCP1, a protein directly involved in the lethality of B. pseudomallei, the causative agent of melioidosis, in order to offer an alternative to the currently inadequate repertoire of therapeutic methods persisting in melioidosis-affected areas of the world. Additionally, application of this aptamer within the realm of basic science may cast further comprehension upon the nature of T6SS and the specific, now speculative, role occupied by the proteins endemic to them, specifically, Hcp1.
1. Mongkol Vesaratchavest et. al, Nonrandom Distribution of Burkholderia pseudomallei Clones in Relation to Geographical Location and Virulence J. Clin. Microbiol., Jul 2006; 44: 2553 - 2557.
2. Bingle, Lewis et. al. Type VI secretion: a beginner’s guide Curr. Op. in Microbiol., February 2008, 11(1): 3 - 8.
3. Schell, Mark A. et. al. Type VI secretion is major virulence determinant in Burkholderia mallei Mol. Micro., 2007, 64(8): 1466 - 1485
Nucleic Aptamer Selection Against S100A4 for Prevention of Cancer Metastasis
The full proposal has been posted on Dropbox.
S100A4 (also known as calvasculin) belongs to the S100 family of Ca2+-binding proteins. S100 proteins are localized in the cytoplasm and/or nucleus of a wide range of cells, and involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. Calvasculin is a polypeptide of 100 amino acids, excluding the initiating N-terminal methionine. The molecular mass of the natural protein, determined by electrospray ionisation mass spectrometry, is 11,646 Da. (Barraclough 1998). The protein has many functions, such as induction of angiogenesis, stimulation of neurite outgrowth and protection of cells from proapoptotic stimuli (Hua et al. 2009). S100A4 may also function in motility, invasion, and tubulin polymerization. Chromosomal rearrangements and overexpression of this protein have been implicated in the motility and metastasis of cancer through interactions with cytoskeletal constituents.
S100A4 signaling can focus on factors associated with normal and abnormal proliferation, apoptosis and growth, and differentiation. Therefore, it is arguably a molecular target of considerable potential possessing a wide ranging biological activity that can alter and regulate the major phenotypic features of cancer (Sherbet 2008). RNA ligands, called aptamers act as a promising means to inhibit S100A4. Aptamers bind with high specificity and high affinity to their protein targets and are powerful tools in diagnostics and therapeutics (Stoltenburg et al. 2008). Aptamers against S100A4 are expected to provide a major defense against metastasis of cancer and tumor cells.Specific Aim 1: Selection of RNA aptamers against S100A4
S100A4 promotes cell progression through angiogenesis and allows tumor neovascularization and cell progression of cancer cells (Sherbet 2008). Therefore decreased levels of this protein correlate to decreased metastasis. Selection of RNA aptamers against S100A4 will decrease metastasis of cancer and tumor cells and since S100A4 also protects from apoptotic stimuli may also induce apoptosis in these malignant cells.
Barraclough, R. 1998. Calcium-binding protein S100A4 in health and disease. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1448(2): 190-199.
Hua J., D. Chen, H. Fu, R. Zhang, W. Shen, S. Liu, K. Sun, and X. Sun. 2009. Short hairpin RNA–mediated inhibition of S100A4 promotes apoptosis and suppresses proliferation of BGC823 gastric cancer cells in vitro and in vivo. Cancer Letters, 292 (1): 41-47.
Sherbet, G.V. 2008. Metastasis promoter S100A4 is a potentially valuable molecular target for cancer therapy. Cancer Letters, 280(1): 15-30.
Stoltenburg, R., C. Reinemann, and B. Strehlitz. SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomolecular Engineering 24 (4): 381-403.
Hua J., D. Chen, H. Fu, R. Zhang, W. Shen, S. Liu, K. Sun, and X. Sun. 2009. Short hairpin RNA–mediated inhibition of S100A4 promotes apoptosis and suppresses proliferation of BGC823 gastric cancer cells in vitro and in vivo. Cancer Letters, 292 (1): 41-47.
Sherbet, G.V. 2008. Metastasis promoter S100A4 is a potentially valuable molecular target for cancer therapy. Cancer Letters, 280(1): 15-30.
Stoltenburg, R., C. Reinemann, and B. Strehlitz. SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomolecular Engineering 24 (4): 381-403.
Nucleic Acid Aptamer Selection Against MUC-1 for the Early Diagnosis of Epithelial tumors.
Fall 2010 Proposal for MUC1 Selection
Some of the most prominent carcinomas of the breast, lungs, stomach, pancreas, prostate and ovaries are categorized in a group of epithelial tumors (3). Mucins are glycoproteins that provide a protective layer on epithelial surfaces and are involved in cell-cell interactions, signaling, and metastasis (1). Because
mucins and specifically the MUC1 are such great tum
or markers for epithelial malignancies, they are more readily being used for immunotherapeutic and diagnostic approaches (2). Using aptamer binding techniques to target the specific MUC1 glycoprotein would allow for the formation of new diagnostic assays against the tumor to help with early diagnosis of several types of epithelial malignancies (4).
Figure 1: MUC1 structure
Aptamers are nucleic acid oligonucleotides that bind with specificity and tightness to areas of interest. Developing aptamers against certain targets makes it possible to inhibit the target’s f
unction, consequently reducing the effects of a disease they are linked to (5). Other research has shown that using the SELEX methodology to choose aptamers against MUC1 provided insight to the exact binding site between the aptamer and MUC1 complexes which gives hope to the possibility to improving this detection methodology for further use in early diagnosis techniques (6).
Specific Aim 1:
MUC1 is an excellent epithelial tumor antigen in several different carcinomas around the body. It has been shown that by binding aptamers to MUC1, isolating cancer cells at early stag
es of malignancies can be greatly beneficial to the populace. Thus the selection of RNA aptamers against MUC1 will make cancer cells more apparent at an early stage and destroying those specific cancer cells will become easier.
