title: Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.
abstract: The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.
authors.affiliation: Department of Biochemistry, ‡Genetics Area Program, §Department of Biological Engineering, ∥Department of Molecular Microbiology and Immunology, and ⊥Bond Life Sciences Center, University of Missouri Columbia , Coumbia, Missouri, 65211, United States.
authors.forename: Raghav R
authors.identifier: 0000-0001-8911-6867
authors.initials: RR
authors.lastname: Poudyal
authors.affiliation: Department of Biochemistry, ‡Genetics Area Program, §Department of Biological Engineering, ∥Department of Molecular Microbiology and Immunology, and ⊥Bond Life Sciences Center, University of Missouri Columbia , Coumbia, Missouri, 65211, United States.
authors.forename: Malak
authors.initials: M
authors.lastname: Benslimane
authors.affiliation: Department of Biochemistry, ‡Genetics Area Program, §Department of Biological Engineering, ∥Department of Molecular Microbiology and Immunology, and ⊥Bond Life Sciences Center, University of Missouri Columbia , Coumbia, Missouri, 65211, United States.
authors.forename: Melissa P
authors.initials: MP
authors.lastname: Lokugamage
authors.affiliation: Department of Biochemistry, ‡Genetics Area Program, §Department of Biological Engineering, ∥Department of Molecular Microbiology and Immunology, and ⊥Bond Life Sciences Center, University of Missouri Columbia , Coumbia, Missouri, 65211, United States.
authors.forename: Mackenzie K
authors.initials: MK
authors.lastname: Callaway
authors.affiliation: Department of Biochemistry, ‡Genetics Area Program, §Department of Biological Engineering, ∥Department of Molecular Microbiology and Immunology, and ⊥Bond Life Sciences Center, University of Missouri Columbia , Coumbia, Missouri, 65211, United States.
authors.forename: Seth
authors.initials: S
authors.lastname: Staller
authors.affiliation: Department of Biochemistry, ‡Genetics Area Program, §Department of Biological Engineering, ∥Department of Molecular Microbiology and Immunology, and ⊥Bond Life Sciences Center, University of Missouri Columbia , Coumbia, Missouri, 65211, United States.
authors.forename: Donald H
authors.initials: DH
authors.lastname: Burke
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000378:metabolism; D002384:Catalysis; D008274:Magnesium / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D009696:Nucleic Acids; D016337:RNA, Catalytic; D012313:RNA; D000255:Adenosine Triphosphate; D008274:Magnesium