This page shows some of the applications of the collected ribozymes. We listed three kinds of applications, ‘Gene Expression System’, ‘Detection System’, and ‘Gene Editing System’ as shown in the following table. Two specific examples are also presented, as shown in the figures below.
Twister
To establish RNA aptamers with high stability and expression levels, Jaffrey’s group designed the Tornado (twister-optimized RNA for durable overexpression) expression system for rapid RNA circularization. They use a P3 twister U2A ribozyme and a P1 twister ribozyme to generate RNAs of interest containing a 5’ hydroxyl and 2’,3’-cyclic phosphate. Then the 5’ and 3’ ends would become substrates for RtcB-mediated ligation.Finally, the RNAs of interest would be circularized.
HDV
Schematic diagram for simple and effective generating in vitro transcripts with homogenous 5’ ends and 3’ ends. As show in this diagram, we use hammerhead and HDV ribozymes to produce homogenous RNA with any 5’- and 3’- terminal sequence.
Gene Expression system
Several ribozymes have been used for gene expression regulation. For instance, twister ribozyme can be engineered to the Tornado (‘Twister-optimized RNA for durable overexpression’) expression system for efficient expression of circular RNA. The hammerhead ribozyme was used to digest viral, oncogene or mutant mRNA. Other self-cleaving ribozymes have been designed to regulate gene expression in a ligand-dependent way.
| ribozyme name | Description | First author | Corresponding author | Time | reference | Journal | pubmed ID |
|---|---|---|---|---|---|---|---|
| twister ribozyme | This article established an outstandingly flexible expression platform, which in conjugation with three different aptamer domains, enables the construction of many different one- and two-input regulators of gene expression in both bacteria and yeast. | Felletti, M | J. S. Hartig | 2016 | Twister ribozymes as highly versatile expression platforms for artificial riboswitches | Nat Commun | 27670347 |
| twister ribozyme | This article established the Tornado (‘Twister-optimized RNA for durable overexpression’) expression system for rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. | Litke, J. L | S. R. Jaffrey | 2019 | Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts | Nat Biotechnol | 30962542 |
| pistol ribozyme | This article investigated circularly permuted variants of the pistol ribozyme class (CPP) as a synthetic ribozyme scaffold for mammalian riboswitch, which allows chemical regulation of gene expression in mammalian cells. | Mustafina, K | Y. Yokobayashi | 2021 | Circularly-Permuted Pistol Ribozyme: A Synthetic Ribozyme Scaffold for Mammalian Riboswitches | ACS Synth Biol | 34374523 |
| HDV ribozyme | This article used Hepatitis delta virus (HDV) ribozyme for preparation of homogeneous RNA with any 3′-terminal sequence. | A R Ferré-D'Amaré | J A Doudna | 1996 | Use of cis- and trans-ribozymes to remove 5' and 3' heterogeneities from milligrams of in vitro transcribed RNA | Nucleic Acids Res | 8600468 |
| HDV ribozyme | This article designed a small molecule responsive allosteric ribozymes based on the genomic hepatitis delta virus (HDV) ribozyme by replacing the P4-L4 stem-loop with an RNA aptamer through a connector stem，which can regulate cis-gene expression by theophylline and guanine by up to 29.5-fold in mammalian cell culture. | Nomura, Y | Y. Yokobayashi | 2013 | Controlling mammalian gene expression by allosteric hepatitis delta virus ribozymes | ACS Synthetic Biology | 23697539 |
| HDV ribozyme | This article used HDV ribozyme as cis-cleaving autocatalytic cassettes attached to transcript 3' ends to generate homogenous 3' ends. | Wichlacz, A | J. Ciesiolka | 2004 | Generating in vitro transcripts with homogenous 3' ends using trans-acting antigenomic delta ribozyme | Nucleic Acids Res | 14973333 |
| HDV ribozyme | This article designed a RNAi-regulatory system integrating a theophylline aptamer-fused Hepatitis delta virus (HDV) ribozyme with a pri-miRNA analogue to control gene expression mediated by RNAi.The system can control gene expression by adding theophylline into the culture media in dose dependent fashion. | Zhang, Y | R. Pei | 2017 | Inducible Bcl-2 gene RNA interference mediated by aptamer-integrated HDV ribozyme switch | Integrative Biology | 28548675 |