Figure 2: Mucin acts as an antigen on the surface of most epithelial cancer cells
GenScript USA $54.00 1mg Catalog Number RP20402_1mg
References:
- Ho, J.J.L. "Mucins in the Diagnosis and Therapy of Pancreatic Cancer." Current Pharmaceutical Design 6.18 (2000): 1881-896. Print.
- Parry, S. "Identification of MUC1 Proteolytic Cleavage Sites in Vivo." Biochemical and Biophysical Research Communications 283.3 (2001): 715-20. Print.
- Cheng, Alan K. H., Huaipeng Su, Y. Andrew Wang, and Hua-Zhong Yu. "Aptamer-Based Detection of Epithelial Tumor Marker Mucin 1 with Quantum Dot-Based Fluorescence Readout." Analytical Chemistry 81.15 (2009): 6130-139. Print.
- Ferreira CS, and Papamichael K. "DNA Aptamers against the MUC1 Tumour Marker: Design of Aptamer-antibody Sandwich ELISA for the Early Diagnosis of Epithelial Tumours." Anal Bioanal Chem (2007): 1039-050. Print.
- Cerchia L, and De Franciscis V. "Targeting Cancer Cells with Nucleic Acid Aptamers." Trends Biotechnol. (2010). Print.
- Cheng AK,, Su H, Wang YA, and Yu HZ. "Aptamer-based Detection of Epithelial Tumor Marker Mucin 1 with Quantum Dot-based Fluorescence Readout." Anal Chem (2009): 6130-139. Print.
Nucleic Acid Aptamer Selection Against S100B for the Detection and Progression of Alzheimer’s Disease
Full proposal is in Dropbox.
Final Manuscript can be found here.
Alzheimer’s, a disease in which the brain tissue slowly deteriorates, induces memory loss in millions of people worldwide. Although the cause of this fatal dementia is not yet known, S100B, a protein, could serve as a potential diagnostic or biochemical marker of brain cell damage (Raabe, 2003). S100B is expressed primarily by mature astrocytes and aid in “neurite extension, proliferation of melanoma cells, stimulation of calcium fluxes, inhibition of PKC-mediated phosphorylation, astrocytosis and axonal proliferation, and inhibition of microtubule assembly” (Raybiotech). At nanomolecular concentrations, S100B stimulates neuron growth and survival. In contrast, micromolar concentrations can lead to apoptosis, or cell death (Rothermundt, 2003). Alzheimer’s patients demonstrate an increased level of the protein in areas such as the hippocampus and temporal lobe (Eldik, 1994). Therefore, S100B levels can be used as a diagnostic marker to detect and monitor the progression of Alzheimer’s in an individual.
Figure 1) S100B protein
RNA aptamers are short strands of nucleic acids that can bind to a specific target and have the ability to accurately differentiate between related targets by a single functional group. Thus, an aptamer could bind to S100B and measure how much of the protein is contained in cerebrospinal fluid. There is growing evidence that serum levels of S100B in Alzheimer’s patients are significantly higher than those of healthy individuals (Chaves, 2010). In addition, recent studies also suggest that the level of S100B in cerebrospinal fluid can serve as a marker for brain functional condition and progression of Alzheimer’s disease (Peskind, 2001). As a result, medical examiners can utilize an aptamer to diagnose Alzheimer’s disease at an early stage, track the phases of the dementia, and possibly even improve the quality of life for these infected individuals.
The quantity of S100B found in cerebrospinal fluid can be harmless or deadly. Adequate amounts of the protein can be normal while excessive amounts lead to the on set of Alzheimer’s and eventually memory loss. An aptamer has the ability to attach precisely to the S100B protein helping doctors to monitor the protein levels and diagnose patients. The number of proteins can further tell doctors how severe the disease may be. Consequently, the selection of RNA aptamers against S100B will allow physicians to diagnose and aid their patients in ways that they have never been able to before.
Raybiotech: 100ug $149 Catalog Number: 230-000 02
Figure 2) Ample amounts of S100B can lead to the distruction of brain tissue causing memory loss and eventually Alzheimer’s. An aptamer can serve as a diagnostic marker for the diesase and even help doctors track its stages.
Chaves M., Camozzato A., Ferreira E., Piazenski I., Kochhann R., Dall’Igna O., Mazzini G., Souza D., Portela L. (2010) “Serum levels of S100B and NSE proteins in Alzheimer’s disease patients” Journal of Neuroinflammation.
Eldik L., Griffin W. (1994) “S100B expression in Alzheimer’s disease: Relation to neuropathology in brain regions” Biochimica et Biophysica Acta (BBA) – Molecular Cell Research. 1223(3): 398-403.
Peskind E., Griffin S., Akama K., Raskind M., Eldik L. (2001) “Cerebrospinal fluid S100B is elevated in the earlier stages of Alzheimer’s disease” Neurochemistry International. 39(5-6): 409-413.
Raabe, A., Kopetsh, O., Woszczyk, A., Lang, J., Gerlach, R., Zimmermann, M., Seifert, V. (2003) “Serum S-100B protein as a molecular marker in severe traumatic brain injury.” Restor Neurol Neurosci. 21(3-4): 159-169.
Rothermundt M., Peters M., Prehn JH., Arolt V. (2003) “S100B in brain damage and neurodegeneration.” Microsc Res Tech. 60(6): 614-632.
The Selection of RNA Aptamers against S100 Calcium Binding Protein B for Inhibition of Inflammatory Cytokines in Alzheimer’s.
The full proposal can be seen here
Progress Report 1 can be seen here
Progress Report 2 can be seen here
Final Report can be seen here
S100B is a calcium binding protein produced mainly by glial cells in the Central Nervous System (1). As calcium binds to the EF-hands of the protein, S100B undergoes different conformational changes, allowing the protein to bind to several other molecules in the body, making it a multi-functional target (2). Because of the variety of binding capabilities for S100B, these proteins participate in several neurological diseases including Alzheimer’s, epilepsy, schizophrenia, Down’s syndrome, and melanoma (3).
