Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• The physiological function of cellular prion protein (PrP) remains largely unknown, despite its misfolded isoform being central to prion diseases.
• Identifying molecules that bind to PrP is crucial for understanding its role and the pathology of transmissible spongiform encephalopathies.
• Nucleic acids are known to interact with PrP, suggesting a potential role in PrP function.

Purpose of the Study:

• To identify preferred RNA sequences that bind to ovine recombinant PrP.
• To elucidate the binding sites and characteristics of PrP-RNA interactions.

Main Methods:

• Utilized an in vitro selection approach (SELEX) with a pool of 80-nucleotide (nt) RNAs containing a randomized 40-nt central region.
• Employed surface plasmon resonance and filter binding assays to quantify binding affinity (KD value) and interaction kinetics.
• Determined the minimal RNA sequence required for binding and identified specific protein domains involved in nucleic acid interaction.

Main Results:

• Isolated a high-affinity RNA aptamer, RM312, with a dissociation constant (KD) of 20 nM.
• RM312 exhibited fast association and dissociation rates with immobilized PrP, indicative of biologically relevant interactions.
• Identified two lysine clusters in the N-terminal region of PrP as the primary binding sites for nucleic acids.

Conclusions:

• The identified RNA aptamers and their binding characteristics suggest a physiological role for PrP in interacting with cellular nucleic acids.
• The specific RNA binding preferences and identified lysine clusters provide new insights into PrP's molecular interactions.
• This research contributes to understanding the fundamental function of PrP and the mechanisms underlying prion diseases.