| HDV ribozyme | This article designed an alternatively processed short hairpin RNA (shRNA) molecule termed AgoshRNA with the HDV ribozyme at the 3' terminus to enhance the silencing activity of the AgoshRNA molecules. | Berkhout, B | E. Herrera-Carrillo | 2021 | Design and Evaluation of AgoshRNAs with 3'-Terminal HDV Ribozymes to Enhance the Silencing Activity | Methods in Molecular Biology | 32712923 |
| VS ribozyme | This paper describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates. | Lacroix-Labonte, J | P. Legault | 2016 | Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions | Nucleic Acids Res | 27166370 |
| LC ribozyme | Lariat capping ribozyme functions in vivo in the budding yeast Saccharomyces cerevisiae presumably without cofactors and that lariat capping occurs cotranscriptionally. | Krogh, N | H. Nielsen | 2017 | Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo | RNA | 28159804 |
| LC ribozyme | Lariat capping is thus a versatile tool for studying the importance of the 5' end structure of RNA molecules. | Pietschmann, M | H. Nielsen | 2021 | Use of a Lariat Capping Ribozyme to Study Cap Function In Vivo | Methods Mol Biol | 32712925 |
| RNase P | A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping exon 3 region of the mRNAs encoding the major transcription regulatory proteins IE1 and IE2 of human cytomegalovirus. This study is the first to use M1GS ribozyme for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications. | Trang, P | F. Liu | 2000 | Effective inhibition of human cytomegalovirus gene expression and replication by a ribozyme derived from the catalytic RNA subunit of RNase P from Escherichia coli | Proc Natl Acad Sci U S A | 10811889 |
| hammerhead ribozyme | Incorporating a hammerhead ribozyme on the 5′-end of d56 together with the VSsl at the3′-terminus to overcome 5′ heterogeneity. | A R Ferré-D'Amaré | J A Doudna | 1996 | Use of cis- and trans-ribozymes to remove 5′ and 3′ heterogeneities from milligrams of in vitro transcribed RNA | Nucleic Acids Res |
8600468 |
| hammerhead ribozyme | The catalytic activity of the hammerhead ribozyme can be brought under the control of ATP and theophylline by designing conjoined aptamer-ribozyme complexes. | J Tang | R R Breaker | 1997 | Rational design of allosteric ribozymes | Chem Biol | 9224568 |
| hammerhead ribozyme | The hammerhead ribozyme is established RNA switches as designer molecular sensors of FMN. | G A Soukup | R R Breaker | 1999 | Engineering precision RNA molecular switches | Proc Natl Acad Sci U S A |
10097080 |
| hammerhead ribozyme | Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products. | T P Shields | A Pardi | 1999 | High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme | RNA |
10496226 |
| hammerhead ribozyme | The hammerhead ribozyme is integrated into the transcription unit of a gene or vector leading to potent inhibition of gene or vector expression. | Yen, L | R. C. Mulligan | 2004 | Exogenous control of mammalian gene expression through modulation of RNA self-cleavage | Nature | 15386015 |
| hammerhead ribozyme | The hammerhead ribozyme constructs an aptazyme with a theophylline aptamer and a communication module,whose conformational switches inhibit or promote ribosome binding for regulating downstream gene expression. | Ogawa, A | M. Maeda | 2008 | An artificial aptazyme-based riboswitch and its cascading system in E. coli | Chembiochem |
18098257 |
| hammerhead ribozyme | This paper describes a general approach for the construction of portable and scalable gene-regulatory systems. | Win, M. N | Win, M. N | 2007 | A modular and extensible RNA-based gene-regulatory platform for engineering cellular function | Proc Natl Acad Sci U S A | 17709748 |
| hammerhead ribozyme | This article used hammerhead ribozyme to develop artificial genetic switches in order to control the regulation of 16S rRNA function in Escherichia coli. | Wieland, M., B | J. S. Hartig | 2010 | Aptazyme-mediated regulation of 16S ribosomal RNA | Chem Biol | 20338515 |
| hammerhead ribozyme | This paper shows the theophylline-induced activation of an mRNA-based hammerhead ribozyme resulting in an off-switch of gene expression. | Auslander, S., P | Auslander, S., P | 2010 | A ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression | Mol Biosyst | 20567766 |
| hammerhead ribozyme | Designed tetracycline inducible self-cleaving ribozymes to reduce reporter gene expression in yeast. | Wittmann, A | B. Suess | 2011 | Selection of tetracycline inducible self-cleaving ribozymes as synthetic devices for gene regulation in yeast | Mol Biosyst | 21603688 |
