Biomod/2015/OhioMOD/background

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BACKGROUND
Introduction
Misfolded protein aggregates cause numerous diseases including Alzheimer's, Parkinson's and Macular Degeneration. The ability to detect the protein aggregates, which are often misfolded proteins, provide crucial early-stage diagnostics and monitoring to ultimately improve treatment outcomes. Over the past several decades, a myriad of diagnostic approaches have emerged to treat these diseases including PEG conjugations and solubility assays[1]. While several approaches have yielded promising results, strong limitations still exist including hydrophobic aggregation and the hindrance of current methods due to complex kinetic monitoring[1]. Thus, novel diagnostic approaches are required. One such novel approach is via the employment of structural DNA nanotechnology, namely DNA origami, which offers control over the design and functional placement of diagnostic/therapeutic molecular modifications on 2- and 3- dimensional objects at the nanoscale. Our goal is to design, fabricate, and characterize a novel DNA nanodevice that will possess both diagnostic and therapeutic potential. We aim to build a device that not only detects the target of interest but also aids to clear the target protein or treat the disease. We hypothesize that a hinge-like device (Biosensing Unit Structure, BUS) constructed via DNA origami may be functionalized with specific aptamers to detect misfolded protein aggregates allowing for diagnosis.
Alzheimer's

Alzheimer's Disease (AD) is a progressive neurodegenerative disease that is classified as a subset of dementia that directly affects memory, thinking, and behavior. In addition, AD is the most common type of dementia accounting for 60 to 80 percent of all dementia cases [3] as dementia is only classified as AD if the diagnosed patient is past the age of 65.

In the 1900s, 4% of the population was above the age of 65 in the U.S. By 1980, the population of people in the U.S. above the age of 65 had risen to 10.3% [2]. In 2015, the number is currently hovering around 15% [2]. With this demographic trend expected to continue, as life expectancy continues to increase, this age-associated cognitive decline is starting to become a larger threat. There are more than 3 million cases of Alzheimer's disease per year and it also is one of the most financially costly diseases in developed countries [3]. In 2015, in the U.S alone Alzheimer’s will cost the nation about 220 billion dollars [3].

Figure 1 (2015 Alzheimer's Facts and Figures)
Currently, AD cannot be cured or attenuated. The actual cause of AD is debatable as a single mechanism has not yet been identified. However, it is believed AD is caused by the aggregation of beta amyloid peptides in the brain that eventually lead to neuron apoptosis (programmed cell death) [4]. Beta amyloids are seen as one of the key elements in the progression of the disease as a majority of AD patients have extremely high concentrations of beta amyloid peptides in the brain [5]. A beta amyloid is a peptide constructed up of ~40 amino acids that are produced by Amyloid Precursor Protein (APP) via a mutation that causes the amino acid, valine, to be replaced with amino acid, isoleucine, at protein position V717I. This allows the peptide to be slightly longer and “stickier,” creating higher affinity for other beta amyloid peptides [6]. These beta amyloid peptides then bind with one another to form oligomers that aggregate to form fibrils, which finally gather to form a cloud of dead particles known as a plaque [6].
Figure 2 (PET scans of three Alzheimer’s patients on the left compared to three healthy patients on the right. Yellow regions represent higher concentrations of beta amyloids)
Figure 3 (Biomolecular activity of APP, beta amyloid peptides, and oligomers)
DNA Origami & Biosensors

DNA origami is the programmed restructuring of a circular single stranded DNA plasmid, “scaffold”, by exploiting Watson-Crick base pairing to an exact predetermined shape through the use of complementary, coded staple strands. Through the use of unique preprogrammed oligonucleotides, “staples”, a single scaffold and hundreds of staples can bind in a myriad of ways to form 2D and 3D shapes [7].

Although the initial idea of using DNA as construction material was developed by Nadrian Seeman in 1982 [8], the DNA origami molecular self-assembly process was created by Rothemund in 2006 [9]. He found that he could use staples and a single scaffold to the make many 2D creations, which led to the iconic creation of the smiley face. In 2009, Douglas, et. al. expedited the DNA origami design process by creating caDNAno [10], which automatically generated staple sequences. Furthermore, Castro, et. al. provided a clear step-by-step guide to create custom DNA nanostructures via DNA origami that included canDO as an experimental resource [7]. Castro and colleagues further optimized and expedited the DNA origami process, making the procedure more cost-effective and allowing for more variation in structures [7]. Douglas and colleagues created the first functional biological system application for DNA origami nanostructures as a logic-gated nanorobot. This was “loaded with a variety of materials in a highly organized fashion and is controlled by an aptamer-encoded logic gate, enabling it to respond to a wide array of cues” (Douglas 2012).

