An efficient method to evaluate experimental factor influence on in vitro binding of aptamers

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemistry

Background:

• Nucleic acid aptamers offer a promising alternative to antibodies but face challenges in their selection process (SELEX).
• Previous studies on SELEX experimental factors (EFs) lacked experimental confirmation or considered limited EFs.
• A more efficient method for probing EFs in aptamer selection is needed.

Purpose of the Study:

• To develop a rapid, quantitative method for assessing the influence of multiple EFs on aptamer selection.
• To evaluate the impact of both known and novel EFs on aptamer enrichment efficiency.

Main Methods:

• A quantitative method was developed using the binding ratio of specific to nonspecific aptamer binders per bead.
• Three known EFs (surface coverage, buffer composition, DNA concentration) and four novel EFs (surface chemistry, heat treatment, elution methodology, pool purity) were investigated.
• Thrombin and streptavidin were used as model targets.

Main Results:

• The developed method quantitatively assessed the influence of multiple EFs on aptamer enrichment.
• Binding ratios varied significantly across tested EFs, ranging from 0.03 ± 0.03 to 14.60 ± 2.30.
• Results aligned with existing literature, confirming the method's feasibility.

Conclusions:

• The study provides a robust method for optimizing aptamer selection and binding evaluation.
• Guidance is offered for selecting appropriate EFs to enhance aptamer enrichment efficiency.
• This work facilitates the development of more effective aptamer-based applications.