Engineering high-speed allosteric hammerhead ribozymes

Abstract Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg2+. The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (k obs) as high as 8 min-1 in the presence of theophylline and maximal k obs increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.

[1]  M. Fedor,et al.  Kinetics and thermodynamics make different contributions to RNA folding in vitro and in yeast. , 2005, Molecular cell.

[2]  Anastasia Khvorova,et al.  Fast cleavage kinetics of a natural hammerhead ribozyme. , 2004, Journal of the American Chemical Society.

[3]  Andrew D Ellington,et al.  Selecting nucleic acids for biosensor applications. , 2002, Combinatorial chemistry & high throughput screening.

[4]  C. Romero-Lopez,et al.  Inhibition of HIV-1 replication by RNA targeted against the LTR region , 2005, AIDS.

[5]  W. Scott,et al.  Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis , 2006, Cell.

[6]  A. Pardi,et al.  High-resolution molecular discrimination by RNA. , 1994, Science.

[7]  R. D'Amato,et al.  Exogenous control of mammalian gene expression through modulation of RNA self-cleavage , 2004, Nature.

[8]  Ralph Weissleder,et al.  Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries. , 2006, RNA.

[9]  R. Breaker,et al.  Control of gene expression by a natural metabolite-responsive ribozyme , 2004, Nature.

[10]  O. Uhlenbeck,et al.  Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[11]  R. Symons,et al.  Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites , 1987, Cell.

[12]  G. Soukup,et al.  A versatile communication module for controlling RNA folding and catalysis. , 2002, Nucleic acids research.

[13]  R. Breaker.  Engineered allosteric ribozymes as biosensor components. , 2002, Current opinion in biotechnology.

[14]  S. K. Desai,et al.  Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation. , 2004, Journal of the American Chemical Society.

[15]  Ricardo Flores,et al.  Peripheral regions of natural hammerhead ribozymes greatly increase their self‐cleavage activity , 2003, The EMBO journal.

[16]  D. Crothers,et al.  The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. , 2005, Molecular cell.

[17]  Andrew D Ellington,et al.  Group I aptazymes as genetic regulatory switches , 2002, BMC biotechnology.

[18]  S. Woodson.  Recent insights on RNA folding mechanisms from catalytic RNA , 2000, Cellular and Molecular Life Sciences CMLS.

[19]  A. Jäschke,et al.  Controlling the rate of organic reactions: rational design of allosteric Diels-Alderase ribozymes , 2006, Nucleic acids research.

[20]  D. Herschlag.  RNA Chaperones and the RNA Folding Problem (*) , 1995, The Journal of Biological Chemistry.

[21]  R R Breaker,et al.  Relationship between internucleotide linkage geometry and the stability of RNA. , 1999, RNA.

[22]  B. Sullenger,et al.  Emerging clinical applications of RNA , 2002, Nature.

[23]  A. Ellington,et al.  Computational selection of nucleic acid biosensors via a slip structure model. , 2007, Biosensors & bioelectronics.

[24]  O. Uhlenbeck.  Less isn't always more. , 2003, RNA.

[25]  D. Crothers,et al.  The kinetics of ligand binding by an adenine-sensing riboswitch. , 2005, Biochemistry.

[26]  R. Breaker,et al.  Examination of the structural and functional versatility of glmS ribozymes by using in vitro selection , 2006, Nucleic acids research.

[27]  A D Ellington,et al.  Design and optimization of effector-activated ribozyme ligases. , 2000, Nucleic acids research.

[28]  Travis S. Bayer,et al.  Programmable ligand-controlled riboregulators of eukaryotic gene expression , 2005, Nature Biotechnology.

[29]  Ronald R. Breaker,et al.  Natural and engineered nucleic acids as tools to explore biology , 2004, Nature.

[30]  David R. Liu,et al.  In vivo evolution of an RNA-based transcriptional activator. , 2003, Chemistry & biology.

[31]  R. Breaker,et al.  Engineering precision RNA molecular switches. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[32]  A. Karpeisky,et al.  Chemically modified hammerhead ribozymes with improved catalytic rates. , 1996, Biochemistry.

[33]  R R Breaker,et al.  Altering molecular recognition of RNA aptamers by allosteric selection. , 2000, Journal of molecular biology.

[34]  R. Breaker,et al.  Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes , 2005, Nature Biotechnology.

[35]  Andrea Barta,et al.  Strategies for RNA folding and assembly , 2004, Nature Reviews Molecular Cell Biology.

[36]  R R Breaker,et al.  Rational design of allosteric ribozymes. , 1997, Chemistry & biology.

[37]  R R Breaker,et al.  Allosteric nucleic acid catalysts. , 2000, Current opinion in structural biology.

[38]  R. Breaker,et al.  Engineered allosteric ribozymes that respond to specific divalent metal ions , 2005, Nucleic acids research.

[39]  S. Silverman,et al.  Rube Goldberg goes (ribo)nuclear? Molecular switches and sensors made from RNA. , 2003, RNA.

[40]  R R Breaker,et al.  Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes. , 2001, RNA.

[41]  Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity , 2003, Nature Structural Biology.

[42]  B. Suess,et al.  A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. , 2004, Nucleic acids research.