Continuing with these biological applications, we have created an aptamer-functionalized DNA origami device that induces a functional response that aids to the clear the target protein or to treat the disease. This DNA nanostructure referred to as the Biosensing Unit Structure, BUS, is comprised of two plates connected by a DNA hinge. Once BUS units have bound to their respective proteins via an aptamer, exposed internal overhangs bind to other complementary BUS units to form dimers and trimers. These higher order structures will be utilized to identify the presence of misfolded protein aggregates to allow for accurate diagnosis.

OhioMOD is currently focused on producing a proof-of-concept biosensor targeting proteins using an interchangeable aptamer. Our future goals will entail the novel approach of using cryptic binding sites initially buried in the inner part of the device and exposed after the protein-driven conformational change. Our functional response will either expose signaling molecules to drive trafficking of aggregate sensors into defined geometries. Future studies will focus on using our “BUS” DNA nanodevice to recognize and clear vascular endothelial growth factor (VEGF) in the context of Macular Degeneration.

Figure 4 (Progression of DNA origami)
VEGF

Vascular Endothelial Growth Factor (VEGF) is a signaling protein that triggers vasculogenesis in the body, which is the essential process of blood vessel formation especially during embryonic development [11]. However, if VEGF becomes overexpressed it can cause a vascular disease in the retina known as Macular Degeneration [12]. In addition, cancers such as macular degeneration that express VEGF can continue to metastasize due to the continued influx of oxygen rich blood supply as a result of vasculogenesis [12]. Thus, VEGF is a critically important target to sequester and clear in order to treat Macular Degeneration and a variety of human cancers.

The OhioMOD team is using VEGF as a proof-of-concept for our nanobiosensor, Biosensing Unifying Structure (BUS) due to high costs of a quality beta amyloid aptamer. BUS will bind to a VEGF protein in place of beta amyloid peptides to embody the process in which the BUS will diagnose diseases. Upon binding to VEGF, BUS will unfold and expose its internal overhangs on the interior of the structure allowing BUS to fasten itself to two other unique BUS structures successfully forming a trimer. These high order DNA nanostructures will be visualized and detected in a very robust manner ultimately allowing for rapid detection of VEGF.

Figure 4 (2VPF: Vascular Endothelial Growth Factor refined to 1.93 angstroms resolution)

References

[1] Bondos, Saah E., and Alicia Bicknell. "Detection and Prevention of Protein Aggregation Before, During, and after Purification." Analytical Biochemistry 316.2 (2003): 223-31. Web.

[2] Nitsch, Roger M. Alzheimer's Disease: Amyloid Precursor Proteins, Signal Transduction, and Neuronal Transplantation. New York, NY: New York Academy of Sciences, 1993. Print.

[3] "Latest Alzheimer's Facts and Figures." Latest Facts & Figures Report. N.p., 17 Sept. 2013. Web. 23 Oct. 2015.

[4] "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 23 Oct. 2015.

[5] "New Clue in the Search to Predict Alzheimer's Disease | Berkeley Lab."News Center. N.p., 16 Dec. 2008. Web. 23 Oct. 2015.

[6] "APP Gene." Genetics Home Reference. N.p., n.d. Web. 23 Oct. 2015.

[7] Castro, Carlos Ernesto, Fabian Kilchherr, Do-Nyun Kim, Enrique Lin Shiao, Tobias Wauer, Philipp Wortmann, Mark Bathe, and Hendrik Dietz. "A Primer to Scaffolded DNA Origami." Nature Methods Nat Meth 8.3 (2011): 221-29. Web.

[8] Seeman, N (1982). "Nucleic acid junctions and lattices". Journal of Theoretical Biology 99 (2): 237–47. doi:10.1016/0022-5193(82)90002-9. PMID 6188926

[9] Rothemund, Paul W. K. "Folding DNA to Create Nanoscale Shapes and Patterns." Nature 440.7082 (2006): 297-302. Web.

[10] Douglas, S. M., A. H. Marblestone, S. Teerapittayanon, A. Vazquez, G. M. Church, and W. M. Shih. "Rapid Prototyping of 3D DNA-origami Shapes with CaDNAno." Nucleic Acids Research 37.15 (2009): 5001-006. Web.

[11] Duffy, Angela M., David J. Bouchier-Hayes, and Judith H. Harmey. "Vascular Endothelial Growth Factor (VEGF) and Its Role in Non-Endothelial Cells: Autocrine Signalling by VEGF." VEGF and Cancer (2004): 133-44. Web.

[12] Patan, Sybill. "Vasculogenesis and Angiogenesis." Cancer Treatment and Research Angiogenesis in Brain Tumors (2004): 3-32. Web.

[13] "2VPF." RCSB PDB. N.p., n.d. Web. 24 Oct. 2015.