Wet-Dry-Wet Experiments Enable Simultaneous Aptamer Isolation and Critical Binding Domain Identification.

Area of Science:

• Biotechnology
• Molecular Biology
• Chemical Biology

Background:

• Identifying aptamer critical binding domains (CBDs) typically requires extensive affinity screening.
• Current methods for aptamer isolation and CBD identification are often time-consuming and labor-intensive.

Purpose of the Study:

• To develop and validate an efficient wet-dry-wet experimental strategy for the simultaneous isolation of aptamers and identification of their CBDs.
• To apply this method for the isolation of rapamycin-binding aptamers and characterization of their CBD.

Main Methods:

• A 15-round Capture-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was performed for initial library enrichment.
• High-throughput sequencing data analysis, including K-mer assembly, RBM (Restricted Boltzmann Machine), and mFold simulations, identified key aptamer structural motifs (3S3L and 3S3L-A).
• A second Capture-SELEX (6 rounds) was conducted on a structure-confined library, followed by sequencing to identify high-affinity aptamers and their CBDs.

Main Results:

• The study successfully isolated rapamycin-binding aptamers with nanomolar dissociation constants and high specificity.
• An 11-nucleotide CBD was identified, and mutations within this domain confirmed its critical role in binding.
• A strand-displacement fluorescence sensor was developed, achieving nanomolar detection limits for rapamycin in human serum.

Conclusions:

• The developed wet-dry-wet strategy offers an efficient and simultaneous approach for aptamer isolation and CBD identification.
• This method significantly reduces the need for laborious affinity testing of numerous sequences.
• The approach is potentially applicable to the discovery and characterization of aptamers for various molecular targets.