DNA aptamers selected against potato spindle tuber viroid bind the viroid and differentially modulate infection

DNA aptamers selected against PSTVd inhibited or promoted PSTVd infection in co-inoculated assays, correlating with their specific binding sites predicted in silico. Viroids are noncoding plant pathogens consisting of highly structured, single-stranded, circular RNA approximately 230-430 nucleotides. To develop DNA aptamers that specifically bind to potato spindle tuber viroid (PSTVd), a systematic evolution of ligands by exponential enrichment (SELEX) was performed from a random population of 30-nucleotide single-stranded DNAs. Amplicon analysis by next-generation sequencing of every five rounds up to 15 SELEX rounds revealed that the random DNA population became progressively enriched and less diverse at each sampling point; that is, the number of unique sequences detected in the 15th round was 1/18th of that in the 5th round. Sixteen sequences ranked high in 5th, 10th, and 15th rounds, as well as two low abundance sequences in 5th round, were selected to analyze the affinities to PSTVd by pull-down assay. Among them, seven bound either in vitro-transcribed or native PSTVd. The strongest binder was the one from low abundance in 5th round, while five were those ranked high in 15th round. Four sequences, including the one previously reported, were selected and further analyzed for their affinities to PSTVd by co-inoculation assay on tomato plants with infectious PSTVd transcripts. Some significantly inhibited PSTVd infection, whereas others promoted it, suggesting that aptamers may affect viroid infectivity.