Automated high-throughput selection of DNA aptamers using a common optical next-generation sequencer

Area of Science:

• Biotechnology
• Molecular Biology
• Genomics

Background:

• Conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for aptamer selection is inefficient, time-consuming, and labor-intensive.
• Existing SELEX methods have limitations in efficacy and throughput for identifying high-affinity aptamers.

Purpose of the Study:

• To develop and validate a novel automated high-throughput screening platform to augment DNA aptamer selection.
• To enhance the efficiency and speed of aptamer discovery compared to traditional SELEX.

Main Methods:

• Modification of an optical next-generation sequencer's software to enable automated fluorescence-based binding assays.
• Integration of sequencing with binding assays for real-time screening of displayed DNA sequences.
• Application of the platform for aptamer selection against Pseudomonas aeruginosa proteins (LecA, LecB, PEA) and other targets.

Main Results:

• High-affinity DNA aptamers for LecA, LecB, and PEA were identified in only 3-5 SELEX rounds.
• The automated method yielded aptamers with higher affinity compared to 12 rounds of conventional SELEX.
• Demonstrated versatility of the platform for aptamer selection against small molecules and whole cells, including a kanamycin-binding aptamer and Escherichia coli.

Conclusions:

• The automated high-throughput platform significantly enhances SELEX efficacy and reduces selection time to approximately one week.
• This novel approach offers deeper insights into the aptamer selection process.
• The platform is adaptable for selecting aptamers against diverse targets, from small molecules to whole cells.