Functional Aptamers In Vitro Evolution for Protein-Protein Interaction Blockage.

Area of Science:

• Biotechnology and Molecular Biology
• Biochemistry and Biophysics

Background:

• Aptamer applications have expanded beyond high affinity to functional capabilities.
• Traditional SELEX methods for functional aptamer identification are time-consuming and labor-intensive.
• High aptamer affinity does not guarantee optimal functionality.

Purpose of the Study:

• To introduce a novel screening method, functional aptamers in vitro evolution (FAIVE), to efficiently identify functional aptamers.
• To demonstrate FAIVE's effectiveness in obtaining aptamers that inhibit protein-protein interactions.
• To provide theoretical insights into validating bead library quality for aptamer selection.

Main Methods:

• Developed and implemented functional aptamers in vitro evolution (FAIVE), a method linking aptamer sequence functionality to fluorescence intensity.
• Applied FAIVE to obtain modified DNA aptamers targeting the SARS-CoV-2 spike receptor-binding domain (RBD) and hACE2 interaction.
• Modeled the emulsion PCR process to investigate criteria for validating bead library quality.

Main Results:

• FAIVE successfully identified modified DNA aptamers capable of disrupting the SARS-CoV-2 RBD-hACE2 interaction, demonstrating protein-protein interaction inhibition.
• The study provides theoretical insights into bead library quality validation through emulsion PCR modeling.
• FAIVE facilitates the identification of aptamers with diverse functionalities.

Conclusions:

• FAIVE offers a significant advancement in aptamer screening, directly linking sequence functionality to a measurable signal.
• This method accelerates the discovery of functional aptamers, including those for protein-protein interaction inhibition relevant to viral targets like SARS-CoV-2.
• The FAIVE concept is adaptable to various research contexts for identifying aptamers with diverse functional capabilities.