Alzheimer’s disease (AD) is the predominant form of dementia, where patients suffer from a decline of cognitive functions such as language, personality, and memory (4). S100B plays a substantial role in AD as suggested by the elevated levels of the protein in the brain or CNS after diagnosis. S100B can act intracellularly or can be secreted into the extracellular space of glial cells and act as a cytokine, which binds to nearby cells via receptors (5). S100B activates the Receptor for Advanced Glycation Endproducts (RAGE), an immunoglobulin-like cell surface receptor , and in turn, triggers NF-kB and MEK signaling pathways (6). Although nanomolar concentrations of S100B can have trophic and neuroprotective effects, any over expression of the S100B protein can lead to neuroinflammation, neuronal dysfunction, and cell apoptosis that contributes to the degenerative affects of Alzheimer’s (4).
RAGE is activated initially by S100B, that starts a positive feedback loop to continually trigger S100B proteins that activate NF-ĸB gene transcription which release inflammatory cytokines.
Although elevated levels of S100B can indicate brain trauma and Alzheimer’s, no preventative measures to actually delay the onset of neurological damage have been developed. An aptamer is a sequence of nucleic acid that binds specifically to target molecules, primarily proteins (7). An aptamer selected for S100B protein can potentially be used as both a therapeutic and diagnostic tool in combating Alzheimer’s.
Specific Aim 1: Selection of RNA aptamer against S100B protein High concentrations of S100B in the brain and CNS lead to over stimulation of RAGE leading to neurological damage. The selection of an aptamer for S100B that inhibits binding, either directly or allosterically, to the V domain of RAGE will impede further signal transduction pathways that hinder neurological function. The inhibition of the S100B/RAGE interaction would provide a therapeutic method to slow down the devastating effects of Alsheimer’s.
References
1. Zhang, X.Y., et. Al. (2010) “Increased Serum S100B in never-medicated and medicated Schizophrenic patients.” Journal of Psychiatric Research 10:1-5.
2. Razvanpour, A., and Shaw, G. (2009) “Unique S100 Target protein interactions.” Gen. Phsyiol. Biophys. 28: 39-46.
3. http://en.wikipedia.org/wiki/S100B
4. Leclerc,E., et. al. (2010) “The S100B/RAGE Axis in Alzheimer’s Disease.” Cardiovasc. Psychiatry Neurol. P. 1-20.
5. Huttunen, H. et al. (2000) “Coregulation of Neurite Outgrowth and cell survival by Amphoterin and S100 proteins through Receptor for Advanced Glycation Endproducts (RAGE) Activation.” Journal of Biological Chemistry. 275(51): 40096-40105.
6. Sorci, G. et al. (2003) “S100B inhibits Myogenic differentiation and Myotube formation in a RAGE-independent manner.” Molecular and Cellular Biology. 23(14): 4870-4881.
7. http://en.wikipedia.org/wiki/aptamer
Image: Leclerc,E., et. al. (2010) “The S100B/RAGE Axis in Alzheimer’s Disease.” Cardiovasc. Psychiatry Neurol. P. 1-20.
S100B RayBiotech, Inc.
$149/100ug
HIS tag
PBS buffer
RNA Aptamer Selection Against Interferon-gamma to Reduce T-cell Proliferation in Transplant Rejection (Ashley Dawson)
The full proposal has been posted on DropBox
The final manuscript has now been posted on Dropbox as well.
Organ transplantation has the ability to extend the life-expectancy of many patients with severe illnesses. However, if the Human Leukocyte Antigens (HLA) of the donor and recipient do not match almost exactly, the recipient’s own immune system can jeopardize the health of the transplant. While the immune system is extremely complex, several immunosuppressant drugs have been developed that inhibit this negative response [1]. As many of these drugs target T-cell proliferation in general, this results in a greatly reduced ability to respond to infections. Targeting only a specific step in the immune pathway would allow for the continuation of many other aspects of the immune response and decrease the likelihood of acquiring an opportunistic infection.
The final manuscript has now been posted on Dropbox as well.
Organ transplantation has the ability to extend the life-expectancy of many patients with severe illnesses. However, if the Human Leukocyte Antigens (HLA) of the donor and recipient do not match almost exactly, the recipient’s own immune system can jeopardize the health of the transplant. While the immune system is extremely complex, several immunosuppressant drugs have been developed that inhibit this negative response [1]. As many of these drugs target T-cell proliferation in general, this results in a greatly reduced ability to respond to infections. Targeting only a specific step in the immune pathway would allow for the continuation of many other aspects of the immune response and decrease the likelihood of acquiring an opportunistic infection.
Figure 1. There are many steps in the immune system pathway. Inhibiting something specific, such a cytokine, could lessen the intensity of the response.
Interferon-gamma (IFN-γ) is a 16 kDa cytokine that is 143 amino acids in length [2]. It is released when T-cells are stimulated by Interleukin-18 and has been found to induce expression of HLA-DR (a major histocompatibility complex cell surface receptor) which in turn mediates graft-versus-host disease. This is due to non-matching HLA’s and ultimately contributes to transplant rejection [3]. To decrease the levels of IFN-γ without entirely inhibiting its production, an aptamer could be developed to specifically bind to the cytokine. This would change its formation and prevent it from interacting with its receptor (CXCR3), reducing the amount of signals that would continue along the immune response pathway.
Figure 2: Interleukin 18 stimulates T-cells to release IFN-γ. The aptamer would then bind to the cytokine and change its shape, rendering it unable to bind to its receptors (CXCR3).
Many experiments have been performed involving the inhibition of IFN-γ and its relation to transplant rejection. For instance, Skurkovich B. et. al determined that treatment with anti-interferon gamma antibodies improved corneal transplant rejection [3]. While most of the other inhibiting agents were also antibodies; one paper (Lee et. al) did describe an oligonucleotide sequence that was determined to inhibit the interaction between IFN-γ and its receptor [4]. This proposal plans to select an aptamer instead of using antibodies due to the high level of specificity necessary as there are several different types of Interferon.
