Characterization and application of a novel RNA aptamer against the mouse prion protein

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• The mouse prion protein (mPrP) is implicated in neurodegenerative diseases.
• Developing specific detection tools for mPrP is crucial for research.

Purpose of the Study:

• To isolate and characterize RNA aptamers with high affinity for mouse prion protein (mPrP).
• To evaluate the potential of these aptamers as diagnostic or research tools.

Main Methods:

• In vitro selection (SELEX) was used to generate RNA aptamers from pools with a randomized region.
• Binding affinity was measured using dissociation constants (K(d)).
• RNase resistance was conferred using 2'-fluoro-pyrimidine modifications.
• Detection capability was assessed via Northwestern blotting on mouse brain homogenate.
• Protein-deletion-mutant and competition-binding analyses identified the mPrP binding site.

Main Results:

• Aptamer 60-3 demonstrated high affinity for mPrP (K(d) = 5.6 +/- 1.5 nM).
• Modified aptamer 60-3 retained binding activity (K(d) = 22 +/- 4 nM) and detected mPrP in brain homogenate.
• Binding sites were localized to amino acids 23-108 of mPrP.

Conclusions:

• RNA aptamer 60-3 is a high-affinity, specific binder for mouse prion protein.
• The modified aptamer is suitable for detecting mPrP in biological samples.
• The identified binding region provides insights into mPrP-aptamer interactions.