High-Throughput Sequencing and SELEX-Based Protocol for Selecting Aptamers Against Potato Spindle Tuber Viroid.

Area of Science:

• Molecular Biology
• Biotechnology
• RNA Biology

Background:

• Aptamers are nucleic acid or protein molecules used for biomolecule detection.
• Viroids, structured self-replicating RNAs, lack explored aptamer applications.
• Potato spindle tuber viroid (PSTVd) is a significant plant pathogen.

Purpose of the Study:

• To develop and present a novel protocol for selecting aptamers against PSTVd.
• To identify potential PSTVd-binding aptamers using a high-throughput method.
• To reduce the time and cost associated with aptamer production for viroid detection.

Main Methods:

• A magnetic bead- and high-throughput sequencing-based SELEX (MB-HTS-SELEX) protocol was employed.
• Full-length biotinylated PSTVd RNA was immobilized on magnetic beads and incubated with oligo-DNA libraries.
• High-throughput sequencing and bioinformatics analysis were used to identify enriched aptamer sequences.

Main Results:

• Several enriched DNA sequences with putative PSTVd-binding capabilities were identified after 10 selection rounds.
• The most abundant oligo-ssDNA from the L30 library was confirmed to bind PSTVd molecules via pull-down assays.
• The MB-HTS-SELEX method demonstrated efficiency in aptamer selection against PSTVd.

Conclusions:

• The developed MB-HTS-SELEX protocol effectively selects high-affinity aptamers against PSTVd.
• This method offers a faster, more cost-effective approach for aptamer production.
• The findings facilitate future large-scale and economical detection and characterization of viroids.