Genway Bio: 50 µg $165 (IFN-γ with His tag) Catalogue Number: 10-663-45807
[1] Duncan and Wilkes. “Transplant-related Immunosuppression, A Review of Immunosuppression and Pulmonary Infections”. The Proceedings of the American Thoracic Society. 2005; 2:449-455
[2] Horst Ibelgaufts' COPE: Cytokines & Cells Online Pathfinder Encyclopaedia. http://www.copewith cytokines.de/cope.cgi?key=IFN-gamma
[3] Skurkovich B. “Treatment of corneal transplant rejection in humans with anti-interferon-gamma antibodies”. Am J Ophthalmol. 2002; 133(6):829-30.
[4] Lee et. al. “An oligonucleotide blocks interferon-gamma signal transduction”. Transplantation. 1996; 62(9):1297-301.
Figure 1: figure taken from medscape.com “Transplantation Tolerance”. http://cme.medscape.com/ viewarticle/418534_3 .
In Vitro Selection of RNA Aptamers Against CXCL1 to Inhibit Carcinogenesis.
Final Manuscript is available here
CXCL1, chemokine (C-X-C motif) ligand 1, is a protein that has been found to be a responsible party for tumorigenesis. This particular cytokine has been predominately associated with breast and melanoma cancers. As well, CXCL1 has been linked to cancers such as colon, pancreatic, ovarian and melanoma. As CXCL1 and other chemokines regulation is accelerated, the activation of NF-κB(also widely associated with tumors) is positively correlated. [1]
Figure 1 CXCL1 [2]
Specific Aim 1: To select an RNA aptamer to blockade CXCL1 significantly reducing tumor growth.
As CXCL1 is now known to influence metastasis, research with antibodies has begun for breast cancer. Current literature suggests that a combination of chemotherapy as well as an inhibitor to the tumor growth process could potentially be a more effective way of curing cancer. An aptamer for this task would be more preferable as it would be designed far more specific, and would be less likely trigger an immune response. [3]
Other potential uses for an aptamer selected for CXCL1 include regulations of other cancers as well. CXCL1 in epithelial cells has been associated greatly with aging and melanoma.[4]
Recombinant Human Growth-regulated protein alpha is sold through raybiotech.com for $98.00 per 100µl . The catalog number is RB-01-0002P-1.
[1] Dhawan, P., Richmond, A. Role of CXCL1 in tumorigenesis of melanoma. Journal of Leukocyte Biology. 2002;72:9-18.
[2] http://en.wikipedia.org/wiki/CXCL1
[3] Fulmer, T. SciBX 3(4); doi:10.1038/scibx.2010.105 Published online Jan. 28, 2010
[4] Fimmel,S., Devermann, A. H., Zouboulis, C. Gro-a A potential Marker for Cancer and Aging Silenced by RNA Interference. Ann. N.Y. Acad. Sci. 1119: 176–189 (2007).
Selection Against Amyloid Precursor Protein (APP), using nucleic acid aptamers, holds therapeutic and diagnostic potentials for Alzheimers Disease (AD
The full proposal has been posted on Dropbox.
Selection Against Amyloid Precursor Protein (APP), using nucleic acid aptamers, holds therapeutic and diagnostic potentials for Alzheimers Disease (AD).
AD is a neuro-degenerative disease usually affecting individuals in the latter years of life; it is also the most common form of dementia. Common symptoms include short and long term memory loss, speech impediments, and gradually loss of bodily function. Even though AD was first diagnosed over 100 years ago, there exists no definitive cure for it, and diagnostics are based on expensive and time consuming imaging technologies and neuropsychological tests [1].
Amyloid beta is a protein that is key in the progression of AD, although the specifics of the mechanism are still being researched, the hypothesis that accumulation of this peptide leads to neural degeneration is considerably valid. An effective diagnostic tool, then, would be some sort of biomarker that could target and thus detect amyloid beta. Recent studies, conducted by Rahimi and Murakami of the David Geffen School of Medicine, however, investigated the practicality of targeting beta amyloid with such aptamers, but unfortunately, did not conclude with positive results [2].
The only information gleaned from this study was the fact that aptamer’s did show some affinity towards 40 and 42 Abeta concentrations. The conclusion however was that, “aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils” [2].
Figure I: The Abeta formation pathway [3]
Even though Abeta binding Aptamers weren’t exactly successful, a second prevalent protein, the precursor to Abeta in fact, also has high potential for diagnostic or perhaps even therapeutic research with aptamers. As seen in Figure I above, there is a stage in the Abeta formation pathway where APP is soluble and free of amyloid fibrils. If an aptamer, with high enough affinity for APP were to be found, it could be used to signal abnormally high levels of the same, or even halt the formation of Abeta fibrils in general: essentially, either eliminating Abeta plaque formation in general, or becoming a highly effective diagnostic tool.
APP is available for order through GenScript, catalogue number RP20165, for 189.00 dollars per milligram. [4]
[1] http://en.wikipedia.org/wiki/Alzheimer's_disease#Diagnosis
[2] Rahimi, F, et al. (2010), “RNA aptamers generated against oligomeric Abeta40 recognize common amyloid aptatopes with low specificity but high sensitivity”, David Geffen School of Medicine, Department of Neurobiology, Los Angeles, California.
[3] The Trafficking and Metabolism of Amyloid Precursor Protein (APP). Digital image. Coffee Break. NCBI, 15 Sept. 1999. Web. 25 Aug. 2010..
[4] "Beta Amyloid Peptides/ Beta Amyloid Peptide/ β Amyloid Peptide - GenScript." GenScript - Your Innovation Partner in Drug Discovery! Web. 31 Aug. 2010..
