Haley Wolf's GUS aptamer project (2016)

Nucleic Acid Aptamer Selection Against Beta-glucoronidase for the Treatment of Alzheimer’s Disease

Introduction/Background

Alzheimer’s Disease (AD) is a neurodegenerative disease that has been associated with progressive deficits in both verbal and visuospatial memory (Hirao et. al., 2014). Alzheimer’s is the most common cause of dementia, a group of neurological diseases that cause progressive loss of memory and brain function, and worsens even more so if not treated (NIH, 2016 and Mayo Clinic, 2015). AD currently has no known cure, and continues to affect millions of Americans with more and more people diagnosed each year. In fact, one in every nine Americans age 65 and older are diagnosed with AD, and the prevalence only increases with age (Alzheimer’s Association, 2016). In the brains of those who suffer from AD, as can be seen in Figure 3, “abnormal deposits of proteins form amyloid plaques and tau tangles throughout the brain”, and neurons begin to lose their ability to function and their connections with other neurons become disabled, causing them to die (NIH, 2016). Today, PET scans are being used to diagnose AD before death by looking at the glucose metabolism data that can be deduced from the images much like the ones in Figure 3, but these scans do not provide an absolute answer (Calsolaro et. al., 2016). Currently, post-mortem autopsies are the only conclusive way to diagnose AD because there is not an accurate test to definitively diagnose patients during their lifetime. There are treatments available for people suffering from this disease but, without a cure, the disease will continue to result in fatality until a viable therapeutic cure can be developed.

Deficits in glucose metabolism have been reported “in temporoparietal cortical association areas” of the brains of patients suffering from AD (Hirao et. al., 2014). Strong correlations between areas of poor glucose metabolism and neuropathological markers have been reported in the brains of deceased patients, which have lead researchers to believe that this poor glucose metabolism could be causing the formation of amyloid plaques and tau tangles observed in Alzheimer’s patients (Hirao et. al., 2014). Beta-glucuronidase (GUS) is a lysosomal enzyme that aids “in the degradation of glucuronate-containing glycosaminoglycan” (GAGs), which are composed of sugar molecules linked together in a long string (Naz et. al., 2013 and NIH, 2016). GUS can also be found in E. coli in the gut and the breast milk of nursing women, but these locations are not the focus of this research. Figure 4 shows where GUS resides in the lysosomes of brain cells (shown as hydrolytic enzyme mixture in the figure). When GUS does not function properly in human brain cells, it causes lysosomal storage that can lead to the degradation seen in Alzheimer’s patients.

An aptamer that binds to beta-glucuronidase could be used to help treat Alzheimer’s disease by aiding in drug delivery. An aptamer is an oligonucleotide species that binds with high specificity and affinity to a given target which, in this case, would be GUS. In-vitro nickel bead-based aptamer selection will be performed in rounds to find an aptamer that binds to GUS. A round of aptamer selection includes incubating the target protein with the large RNA pool, washing away the unbound species, eluting the bound species, and amplifying the eluted bound species to use the amplified RNA as the new, smaller pool for the next round of selection. At least six rounds of selection will be needed before sequencing the RNA and running a binding assay with the minimized pool of RNA to determine the presence of an aptamer. An ideal application would be an aptamer that attaches to GUS on one end and to the drug being delivered on the other end that would be specifically delivered to GUS. A drug that could be specifically targeted to repair the function of inactive or poorly functioning GUS could help prevent or stop the build up of glucose in the brain and, hopefully, help to prevent the progression of Alzheimer’s or stop the disease entirely.

Beta-glucuronidase (GUS), as seen in Figure 1, is a 57 kDa enzyme that functions as a tetramer and comes from E. coli for use in the FRI Aptamer Lab. GUS is not presently known to bind to any nucleic acids, and can be found in human lysosomes as well as E. coli in human guts. GUS was suspended in PBS buffer, which can be used as the selection buffer with added MgCl2 in order to help the negatively charged GUS “stick” better to RNA. It has been used in the past as an aid in detection of neoplastic meningitis in cerebrospinal fluid, and has a wide array of other possible diagnostic and therapeutic applications (Tallman et. al, 1985). The structure and function of GUS makes it an ideal target for an aptamer to be used to treat AD by specifically delivering a drug directly to the enzyme.

The project remains in round one of aptamer selection against GUS due to difficulties in the early steps of round 2, causing a needed backtrack. The project remains in round one despite weeks of work last semester due to slow progress with the selection process. The amplification of the selected RNA from round one had many unsuccessful cycle course PCR gels, and it was eventually discovered that the reagents were not being mixed in the correct way due to procedural confusion. After several rounds of selection, an aptamer that binds specifically binds to GUS could help create a specialized drug for patients with Alzheimer’s Disease, thus, slowing the progression of this degenerative disease.

Click here for the Final Report

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