Multiplex Aptamer Discovery through Apta-Seq and Its Application to ATP Aptamers Derived from Human-Genomic SELEX

5 years ago

Multiplex Aptamer Discovery through Apta-Seq and Its Application to ATP Aptamers Derived from Human-Genomic SELEX

Michael M. K. Vu, Bao T. Ho, Robert C. Spitale, Julio Polanco, Michael M. Abdelsayed, Andrej Lupták

Laboratory-evolved RNAs bind a wide variety of targets and serve highly diverse functions, including as diagnostic and therapeutic aptamers. The majority of aptamers have been identified using in vitro selection (SELEX), a molecular evolution technique based on selecting target-binding RNAs from highly diverse pools through serial rounds of enrichment and amplification. In vitro selection typically yields multiple distinct motifs of highly variable abundance and target-binding affinities. The discovery of new aptamers is often limited by the difficulty of characterizing the selected motifs, because testing of individual sequences tends to be a tedious process. To facilitate the discovery of new aptamers within in vitro selected pools, we developed Apta-Seq, a multiplex analysis based on quantitative, ligand-dependent 2′ acylation of solvent-accessible regions of the selected RNA pools, followed by reverse transcription (SHAPE) and deep sequencing. The method reveals, in a single sequencing experiment, the identity, structural features, and target dissociation constants for aptamers present in the selected pool. Application of Apta-Seq to a human genomic pool enriched for ATP-binding RNAs yielded three new aptamers, which together with previously identified human aptamers suggest that ligand-binding RNAs may be common in mammals.

Publisher URL: http://dx.doi.org/10.1021/acschembio.7b00001

DOI: 10.1021/acschembio.7b00001