Engineering High-Performance Aptamers via Optimized SELEX and Structure-Guided Dimer Assembly for Clinical Malaria...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Parasitology

Background:

• Malaria diagnostics are critical due to high transmissibility and mortality.
• Existing aptamer-based detection methods for malaria face limitations in affinity and nuclease stability.

Purpose of the Study:

• To develop high-performance aptamers targeting Plasmodium falciparum lactate dehydrogenase (PfLDH) for improved malaria diagnostics.
• To overcome limitations of existing aptamers by enhancing affinity and nuclease stability.

Main Methods:

• A two-step strategy combining optimized SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and structure-guided dimer engineering.
• Utilized bead-based and gel-based SELEX for initial enrichment to preserve aptamer structure and reduce bias.
• Engineered monomeric aptamers into symmetric inverted dimeric aptamers (IDAs) with a 3'-3' linkage for enhanced stability and affinity.

Main Results:

• Developed IDAs with picomolar affinity, over 800-fold higher than existing aptamers, and enhanced nuclease resistance.
• Achieved a limit of detection of 3.3 ng/mL for PfLDH in human serum using a cascading colorimetric amplification system.
• Clinical evaluation showed 81.8% sensitivity and 100% specificity in 52 human blood samples.

Conclusions:

• The novel IDA aptamers demonstrate superior performance for PfLDH detection.
• These aptamers hold significant translational potential for developing accurate point-of-care malaria diagnostics.
• The developed method offers a promising alternative to current commercial rapid diagnostic tests.