Selection Against Amyloid Precursor Protein (APP), using nucleic acid aptamers, holds therapeutic and diagnostic potentials for Alzheimers Disease (AD).
AD is a neuro-degenerative disease usually affecting individuals in the latter years of life; it is also the most common form of dementia. Common symptoms include short and long term memory loss, speech impediments, and gradually loss of bodily function. Even though AD was first diagnosed over 100 years ago, there exists no definitive cure for it, and diagnostics are based on expensive and time consuming imaging technologies and neuropsychological tests [1].
Amyloid beta is a protein that is key in the progression of AD, although the specifics of the mechanism are still being researched, the hypothesis that accumulation of this peptide leads to neural degeneration is considerably valid. An effective diagnostic tool, then, would be some sort of biomarker that could target and thus detect amyloid beta. Recent studies, conducted by Rahimi and Murakami of the David Geffen School of Medicine, however, investigated the practicality of targeting beta amyloid with such aptamers, but unfortunately, did not conclude with positive results [2].
The only information gleaned from this study was the fact that aptamer’s did show some affinity towards 40 and 42 Abeta concentrations. The conclusion however was that, “aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils” [2].
Figure I: The Abeta formation pathway [3]
Even though Abeta binding Aptamers weren’t exactly successful, a second prevalent protein, the precursor to Abeta in fact, also has high potential for diagnostic or perhaps even therapeutic research with aptamers. As seen in Figure I above, there is a stage in the Abeta formation pathway where APP is soluble and free of amyloid fibrils. If an aptamer, with high enough affinity for APP were to be found, it could be used to signal abnormally high levels of the same, or even halt the formation of Abeta fibrils in general: essentially, either eliminating Abeta plaque formation in general, or becoming a highly effective diagnostic tool.
APP is available for order through GenScript, catalogue number RP20165, for 189.00 dollars per milligram. [4]
[1] http://en.wikipedia.org/wiki/Alzheimer's_disease#Diagnosis
[2] Rahimi, F, et al. (2010), “RNA aptamers generated against oligomeric Abeta40 recognize common amyloid aptatopes with low specificity but high sensitivity”, David Geffen School of Medicine, Department of Neurobiology, Los Angeles, California.
[3] The Trafficking and Metabolism of Amyloid Precursor Protein (APP). Digital image. Coffee Break. NCBI, 15 Sept. 1999. Web. 25 Aug. 2010.
[4] "Beta Amyloid Peptides/ Beta Amyloid Peptide/ β Amyloid Peptide - GenScript." GenScript - Your Innovation Partner in Drug Discovery! Web. 31 Aug. 2010.
RNA Aptamer Selection Against Beta Lactamase to Inhibit Growth of Multi-Drug Resistant Bacterial Strains
The full proposal has been posted on Dropbox.
The advent of antibiotics was an extremely important step in the progress of human society. Yet with the constant usage of these antibiotics, bacteria have developed resistance. One of the ways that bacteria have gained resistance is through the protein β-lactamase.
The process of evolution takes place constantly; as a result, certain bacterial strains have developed resistance to antibiotics. Antibiotics, such as penicillin (a β-lactam) stop bacterial growth by interfering with the synthesis of the peptidoglycan layer of cell walls. These antibiotics disrupt DD-peptidases that form the cross-links in peptidoglycan [1]. However, through repeated exposure to these antibiotics, bacteria have evolved to counteract this by synthesizing the protein β-lactamase. This particular enzyme attacks the β-lactam ring of antibiotics, thereby rendering the antibiotic ineffective [2].
Figure 1: Ribbon Structure of Beta Lactamase, Adapted from Fonze, E. et al (1995)
An aptamer is a short sequence of nucleic acid that binds to a certain target, usually protein, very tightly. Using the SELEX method of aptamer selection incorporates the same driving force of evolution that produced β-lactamase in bacteria. By exponentially multiplying the nucleic acid sequences that bind most tightly to the protein of interest, only the most “fit” nucleic acids remain. This tightly binding nucleic acid can subsequently interfere with protein function. Because β-lactamase confers bacterial resistance to many antibiotics, introduction of a tightly binding aptamer along with the normal antibiotic would then be sufficient to stop growth of the bacteria. Without this aptamer, the antibiotic alone would be broken down by the bacteria.
Consequently, by finding a therapeutic aptamer that binds tightly to β-lactamase, traditional antibiotics can be used instead of constantly developing new antibiotics. Development of new antibiotics is not only a lengthy process, but also very costly. Many new bacterial strains evolve every year with increased resistance towards many of the antibiotics on the market today. This fact emphasizes the need for a method that allows the use of antibiotics already in circulation. Aptamer selection against β-lactamase cannot solve the problem of multi-drug resistant bacteria, but can help to find new ways of fighting these pathogens.
Specific Aim 1: Selection of RNA Oligonucleotides against β-lactamase
Beta lactamase provides bacteria with antibiotic resistant capacity. Tightly binding RNA aptamer would inhibit β-lactamase, thereby allowing traditional antibiotics to stop growth of and eventually kill antibiotic-resistant bacteria.
Figure 2: Visual Depiction of Specific Aim
There are many types of β-lactamase that can be used for potential targets. Selection has been performed on metallo beta lactamase [3], which can break down penicillin, cephalosporin, and carbapenem. However, different findings may come of this particular proposal, as a different RNA sequence could be found. This sequence could work on different strains of bacteria than originally thought.
AbD Serotec sells five units of penicillinase (β-lactamase) from Enterobacter cloacae for $409: Catalog Number-7220-1556. Penicillinase specifically breaks down penicillin.
Another vendor, Cell Sciences, sells 1 mg recombinant E. coli β-lactamase for $235. Catalog number: CSI12795
In addition, the protein is large enough upon which to perform selection. Most of this class of proteins tends to be around 50 kD [4] in molecular weight.
