title: High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase.
abstract: Systematic evolution of ligands through exponential enrichment (SELEX) is a well-established method for generating nucleic acid populations that are enriched for specified functions. High-throughput sequencing (HTS) enhances the power of comparative sequence analysis to reveal details of how RNAs within these populations recognize their targets. We used HTS analysis to evaluate RNA populations selected to bind type I human immunodeficiency virus reverse transcriptase (RT). The populations are enriched in RNAs of independent lineages that converge on shared motifs and in clusters of RNAs with nearly identical sequences that share common ancestry. Both of these features informed inferences of the secondary structures of enriched RNAs, their minimal structural requirements and their stabilities in RT-aptamer complexes. Monitoring population dynamics in response to increasing selection pressure revealed RNA inhibitors of RT that are more potent than the previously identified pseudoknots. Improved potency was observed for inhibition of both purified RT in enzymatic assays and viral replication in cell-based assays. Structural and functional details of converged motifs that are obscured by simple consensus descriptions are also revealed by the HTS analysis. The approach presented here can readily be generalized for the efficient and systematic post-SELEX development of aptamers for down-stream applications.
authors.affiliation: Molecular Microbiology and Immunology, University of Missouri, Columbia, MO, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Ditzler
authors.forename: Margaret J
authors.initials: MJ
authors.lastname: Lange
authors.forename: Debojit
authors.initials: D
authors.lastname: Bose
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Bottoms
authors.forename: Katherine F
authors.initials: KF
authors.lastname: Virkler
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Sawyer
authors.forename: Angela S
authors.initials: AS
authors.lastname: Whatley
authors.forename: William
authors.initials: W
authors.lastname: Spollen
authors.forename: Scott A
authors.initials: SA
authors.lastname: Givan
authors.forename: Donald H
authors.initials: DH
authors.lastname: Burke
mesh_terms: D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000494:pharmacology; D001483:Base Sequence; D016384:Consensus Sequence; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D059372:Nucleotide Motifs; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D052156:SELEX Aptamer Technique; D017423:Sequence Analysis, RNA / Q000379:methods*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D019380:Anti-HIV Agents; D052157:Aptamers, Nucleotide; D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase