{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T17:46:26Z","timestamp":1788630386579,"version":"build-2803163510"},"reference-count":46,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T00:00:00Z","timestamp":1771027200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japanese Society for the Promotion of Science","doi-asserted-by":"crossref","award":["14F04078"],"award-info":[{"award-number":["14F04078"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Grant-in-Aid for Scientific Research","award":["18K19196"],"award-info":[{"award-number":["18K19196"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Aptamers are powerful tools for detecting and analyzing biomolecules that consist of proteins or nucleic acids. However, their application to aptamers against viroids\u2014highly structured self-replicating RNAs\u2014has 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 \u00d7 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.<\/jats:p>","DOI":"10.3390\/ijms27041831","type":"journal-article","created":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T10:14:32Z","timestamp":1771236872000},"page":"1831","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["High-Throughput Sequencing and SELEX-Based Protocol for Selecting Aptamers Against Potato Spindle Tuber Viroid"],"prefix":"10.3390","volume":"27","author":[{"given":"Maria S.","family":"Kaponi","sequence":"first","affiliation":[{"name":"Hellenic Organization of Agricultural Insurances (ELGA), Maiandrou and Petrakogiorgi, 71305 Heraklion, Crete, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Teruo","family":"Sano","sequence":"additional","affiliation":[{"name":"Department of Applied Biology and Food Sciences, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Aomori, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takashi","family":"Naoi","sequence":"additional","affiliation":[{"name":"Department of Applied Biology and Food Sciences, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Aomori, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4231-6727","authenticated-orcid":false,"given":"Akiko","family":"Kashiwagi","sequence":"additional","affiliation":[{"name":"Department of Applied Biology and Food Sciences, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Aomori, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1016\/0042-6822(71)90342-4","article-title":"Potato spindle tuber \u2018virus\u2019. 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