Screening of DNA Aptamer Against Mouse Prion Protein by Competitive Selection

Abstract

Prion disease is a neurodegenerative disorder, in which the normal prion protein (PrP) changes structurally into an abnormal form and accumulates in the brain. There is a great demand for the development of a viable approach to diagnosis and therapy. Not only has the ligand against PrP been used for diagnosis, but it has also become a promising tool for therapy, as an antibody. Aptamers are a novel type of ligand composed of nucleic acids. DNA aptamers in particular have many advantages over antibodies. Therefore, we tried to isolate the DNA aptamer for mouse PrP. We developed a competitive selection method and tried to screen the DNA aptamer with it. In the fourth round of selection, several clones of the aptamer with an affinity to PrP were enriched, and clone 4-9 showed the highest affinity of all. The investigation by aptamer blotting and Western blotting showed that clone 4-9 was specifically able to recognize both α-PrP and β-PrP. Moreover, it was indicated that clone 4-9 could recognize the flexible region of the N-terminal domain of PrP. These characteristics suggest that clone 4-9 might be a useful tool in prion-disease diagnosis and research.

Acknowledgements

This work is partly supported by the Grant-in-Aid for the 21st Century COE “Future Nano- Materials” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and also by Industrial Technology Research Grant Project in 2006 from New Energy and Industrial Technology Development Organization (NEDO) of JAPAN.

Figures and Tables

Figure 1 Sequence of operations in the competitive selection method. The protein-blotted membrane was incubated with the DNA library. After the removal of the unbound DNAs, the portion of the membrane on which the target was immobilized was cut out. The target-bound DNAs were eluted and then amplified by PCR to obtain a new library. This cycle was repeated.

Figure 2 Binding assay of DNA pools to target proteins by aptamer blotting. At the top left, the immobilized proteins on the membrane are shown. α-PrP and Zif268 were spotted onto the membrane at 0.9 pmol and 79 pmol, respectively. The other figure shows a chemiluminescent image of the aptamer blotting. DNA at a concentration of 1 µM was incubated with the membrane. The black spots indicate chemiluminescence, which reflects the amount of target-bound DNA. The number of rounds is shown on the left side of the image.

Figure 3 Binding assay of DNA clones by SPR. The constantly appearing DNA clones were injected on a chip, onto which α-PrP was already immobilized. The names of the clones are shown on the right. The clones are ordered according to the strength of the signal.

Figure 4 Evaluation of the specificity of clone 4–9 by aptamer blotting. Images of chemiluminescence detection are shown. On the left, the immobilized proteins are indicated. The upper number shows the quantitative ratio of proteins immobilized on the membrane. “1” means 1 pmol proteins in the case of α-PrP, Zif268 and BSA, whereas “1” means 4 pmol proteins in the case of β-PrP.

Figure 5 Detection of PK-digested β-PrP by Western blotting. Images of chemiluminescence detection using clone 4–9 and the anti-PrP antibody 7D9 are shown. β-PrP at a concentration of 0.2 mg/mL was treated with PK at various concentrations. The numbers show the concentration of PK: (1) no PK, (2) 4 ng/mL, (3) 8 ng/mL, (4) 40 ng/mL, (5) 80 ng/mL.

Figure 6 Predicted folding of clone 4–9. The folding of clone 4–9 was predicted using the Mfold web server (Zuker, 2003).

Figure 7 Application of clone 4–9 to a detection system with fluorescence depolarization. FITC-labeled DNA at a concentration of 1 µM and α-PrP at various concentrations were mixed in PBS buffer. The mixture was incubated at room temperature for more than ten min, and then its fluorescence depolarization was measured. BSA was used as a control.

Table 1 The concentrations of DNA, proteins and tRNA at each round of SELEX

Note

Supplemental information can be found at: www.landesbioscience.com/supplement/Ogasawara PRION1-4-Suppl.pdf