ssDNA Aptamer Selection against Alpha1-antichymotrypsin

Joey Dominguez
September 3, 2012
Fall 2012
R40, DNA, α1-antichymotrypsin

sDNA Aptamer Selection against α1-antichymotrypsin for the Prevention and Degradation of Senile Plaques and Neurofibrillary Tangles

Tauopathies are neurodegenerative diseases characterized by the formation of tau protein aggregates, or neurofibrillary tangles, within nervous cells.  In some tauopathies, as with Alzheimer’s disease, aggregates of protein fragments, or senile plaques, form between neurons.  Though no single protein triggers the formation of tangles and plaques, alpha1‑antichymotrypsin (ACT) both inhibits amyloid beta’s degradation, directly leading to the formation of senile plaques, and exists as a secondary component of these plaques. (Abraham, et. al 2000; Abraham, et. al. 1988)  In addition, elevated levels of ACT have been shown to induce tau hyperphosphorylation characteristic of Alzheimer’s disease, arguably the most famous tauopathy. (J. Padmanabhan, et al., 2006)  Because of ACT’s clear role in the development of tauopathies, several studies have identified it as a potential target for an inhibitory therapeutic mechanism. (Porcellini, et. al, 2008; Padmanabhan, et. al., 2006)
Due to ACT’s ability to bind DNA, ACT is a prime candidate for the development of an aptamer.  Aptamers, nucleic acid oligomers, bind with high specificity to target molecules, serving as tools for labeling particular molecules, delivering chemicals to a target molecule, and, ideally, inhibiting the activity of the target molecule.  Since overexpression of ACT has been linked to major characteristics of tauopathies, the development of an inhibitory aptamer for ACT could antagonize the onset and progression of several tauopathies.  Aptamers are selected from a pool of randomized oligomers through the SELEX process which gradually reduces the variability of the pool until only those aptamers which bind most strongly with the target molecule under a set of predetermined temperature, pH, and salt concentration conditions remain.  An ssDNA SELEX process includes binding and selection under predetermined environmental conditions, asymmetric PCR amplification, and purification.  The resulting pool can then be used for additional rounds of selection.

Specific Aim 1: Aptamer Selection for an Inhibitory Therapy against ACT

 Normally, ACT binds to serine proteases, serving to protect the respiratory tract from proteolytic enzymes; its accumulation within the cerebrospinal fluid and nervous system lead to the formation of tangles and plaques. (Kalsheker, 1996)  Inhibiting ACT activity could both prevent the accumulation of amyloid beta and the hyperphosphorylation of tau, impeding if not completely halting the formation of both the tangles and plaques.    The selection of an inhibitory aptamer against ACT could lead to the development of a therapeutic tool which could then be widely applied to several tauopathies.

Specific Aim 2: Selection and Modification of an Aptamer for ACT Degradation

            The major factor leading to the development of the tangles and plaques is not ACT’s presence in the nervous system, but rather its accumulation.  Even if an inhibitory aptamer against ACT could not be developed, an ubiquitinated aptamer could bind to excessive ACT and, by tagging the ACT for degradation, relieve the stresses caused by the overexpression.  In addition, the ubiquitin tag could serve to reduce the senile plaques since ACT serves as a component.
ACT can be obtained from Abcam for 1 mg at $492, costing roughly $2.00 per round of selection.  It has a molecular weight of 45 kD.  The catalogue number for ACT is ab80517.  Abcam can be contacted at 888-772-2226.
Abraham, Carmela R., et al. “a1-Antichymotrypsin Inhibits Aß Degradation in Vitro and in Vivo.” Annals of the New York Academy of Sciences 920 (2000): 245-48. Wiley Online Library. Web. 03 Sept. 2012.
Abraham, Carmela R., Dennis J. Selkoe, and Huntington Potter. “Immunochemical identification of the serine protease inhibitor a1-antichymotrypsin in the brain amyloid deposits of Alzheimer’s disease.” Cell 52.4 (1988): 487-501. ScienceDirect. Web. 03 Sept. 2012.
Kalsheker, Noor A. “a1-antichymotrypsin.” The International Journal of Biochemistry & Cell Biology 28.9 (1996): 961-64. ScienceDirect. Web. 03 Sept. 2012.
Padmanabhan, Jaya, et al. “Alpha1-antichymotrypsin, an inflammatory protein overexpressed in Alzheimer’s disease brain, induces tau phosphorylation in neurons.” Brain 129.11 (2006): 3020-34. ingentaconnect. Web. 03 Sept. 2012.
Porcellin, E., et al. “Elevated plasma levels of alpha-1-anti-chymotrypsin in age-related cognitive decline and Alzheimer’s disease: a potential therapeutic target.” Curr Pharm Des 14.26 (2008): 2659-64. Abstract. PubMed. Web. 03 Sept. 2012.

1 comment:

Gwen Stovall said...

Dear Joey,
Here are some suggestions:
1. please include the telephone number for abcam in the budget section
2. include molecular weight in the budget section

,..ordering target now!

We'll talk some more soon!