[1] Kelly, J.A., et al. (1988) in Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function (Actor P., et al) American Society of Microbiology, Washington, D.C. p. 261-267.
[2] Livermore, D.M. (1991) “Mechanisms of Resistance to β-lactam Antibiotics.” Scandinavian Journal of Infectious Diseases, Supplement 78, p. 7-16.
[3] Kyu Mee Kim (2004) “Inhibition of Metallo-Beta-Lactamase by RNA” Master’s thesis Texas Tech University.
Image: Fonzé, E., P. Charlier, Y. To'th, M. Vermeire, X. Raquet, A. Dubus, and J-M. Frère. (1995) "TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant". Acta Cryst. 51:682-694.
The advent of antibiotics was an extremely important step in the progress of human society. Yet with the constant usage of these antibiotics, bacteria have developed resistance. One of the ways that bacteria have gained resistance is through the protein β-lactamase.
The process of evolution takes place constantly; as a result, certain bacterial strains have developed resistance to antibiotics. Antibiotics, such as penicillin (a β-lactam) stop bacterial growth by interfering with the synthesis of the peptidoglycan layer of cell walls. These antibiotics disrupt DD-peptidases that form the cross-links in peptidoglycan [1]. However, through repeated exposure to these antibiotics, bacteria have evolved to counteract this by synthesizing the protein β-lactamase. This particular enzyme attacks the β-lactam ring of antibiotics, thereby rendering the antibiotic ineffective [2].
Figure 1: Ribbon Structure of Beta Lactamase, Adapted from Fonze, E. et al (1995)
An aptamer is a short sequence of nucleic acid that binds to a certain target, usually protein, very tightly. Using the SELEX method of aptamer selection incorporates the same driving force of evolution that produced β-lactamase in bacteria. By exponentially multiplying the nucleic acid sequences that bind most tightly to the protein of interest, only the most “fit” nucleic acids remain. This tightly binding nucleic acid can subsequently interfere with protein function. Because β-lactamase confers bacterial resistance to many antibiotics, introduction of a tightly binding aptamer along with the normal antibiotic would then be sufficient to stop growth of the bacteria. Without this aptamer, the antibiotic alone would be broken down by the bacteria.
Consequently, by finding a therapeutic aptamer that binds tightly to β-lactamase, traditional antibiotics can be used instead of constantly developing new antibiotics. Development of new antibiotics is not only a lengthy process, but also very costly. Many new bacterial strains evolve every year with increased resistance towards many of the antibiotics on the market today. This fact emphasizes the need for a method that allows the use of antibiotics already in circulation. Aptamer selection against β-lactamase cannot solve the problem of multi-drug resistant bacteria, but can help to find new ways of fighting these pathogens.
Specific Aim 1: Selection of RNA Oligonucleotides against β-lactamase
Beta lactamase provides bacteria with antibiotic resistant capacity. Tightly binding RNA aptamer would inhibit β-lactamase, thereby allowing traditional antibiotics to stop growth of and eventually kill antibiotic-resistant bacteria.
Figure 2: Visual Depiction of Specific Aim
There are many types of β-lactamase that can be used for potential targets. Selection has been performed on metallo beta lactamase [3], which can break down penicillin, cephalosporin, and carbapenem. However, different findings may come of this particular proposal, as a different RNA sequence could be found. This sequence could work on different strains of bacteria than originally thought.
AbD Serotec sells five units of penicillinase (β-lactamase) from Enterobacter cloacae for $409: Catalog Number-7220-1556. Penicillinase specifically breaks down penicillin.
Another vendor, Cell Sciences, sells 1 mg recombinant E. coli β-lactamase for $235. Catalog number: CSI12795
In addition, the protein is large enough upon which to perform selection. Most of this class of proteins tends to be around 50 kD [4] in molecular weight.
[1] Kelly, J.A., et al. (1988) in Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function (Actor P., et al) American Society of Microbiology, Washington, D.C. p. 261-267.
[2] Livermore, D.M. (1991) “Mechanisms of Resistance to β-lactam Antibiotics.” Scandinavian Journal of Infectious Diseases, Supplement 78, p. 7-16.
[3] Kyu Mee Kim (2004) “Inhibition of Metallo-Beta-Lactamase by RNA” Master’s thesis Texas Tech University.
Image: Fonzé, E., P. Charlier, Y. To'th, M. Vermeire, X. Raquet, A. Dubus, and J-M. Frère. (1995) "TEM1 beta-lactamase structure solved by molecular replacement and refined structure of the S235A mutant". Acta Cryst. 51:682-694.
RNA Aptamer Selection Against EphA2 to Decrease Tumor Cell Proliferation
Click here to view the full proposal
Lack of binding between the ephrin A1 ligand and the EphA2 receptor, 130 kDa tyrosine kinase receptor found in adult human epithelial cells, causes unstoppable cell growth, and subsequently, development of tumors associated with epithelial cancers (Kinch, 2005; Ansuini et al, 2009). In a normal cell, EphA2 receptor kinase activity is inhibited when the cell-membrane EphA2 receptor can bind to ephrin A ligands, as shown in figure 1. Conversely, in a cancerous cell, EphA2 receptor is damaged and cannot bind correctly to ligands causing EphA2 to continually phosphorylate thus increasing number of malignant tumor cells (Walker-Daniels et al, 2002). Inhibiting the kinase activity and phosphorylation of EphA2 protein receptor has been associated with a decrease in the growth of malignant cells (Ansuini et al, 2009).
Figure 1: In diagram A, the EphA2 receptor is modified, preventing ephrinA1 from binding. The lack of binding causes kinase activity in the cell and subsequent growth of tumor cells. However, a normally functioning EphA2 receptor will correctly bind to ephrinA1 inhibiting phosphorylation within the cell and normal cell activity, adapted by Larsen (2007).
