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

## TL;DR

Researchers developed a new method using high-throughput sequencing and SELEX to select aptamers that bind to potato spindle tuber viroid, improving detection and analysis.

## Contribution

A novel MB-HTS-SELEX protocol is introduced for selecting aptamers against viroids, which was previously unexplored.

## Key findings

- A magnetic bead-based SELEX protocol was successfully used to select potential PSTVd-binding aptamers.
- High-throughput sequencing identified enriched sequences after 10 selection rounds.
- Pull-down assays confirmed that the most abundant oligo-ssDNA in L30 binds to PSTVd molecules.

## Abstract

Aptamers are powerful tools for detecting and analyzing biomolecules that consist of proteins or nucleic acids. However, their application to aptamers against viroids—highly structured self-replicating RNAs—has not yet been explored. In this study, a magnetic bead- and high-throughput sequencing-based SELEX (MB-HTS-SELEX) protocol for selecting potential aptamers against potato spindle tuber viroid (PSTVd) is presented. Full-length biotinylated-PSTVd RNA was transcribed in vitro, immobilized on streptavidin-coated magnetic beads, and incubated with a library of ~3.32 × 1014 molecules of random single-stranded oligo-DNAs (oligo-ssDNAs) of 20, 30, or 40 nucleotides (L20, L30, or L40, respectively) flanked by primer binding sites for downstream PCR amplification. Simultaneous biotin labeling of the anti-aptamer strand of the resulting double-stranded DNA (dsDNA) amplicons facilitated strand separation using streptavidin-coated magnetic beads. After 10 selection rounds, high-throughput sequencing, followed by bioinformatics analysis of the generated sequences, allowed for the detection of several enriched sequences, representing putative PSTVd-binding aptamers. Subsequent pull-down assays showed that the most abundant oligo-ssDNA in L30 was docked on PSTVd molecules. This combination method may ameliorate the selection of high-affinity aptamers against PSTVd, reduce the number of selection cycles, time, and other costs of aptamer production, thereby promoting future massive and cost-effective viroid detection and characterization.