| hammerhead ribozyme | This article rationally designed and constructed Boolean logic gates composed of two modular aptazyme-based RNA switches to regulate mRNA translation initiation and tRNA activation. | Klauser, B | J. S. Hartig | 2012 | Post-transcriptional Boolean computation by combining aptazymes controlling mRNA translation initiation and tRNA activation | Mol Biosyst | 22777205 |
| hammerhead ribozyme | This article describes a novel approach for using fluorescence activated cell sorting (FACS) in order to identify functional Aptamer-controlled hammerhead ribozymes in bacteria and their subsequent transfer into mammalian cells. | Wieland, M | M. Fussenegger | 2012 | Engineering of ribozyme-based riboswitches for mammalian cells | Methods | 22305857 |
| hammerhead ribozyme | This article described a temperature-responsive RNA hairpin based on the hammerhead ribozyme, thus,generated a temperature-controlled ribozyme that called thermozyme. | Saragliadis, A | J. S. Hartig | 2013 | Thermozymes: Synthetic RNA thermometers based on ribozyme activity | RNA Biol | 23595083 |
| hammerhead ribozyme | This article introduced an RNA-responsive ribozyme-based expression system to the field of artificial riboregulators that can serve as reprogrammable platform for engineering higher-order genetic circuits. | Klauser, B | J. S. Hartig | 2013 | An engineered small RNA-mediated genetic switch based on a ribozyme expression platform | Nucleic Acids Res | 23585277 |
| hammerhead ribozyme | This article reported the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. | Klauser, B | J. S. Hartig | 2014 | Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae | ACS Synth Biol | 24871672 |
| hammerhead ribozyme | This article described the method to select ribozyme-based switches of gene expression in Saccharomyces cerevisiae that we successfully implemented to engineer neomycin- and theophylline-responsive switches. | Klauser, B | J. S. Hartig | 2015 | Engineering of ribozyme-based aminoglycoside switches of gene expression by in vivo genetic selection in Saccharomyces cerevisiae | Methods Enzymol | 25605392 |
| hammerhead ribozyme | This article developed tetracycline-dependent ribozymes, which can switch on gene expression up to 8.7-fold upon addition of tetracycline | Beilstein, K | B. Suess | 2015 | Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes | ACS Synth Biol | 25265236 |
| twister ribozyme | This paper shows that the ribozyme-mediated cleavage of the bacterial 3'-UTR has major effects on gene expression. | Felletti, M | J. S. Hartig | 2017 | The 3'-untranslated region of mRNAs as a site for ribozyme cleavage-dependent processing and control in bacteria | RNA Biol | 27690736 |
| glmS ribozyme | This article established a dynamic reprogramming strategy for reconstructing metabolic networks in B. subtilis, using a typical nutraceutical, N-acetylglucosamine (GlcNAc), as a model product and the glmS (encoding glucosamine-6-phosphate synthase) ribozyme as an engineering element. | Niu, T | L. Liu | 2018 | Engineering a Glucosamine-6-phosphate Responsive glmS Ribozyme Switch Enables Dynamic Control of Metabolic Flux in Bacillus subtilis for Overproduction of N-Acetylglucosamine | ACS Synth Biol | 30138558 |
| twister ribozyme | This article developed mammalian riboswitches that function as guanine-activated ON-switches based on a novel aptazyme architecture in which an aptamer and twister ribozyme are fused in tandem. | Mustafina, K | Y. Yokobayashi | 2020 | Design of Mammalian ON-Riboswitches Based on Tandemly Fused Aptamer and Ribozyme | ACS Synth Biol | 31820936 |
| hammerhead ribozyme | This article developed ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. | Kennedy, A. B | C. D. Smolke | 2014 | Protein-responsive ribozyme switches in eukaryotic cells | Nucleic Acids Res | 25274734 |
| hammerhead ribozyme | This article used HDV as a replicative vector to deliver hammerhead ribozyme into hepatocytes and analyzed the roles of delivered hammerhead ribozyme on the replication of hepatitis B virus (HBV). | Li, X | F. Yang | 2005 | Efficient inhibition of hepatitis B virus replication by hammerhead ribozymes delivered by hepatitis delta virus | Virus Res | 16054262 |
Detection System
Many ribozyme-based detection systems have been designed. For example, theophylline-dependent ribozymes can detect theophylline molecules in mammalian cells. Other detection targets, such as proteins or viruses, have also been designed. In addition, the temperature can be detected based on the self-cleavage ribozyme.