Specific Aim 1: Selection of RNA aptamers against EphA2
Increased levels of EphA2 phosphorylation due to decreased amount of EphA2/ephrinA1 binding has been linked to increased tumor growth. Thus, selection of RNA aptamers conjugated with a bacterial toxin can be used as a therapeutic tool, degrading the malfunctioning EphA2 protein and thus decreasing kinase activity and tumor proliferation. Such an aptamer would be much more useful than its antibody equivalent due to modifications that can make the aptamer less resistant to enzyme degradation which will increase therapeutic delivery time and potentially decrease number of treatments.
Ansuini, H., et al (2009) “Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer.” Journal of Oncology 2009: 1-10.
Kinch, M. S. (2005). Targeted drug delivery using EphA2 or EphA4 binding moieties. Patent No. 20050153923. Laytonsville, MD, US.
Larsen, A. B., Pedersen, M. W., et al (2007) “Activation of the EGFR Gene Target EphA2 Inhibits Epidermal Growth Factor–Induced Cancer Cell Motility.” Molecular Cancer Research 5: 283.
Phillips, J. A., et al (2008) “Applications of aptamers in cancer cell biology.” Analytica Chimica Acta 621 Review: 101-108.
Walker-Daniels, J., et al (2003) “Differential Regulation of EphA2 in Normal and Malignant Cells.”American Journal of Pathology 162: 1037-1042.
Wykosky, Jill. "The EphA2 Receptor and EphrinA1 Ligand in Solid Tumors: Function and Therapeutic Targeting." Molecular Cancer Research 2008.
RNA Aptamer Selection Against Fluorescent Proteins for Signaling Diagnostics
Click here to read the full proposal.
Click here to read the final report.
Fluorescent proteins (FP’s), naturally found in such sea species as Aequorea victoria, serve to tag and label the various cellular elements present in the biological research field (Shcherbo 2007). As fluorescence implies, excitation with a specific wavelength of light causes the protein to emit a specific color. The most notable of the FP’s is green fluorescent protein (GFP), originating from the previously mentioned Aequorea victoria (Tsien 1998). From GFP, additional proteins have been derived that emit different colors and possess increased fluorescence and photostability (Heim 1995).
While this unique property of noninvasive labeling can be applied to such avenues as the visualization of tumor progression and the death of tissue, the following problems arise (Shcherbo 2007): fusing an FP protein to a protein of interest can impair the function of the target protein, potentially adversely affecting cellular function (Wiedenmann 2009); the lack of specificity between FP’s and the target protein limits the necessary protein-protein interaction. However, small oligonucleotides known as aptamers could allow for reduced or eliminated cytotoxicity as well as more specific and increased protein binding (Stoltenburg 2007).
These short nucleic acid sequences, successfully selected against protein targets in such fields as therapeutics and drug delivery, warrant hope for fluorescent imaging with their high binding affinity and antibody-like specificity (Stoltenburg 2007). While the selection process of an aptamer against FP’s could seemingly be avoided through direct covalent attachment of FP’s to target aptamer’s, there is reason to believe that non-covalent linkages (i.e. aptamer:protein interactions) could yield amplified FP fluorescence versus covalent linkages. Additionally, the research of Dr. Milan Stojanovic on modular aptameric sensors presents the possibility of aptamer:aptamer modules bolstering aptamer:target stability.
Using malachite green (MG), an organic dye, bound to a previously selected MG aptamer (MGA), the conjugate was attached to a flavin mononucleotide (FMN): FMN aptamer (FMNA) conjugate (Stojanovic 2004). Through experimental methods it was determined that the presence of FMN, as opposed to large concentrations of MG alone, resulted in a significantly reduced Kd for the aptameric sensor (from >750 to 30 μM) and a 30 to 50 fold increase in the fluorescence of MG (Stojanovic 2004). These findings demonstrate a detector, FMNA, channeling released energy from the stabilization of the aptameric complex, through the Watson-Crick bases communication module. From here, the energy traveled to an awaiting reporter, MGA, and manifested itself as augmented fluorescence of MG (Stojanovic 2004). In application to FP’s, Dr. Stojanovic hypothesizes that this signaling pathway could result in the altered emission color for each FP, a conclusion stemming from the observation that only a slight change in the conformation of GFP results in the FP color varieties (Tsien 1998). Consequently, if a highly malignant and progressive tumor was examined preliminarily with GFP, its observation 6 months later could yield a bluish color, a color of higher energy on the visible light spectrum, as opposed to the anticipated green.
Through the dealings of Dr. Brad Hall of the University of Texas at Austin and Dr. Vladislav Verkhusha of Albert Einstein College of Medicine, approximately one to two milligrams of three different fluorescent proteins, mTagBFP (blue), mTagGFP (green), and TagRFP(red), will be provided by the latter. In return for the proteins, rounds of in vitro RNA aptamer selection (SELEX) will be performed by Dr. Brad Hall’s students.
References
1. Hasegawa, H. et al. (2008) “Improvement of aptamer affinity by
dimerization.” Sensors. 8:1090-1098.
2. Nutiu, R. et al. (2005) “Fluorescence-signaling nucleic acid-based
sensors.” Landesbioscience.
3. Shcherbo, D. et al. (2007) “Bright far-red fluorescent protein for
whole-body imaging.” Nature
Methods. 4:741-746.
4. Stojanovic, M., Kolpashchikov, D. (2004)”Modular aptameric
sensors.” Journal of the American Chemical
Society. 16(30):9266-9270.
5. Soltenburg, R. et al. (2007) “SELEX – A (r)evolutionary method to
generate high-affinity nucleic acid ligands.” Biomedical Engineering. 24:381-403.
6. Hillisch, A. et al. (2001). “Recent advances in FRET: distance
determination in protein – DNA complexes.” Current Opinion in
Structural Biology. 11(2):201-207.
7. Heim, R. et al. (1995) “Improved green fluorescence.” Nature. 373:663-664.
8. Babendure, J. R. et al. (2003) “Aptamers switch on fluorescence on
triphenylmethane dyes.” Journal of the American Chemical Society.
