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

Abstract

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.

High-throughput methods in aptamer discovery and analysis

“…RT stop sites are mapped using ShapeFinder, a package used to quantitatively map RT stops at nucleotide resolution and when a ligand titration is used, the stops uncover RNA conformational changes due ligand binding (Vasa, Guex, Wilkinson, Weeks, & Giddings, 2008). When this method was applied to a human-genomic in vitro selection for ATP-binders, it revealed three novel humangenome-derived ATP-binding RNA aptamers, and confirmed the existence of two previously identified aptamers (Abdelsayed et al, 2017;Vu et al, 2012). These results demonstrated the robustness of the method to perform structural analysis in a high-throughput pipeline and allowed determination of dissociation rate constants, K D s, for selected pools while eliminating single-clone biochemical assessment.…”

“…High-throughput methods that incorporate biochemical assessment of individual sequences decrease the time requirement of aptamer identification and increases the likelihood of uncovering larger diversity of functional sequences. Current methods have been used to uncover human ATP-binding RNA aptamers, analyze MS2 aptamers, and identify CRISPR-Cas complex specificity (Abdelsayed et al, 2017;Buenrostro et al, 2014;Jung et al, 2017). These techniques are valuable in the advancement of in vitro selection and aid in understanding of the evolution of functional RNAs from random sequence space.…”

“…To increase the experimental throughput of these measurements, one approach characterizes of functional RNAs by probing their dynamic properties against a titration of ligand. The technique, Apta-Seq, utilizes a combinatorial, multiplexed approach focused on RNA chemical modification and deep HTS to evaluate structural interaction of aptamers with their target ligand (Abdelsayed et al, 2017). Apta-Seq utilizes selective 2 0 -hydroxyl acylation with primer extension (SHAPE) to determine whether a 2 0 -hydroxyl is solvent-accessible to allow acylation by a SHAPE reagent, such as 2-(azidomethyl)nicotinic acid acyl imidazole (NAI-N 3 ), under variable ligand concentrations (Merino, Wilkinson, Coughlan, & Weeks, 2005;Spitale et al, 2015).…”

“…Alignments of individual clusters, using tools such as MUSCLE or MAFFT, Finally, enriched sequences identified through counting and clustering can be individually tested for target binding specificity, and determination of binding constants, such as K D and k on /k off . High-throughput analysis methods yield these kinetic and thermodynamic constants for many sequences simultaneously, with some methods, such as Apta-Seq (Abdelsayed et al, 2017), revealing structural information as well.…”

Pseudo aptamer expands aptamer’s applications

“…The major interaction forces in the binding process are defined to be hydrogen bond and charge-charge interaction that are provided from native phosphodiester polymeric linker and the bases on loops. [27][28][29][30] The concept of aptamer ligands binding to proteins was reported early 1980s for the study of human immunodeficiency virus (HIV) and adenovirus. 31,32 In this investigation it had shown that the viruses have a high force of affinity and specificity for the encoding of small numbers of RNAs that bind to viruses or cellular proteins.…”