An RNA aptamer that discriminates bovine factor IX from human factor IX

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Factor IX is a critical protein in the coagulation cascade.
• Specific detection of bovine Factor IX is important for various research and diagnostic applications.
• RNA aptamers offer high specificity and affinity for target proteins.

Purpose of the Study:

• To select and characterize an RNA aptamer that specifically binds to bovine Factor IX.
• To identify the minimal binding motif responsible for the aptamer's specificity.
• To determine the binding affinity and kinetics of the aptamer-bovine Factor IX interaction.

Main Methods:

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for aptamer selection.
• Phosphate interference assays to identify key nucleotide regions for binding.
• Surface Plasmon Resonance (SPR) for quantitative binding analysis.

Main Results:

• An RNA aptamer (Clone 5) was selected that effectively discriminates bovine Factor IX from human Factor IX.
• Specific nucleotide regions (73-78 and 80-83) were identified as crucial for bovine Factor IX binding.
• A minimal aptamer motif (nucleotides 65-106) demonstrated over 1,000-fold discrimination ability.
• The equilibrium dissociation constant (Kd) was determined to be 10 nM, indicating high affinity.

Conclusions:

• The selected RNA aptamer exhibits high specificity and affinity for bovine Factor IX.
• The identified minimal binding motif provides insights into aptamer-target interactions.
• This aptamer represents a valuable tool for research and potential clinical applications involving Factor IX.