Abstract
In many synthetic biology applications, modular and easily accessible tools for controlling gene expression are required. In addition, in vivo biosensors and diagnostic devices will become more important in the future to allow for noninvasive determination of protein, ion, or small molecule metabolite levels. In recent years synthetic RNA-based switches have been developed to act as signal transducers to convert a binding event of a small molecule (input) into a detectable output. Their modular design allows the development of a variety of molecular switches to be used in biochemical assays or inside living cells. RNA switches developed by our group are based on the Schistosoma mansoni hammerhead ribozyme, a self-cleaving RNA sequence that can be inserted into any RNA of interest. Connection to an aptamer sensing a small molecule renders the cleavage reaction ligand-dependent. In the past we have successfully designed and applied such hammerhead aptazymes for the allosteric control of both bacterial and eukaryotic gene expression by affecting transcription elongation, translation initiation, or mRNA stability. In order to yield functional switches optimization of the connecting sequence between the aptamer and the HHR needs to be carried out. We have therefore developed an in vivo screening protocol detailed in this chapter that allows the identification of functional aptazymes in bacteria.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Liu J, Cao Z, Lu Y (2009) Functional nucleic acid sensors. Chem Rev 109:1948–1998
Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510
Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822
Winkler W, Nahvi A, Breaker RR (2002) Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419:952–956
Winkler WC, Nahvi A, Sudarsan N, Barrick JE, Breaker RR (2003) An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat Struct Biol 10:701–707
Mandal M, Boese B, Barrick JE, Winkler WC, Breaker RR (2003) Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113:577–586
Vinkenborg JL, Karnowski N, Famulok M (2011) Aptamers for allosteric regulation. Nat Chem Biol 7:519–527
Paige JS, Nguyen-Duc T, Song W, Jaffrey SR (2012) Fluorescence imaging of cellular metabolites with RNA. Science 335:1194
Breaker RR (2002) Engineered allosteric ribozymes as biosensor components. Curr Opin Biotechnol 13:31–39
Blount KF, Uhlenbeck OC (2002) The hammerhead ribozyme. Biochem Soc Trans 30:1119–1122
Fedor MJ (2009) Comparative enzymology and structural biology of RNA self-cleavage. Annu Rev Biophys 38:271–299
Khvorova A, Lescoute A, Westhof E, Jayasena SD (2003) Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nat Struct Biol 10:708–712
Tang J, Breaker RR (1997) Rational design of allosteric ribozymes. Chem Biol 4:453–459
Wieland M, Hartig JS (2008) Improved aptazyme design and in vivo screening enable riboswitching in bacteria. Angew Chem 47:2604–2607
Wieland M, Benz A, Klauser B, Hartig JS (2009) Artificial ribozyme switches containing natural riboswitch aptamer domains. Angew Chem 48:2715–2718
Ogawa A, Maeda M (2008) An artificial aptazyme-based riboswitch and its cascading system in E. coli. Chembiochem 9:206–209
Win MN, Smolke CD (2007) A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. Proc Natl Acad Sci U S A 104:14283–14288
Win MN, Smolke CD (2008) Higher-order cellular information processing with synthetic RNA devices. Science 322:456–460
Wieland M, Gfell M, Hartig JS (2009) Expanded hammerhead ribozymes containing addressable three-way junctions. RNA 15:968–976
Klauser B, Saragliadis A, Auslander S, Wieland M, Berthold MR, Hartig JS (2012) Post-transcriptional Boolean computation by combining aptazymes controlling mRNA translation initiation and tRNA activation. Mol BioSyst 8:2242–2248
Auslander S, Ketzer P, Hartig JS (2010) A ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression. Mol BioSyst 6:807–814
Ketzer P, Haas SF, Engelhardt S, Hartig JS, Nettelbeck DM (2012) Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic adenoviruses. Nucleic Acids Res 40:e167
Berschneider B, Wieland M, Rubini M, Hartig JS (2009) Small-molecule-dependent regulation of transfer RNA in bacteria. Angew Chem 48:7564–7567
Wieland M, Berschneider B, Erlacher MD, Hartig JS (2010) Aptazyme-mediated regulation of 16S ribosomal RNA. Chem Biol 17:236–242
Kumar D, Annna CI, Yokobayashi Y (2009) Conditional RNA interference mediated by allosteric ribozyme. J Am Chem Soc 131:13906–13907
Saragliadis A, Krajewski SS, Rehm C, Narberhaus F, Hartig JS (2013) Thermozymes: Synthetic RNA thermometers based on ribozyme activity. RNA Biol 10(6):1010–6
Klauser B, Hartig JS (2013) An engineered small RNA-mediated genetic switch based on a ribozyme expression platform. Nucleic Acids Res 41(10):5542–52
Soukup GA, Breaker RR (1999) Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96:3584–3589
Reetz MT, Kahakeaw D, Lohmer R (2008) Addressing the numbers problem in directed evolution. Chembiochem 9:1797–1804
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Rehm, C., Hartig, J.S. (2014). In Vivo Screening for Aptazyme-Based Bacterial Riboswitches. In: Ogawa, A. (eds) Artificial Riboswitches. Methods in Molecular Biology, vol 1111. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-755-6_17
Download citation
DOI: https://doi.org/10.1007/978-1-62703-755-6_17
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-754-9
Online ISBN: 978-1-62703-755-6
eBook Packages: Springer Protocols