title: Deep Sequencing Analysis of Aptazyme Variants Based on a Pistol Ribozyme.
abstract: Chemically regulated self-cleaving ribozymes, or aptazymes, are emerging as a promising class of genetic devices that allow dynamic control of gene expression in synthetic biology. However, further expansion of the limited repertoire of ribozymes and aptamers, and development of new strategies to couple the RNA elements to engineer functional aptazymes are highly desirable for synthetic biology applications. Here, we report aptazymes based on the recently identified self-cleaving pistol ribozyme class using a guanine aptamer as the molecular sensing element. Two aptazyme architectures were studied by constructing and assaying 17 728 mutants by deep sequencing. Although one of the architectures did not yield functional aptazymes, a novel aptazyme design in which the aptamer and the ribozyme were placed in tandem yielded a number of guanine-inhibited ribozymes. Detailed analysis of the extensive sequence-function data suggests a mechanism that involves a competition between two mutually exclusive RNA structures reminiscent of natural bacterial riboswitches.
authors.affiliation: Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna , Okinawa 904 0495, Japan.
authors.forename: Shungo
authors.initials: S
authors.lastname: Kobori
authors.affiliation: Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna , Okinawa 904 0495, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna , Okinawa 904 0495, Japan.
authors.forename: Yohei
authors.identifier: 0000-0002-2417-1934
authors.initials: Y
authors.lastname: Yokobayashi
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics; D005818:Genetic Engineering; D059014:High-Throughput Nucleotide Sequencing; D016337:RNA, Catalytic / Q000235:genetics*; D058928:Riboswitch / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D052157:Aptamers, Nucleotide; D016337:RNA, Catalytic; D058928:Riboswitch
keywords: RNA engineering; aptamer; aptazyme; high-throughput sequencing; riboswitch; ribozyme