125(48):14716-14717.
9. Famulok, M. (2004) “Chemical biology: green fluorescent RNA.”
Nature. 430:976-977.
10. Tsien, R. (1998) “The green fluorescent protein.” Annual Review of
Biochemistry. 67:509-544.
11. Pu, Y. et al. (2009) “Aptamers for circulating tumour cells.”
Clinical Laboratory International.
12. Payne, H. (2009) “Nobel prize in chemistry: green fluorescent
protein.” Dartmouth Undergraduate Journal of Science.
13. Wiednenmann, J. et al. (2009) “Fluorescent proteins for live cell
imaging: opportunities, limitations, and challenges.” IUBMB Life.
61(11):1029-1042.
Click here to read the final report.
Fluorescent proteins (FP’s), naturally found in such sea species as Aequorea victoria, serve to tag and label the various cellular elements present in the biological research field (Shcherbo 2007). As fluorescence implies, excitation with a specific wavelength of light causes the protein to emit a specific color. The most notable of the FP’s is green fluorescent protein (GFP), originating from the previously mentioned Aequorea victoria (Tsien 1998). From GFP, additional proteins have been derived that emit different colors and possess increased fluorescence and photostability (Heim 1995).
While this unique property of noninvasive labeling can be applied to such avenues as the visualization of tumor progression and the death of tissue, the following problems arise (Shcherbo 2007): fusing an FP protein to a protein of interest can impair the function of the target protein, potentially adversely affecting cellular function (Wiedenmann 2009); the lack of specificity between FP’s and the target protein limits the necessary protein-protein interaction. However, small oligonucleotides known as aptamers could allow for reduced or eliminated cytotoxicity as well as more specific and increased protein binding (Stoltenburg 2007).
These short nucleic acid sequences, successfully selected against protein targets in such fields as therapeutics and drug delivery, warrant hope for fluorescent imaging with their high binding affinity and antibody-like specificity (Stoltenburg 2007). While the selection process of an aptamer against FP’s could seemingly be avoided through direct covalent attachment of FP’s to target aptamer’s, there is reason to believe that non-covalent linkages (i.e. aptamer:protein interactions) could yield amplified FP fluorescence versus covalent linkages. Additionally, the research of Dr. Milan Stojanovic on modular aptameric sensors presents the possibility of aptamer:aptamer modules bolstering aptamer:target stability.
Using malachite green (MG), an organic dye, bound to a previously selected MG aptamer (MGA), the conjugate was attached to a flavin mononucleotide (FMN): FMN aptamer (FMNA) conjugate (Stojanovic 2004). Through experimental methods it was determined that the presence of FMN, as opposed to large concentrations of MG alone, resulted in a significantly reduced Kd for the aptameric sensor (from >750 to 30 μM) and a 30 to 50 fold increase in the fluorescence of MG (Stojanovic 2004). These findings demonstrate a detector, FMNA, channeling released energy from the stabilization of the aptameric complex, through the Watson-Crick bases communication module. From here, the energy traveled to an awaiting reporter, MGA, and manifested itself as augmented fluorescence of MG (Stojanovic 2004). In application to FP’s, Dr. Stojanovic hypothesizes that this signaling pathway could result in the altered emission color for each FP, a conclusion stemming from the observation that only a slight change in the conformation of GFP results in the FP color varieties (Tsien 1998). Consequently, if a highly malignant and progressive tumor was examined preliminarily with GFP, its observation 6 months later could yield a bluish color, a color of higher energy on the visible light spectrum, as opposed to the anticipated green.
Through the dealings of Dr. Brad Hall of the University of Texas at Austin and Dr. Vladislav Verkhusha of Albert Einstein College of Medicine, approximately one to two milligrams of three different fluorescent proteins, mTagBFP (blue), mTagGFP (green), and TagRFP(red), will be provided by the latter. In return for the proteins, rounds of in vitro RNA aptamer selection (SELEX) will be performed by Dr. Brad Hall’s students.
References
1. Hasegawa, H. et al. (2008) “Improvement of aptamer affinity by
dimerization.” Sensors. 8:1090-1098.
2. Nutiu, R. et al. (2005) “Fluorescence-signaling nucleic acid-based
sensors.” Landesbioscience.
3. Shcherbo, D. et al. (2007) “Bright far-red fluorescent protein for
whole-body imaging.” Nature
Methods. 4:741-746.
4. Stojanovic, M., Kolpashchikov, D. (2004)”Modular aptameric
sensors.” Journal of the American Chemical
Society. 16(30):9266-9270.
5. Soltenburg, R. et al. (2007) “SELEX – A (r)evolutionary method to
generate high-affinity nucleic acid ligands.” Biomedical Engineering. 24:381-403.
6. Hillisch, A. et al. (2001). “Recent advances in FRET: distance
determination in protein – DNA complexes.” Current Opinion in
Structural Biology. 11(2):201-207.
7. Heim, R. et al. (1995) “Improved green fluorescence.” Nature. 373:663-664.
8. Babendure, J. R. et al. (2003) “Aptamers switch on fluorescence on
triphenylmethane dyes.” Journal of the American Chemical Society.
125(48):14716-14717.
9. Famulok, M. (2004) “Chemical biology: green fluorescent RNA.”
Nature. 430:976-977.
10. Tsien, R. (1998) “The green fluorescent protein.” Annual Review of
Biochemistry. 67:509-544.
11. Pu, Y. et al. (2009) “Aptamers for circulating tumour cells.”
Clinical Laboratory International.
12. Payne, H. (2009) “Nobel prize in chemistry: green fluorescent
protein.” Dartmouth Undergraduate Journal of Science.
13. Wiednenmann, J. et al. (2009) “Fluorescent proteins for live cell
imaging: opportunities, limitations, and challenges.” IUBMB Life.
61(11):1029-1042.
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