Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemistry

Background:

• Aptamers, or "artificial antibodies," offer advantages over traditional antibodies but their isolation is challenging.
• Traditional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is time-consuming and resource-intensive.
• Capillary Electrophoresis-SELEX (CE-SELEX) is more efficient but requires laser-induced fluorescence (LIF).

Purpose of the Study:

• To develop a more efficient and cost-effective method for aptamer generation.
• To implement a novel aptamer selection strategy utilizing real-time PCR (RT-PCR) without LIF.
• To isolate and validate a specific aptamer against bovine serum albumin (BSA).

Main Methods:

• Developed a modified SELEX protocol incorporating RT-PCR for indirect detection of aptamer-target complexes.
• Performed three rounds of selection against BSA.
• Validated aptamer specificity using electrophoretic mobility shift assays (EMSAs), unlabeled competitor assays, and supershift assays.

Main Results:

• Successfully generated a specific aptamer for BSA in only three rounds of selection.
• Demonstrated high specificity of the selected aptamer for BSA through various validation assays.
• The RT-PCR based method proved to be highly efficient and cost-effective compared to traditional methods.

Conclusions:

• The implemented RT-PCR based aptamer selection system offers a faster, more economical alternative to existing methods.
• This approach significantly reduces the number of selection rounds required for aptamer generation.
• The validated BSA-specific aptamer holds potential for various diagnostic and therapeutic applications.