Dorothy Nguyen's HRP Aptamer Project

APTAMER AGAINST HORSERADISH PEROXIDASE:

A PROGNOSTIC TOOL FOR INVASIVE BREAST CANCER

Introduction & Background

Breast cancer is one of the leading causes of death and is the most common malignancy among women. Approximately10-15% of patients with breast cancer develop distant invasive cancer within 3 years after the initial detection of the primary tumor (Cheng et al., 2008). The development of metastatic cancer 10 years or more after the initial diagnosis is also not unusual (Cheng et al., 2008). This indefinite time frame of the progression of invasive breast cancer makes it difficult to not only define a cure for the disease, but also to assess the risk factors for metastasis. This calls for new prognostic markers to identify patients who are at the highest risk of developing metastatic breast cancer. In recent studies, it has been demonstrated that the high mRNA expression of MMP-1 in patients directly correlate to unfavorable prognosis of patients with invasive breast carcinoma (Cheng et al., 2008). In order to visually evaluate the expression of this MMP-1 mRNA, an inhibitory aptamer bound to a chemiluminescent enzyme in combination with RNA strand displacement technologies can be used.

Aptamers are short single-stranded oligonucleotides with specific and complex three-dimensional shapes that allow them to bind precisely to target molecules of interest (Ellington and Szostak, 1990). Due to their high selectivity and utility, these molecules are widely used in applications including medical and pharmaceutical basic research, drug development, diagnosis, and therapy (Stoltenburg et al., 2007). In therapeutics, an aptamer could be used to inhibit a protein function or pathway. To diagnose a condition, an aptamer could be modified to signal the presence of its cognate analyte. Furthermore, an aptamer could be functionalized for drug delivery with a drug for specific targeting. In order to develop an aptamer with a high binding affinity to a target of interest, many rounds of a SELEX (systematic evolution of ligands by exponential enrichment) are necessary (Stoltenburg et al., 2007). The bead-based in vitro selection method (Figure 2) is a mechanism of isolating a potential aptamer species from a random sequence pool. In this technique, the target proteins of interest, which are immobilized to beads, are subjected to this RNA random sequence pool and the RNA sequences that bind to the target are then analyzed and prepared for the next round of selection. Multiple cycles of selection and amplification result in favored enrichment of the binding species within the initial pool. An essential feature to the selection process is the target molecule as it provides the key to select for the specific oligonucleotide binding species within the random sequence pool. Following sufficient rounds of selection, Sanger sequencing and a radioactive binding assay can be performed to assess pool enrichment and specificity and affinity for the target molecule.

Horseradish peroxidase (HRP) is a heme-containing enzyme that is produced naturally in the roots of horseradish, a perennial herb. The monomer protein (Figure 3) has a molecular weight of approximately 44 kDa. It has been utilized in numerous biotechnological applications in the environmental, health care, pharmaceutical, and chemical industries (Azevedo et al., 2003). Alone, the HRP enzyme is of little value. In the presence of the proper substrate, on the other hand, the HRP conjugate oxidizes the substrate using hydrogen peroxide to yield a detectable and quantifiable colored or light product. With its characteristic ease in producing visible products, HRP is produced commercially for its use in clinical diagnostic kits and immunoassays (Veitch, 2004). In addition, HRP is frequently utilized in analytical techniques including immunohistochemistry, western blot, and ELISAs (Chau and Lu, 1995). The function of HRP can be utilized in diagnosing breast cancer by selecting for an inhibitory aptamer. Although HRP does not bind nucleic acids naturally at a specific place nor does it have any features that may prove useful in binding nucleic acids, there is still potential for an aptamer to be found as aptamers can be bound to a wide variety of molecules.

Utilizing the chemiluminescent trait of HRP, the expression of mRNA can be visually analyzed and determined using an inhibitory aptamer and RNA strand displacement technologies. This aptamer would bind to the HRP, obstructing the substrate binding area, thus preventing a fluorescence of the enzyme in the presence of its substrate (Bhadra and Ellington, 2015). This inhibitory aptamer would be reengineered by placing an oligonucleotide extension at its 5'- and 3'- ends complementary to the sequence of the target mRNA (in this case, MMP-1 mRNA), forming a nonbinding conformation for the target mRNA to bind (Zhang and Winfree, 2009). In the presence of the MMP-1 mRNA, which would act as the trigger nucleic acid, toehold-mediated strand displacement (Figure 4) would lead to the formation of a binding conformation on the oligonucleotide extension. This binding conformation would lead to the unfolding of the inhibitory aptamer on the chemiluminescent protein, thus unblocking the substrate binding area on the HRP enzyme. Therefore, the HRP enzyme would be able to produce a colored or light product that can be quantified to conclude the expression levels of the MMP-1 mRNA. As a result, a visual and quantifiable product can be analyzed as an accurate method of prognosis for invasive breast cancer.

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Citations

Azevedo, A.M., Martins, V.C., Prazeres, D.M., Vojinović, V., Cabral, J.M., and Fonseca, L.P. (2003). Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol. Annu. Rev. 9, 199–247.

Bhadra, S., and Ellington, A.D. (2015). Chapter Eleven - Design, Synthesis, and Application of Spinach Molecular Beacons Triggered by Strand Displacement. In Methods in Enzymology, D.H. Burke-Aguero, ed. (Academic Press), pp. 215–249.

Chau, Y.P., and Lu, K.S. (1995). Investigation of the Blood-Ganglion Barrier Properties in Rat Sympathetic Ganglia by Using Lanthanum Ion and Horseradish Peroxidase as Tracers. Cells Tissues Organs 153, 135–144.

Cheng, S., Tada, M., Hida, Y., Asano, T., Kuramae, T., Takemoto, N., Hamada, J.-I., Miyamoto, M., Hirano, S., Kondo, S., et al. (2008). High MMP-1 mRNA Expression is a Risk Factor for Disease-Free and Overall Survivals in Patients with Invasive Breast Carcinoma. J. Surg. Res. 146, 104–109.

Ellington, A.D., and Szostak, J.W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822.

Stoltenburg, R., Reinemann, C., and Strehlitz, B. (2007). SELEX—A (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol. Eng. 24, 381–403.

Veitch, N.C. (2004). Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry 65, 249–259.

Welinder, K.G. (1979). Amino Acid Sequence Studies of Horseradish Peroxidase. Eur. J. Biochem. 96, 495–502.

Zhang, D.Y., and Winfree, E. (2009). Control of DNA Strand Displacement Kinetics Using Toehold Exchange. J. Am. Chem. Soc. 131, 17303–17314.