| ribozyme name | Description | First author | Corresponding author | Time | reference | Journal | pubmed ID |
|---|---|---|---|---|---|---|---|
| hammerhead ribozyme | This article reported the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. | Klauser, B | J. S. Hartig | 2015 | Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae | ACS Synth Biol | 24871672 |
| hammerhead ribozyme | This article shows a generally approach to the development of ribozymes capable of monitoring post-translational modification of specific proteins. | Vaish, N. K | S. D. Seiwert | 2002 | Monitoring post-translational modification of proteins with allosteric ribozymes | Nat Biotechnol | 12118241 |
| hammerhead ribozyme | This article demonstrates that ribozymes aimed at the minus-strand replicative intermediate of potato spindle tuber viroid can efficiently block viroid replication and confer high levels of resistance to viroid replication when expressed in transgenic plants. | Yang, X | P. Tien | 1997 | Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes | Proc Natl Acad Sci U S A | 9144155 |
| twister ribozyme | This article stablished a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light. Application of this tool to O. sativa twister achieved the spatial (75 µm) and temporal (≤30 ms) control required to resolve folding and self-cleavage events when combined with single-molecule fluorescence detection of the ribozyme folding pathway. | Korman, A | S. A. Woodson | 2020 | Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space | Proc Natl Acad Sci U S A | 32430319 |
| Diverse ribozymes,include hammerhead ribozyme,twister ribozyme,twister sister ribozyme,pistol ribozyme,hairpin ribozyme,varkud satellite ribozyme | This article stablished an RNA integrator comprising a ribozyme and an unfolded form of the fluorogenic aptamer Broccoli, which enables detection of low-abundance targets in vitro and in living cells. | You, M | S. R. Jaffrey | 2019 | Detection of Low-Abundance Metabolites in Live Cells Using an RNA Integrator | Cell Chem Biol | 30773480 |
| hammerhead ribozyme | This article shows Bis-aptazyme sensors which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme have been designed as an in situ sensor for the detection of HCV virus related proteins. | Cho, S | B. G. Kim | 2005 | Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal | Nucleic Acids Res | 16314308 |
Gene Editing system
To overcome the uncontrolled nature of Cas9-gRNA complex cleavage activity in the current CRISPR/Cas9 system, several labs have designed ribozyme-induced gRNA release to achieve on-demand control of Cas9 activity.
| ribozyme name | Description | First author | Corresponding author | Time | reference | Journal | pubmed ID |
|---|---|---|---|---|---|---|---|
| glmS ribozyme | This article suggests the glmS ribozyme is a very suitable target for antibacterial drug development with antisense oligonucleotides. | Traykovska, M | R. Penchovsky | 2021 | Targeting glmS Ribozyme with Chimeric Antisense Oligonucleotides for Antibacterial Drug Development | ACS Synth Biol | 34734706 |
| HDV ribozyme | This article designed an improved CRISPR-Cas12a system included the Hepatitis delta virus (HDV) ribozyme to facilitate accurate 3'-end processing of the crRNA transcript to produce precise molecules,which enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. | Berkhout, B | E. Herrera-Carrillo | 2021 | Design and Evaluation of Guide RNA Transcripts with a 3'-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation | Methods in Molecular Biology | 32712922 |
| group II intron | This article provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy. | Guo, H | A. M. Lambowitz | 2000 | Group II introns designed to insert into therapeutically relevant DNA target sites in human cells | Science | 10903206 |
| group II intron | This article used mobile group II introns to develop a targetron containing a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial gene disruption at near 100% efficiency after selection. | Zhong, J | A. M. Lambowitz | 2003 | Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker | Nucleic Acids Res | 12626707 |
| group II intron | This article adapted a mutagenesis system based on the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) to function in clostridial hosts. | Heap, J. T | N. P. Minton | 2007 | The ClosTron: a universal gene knock-out system for the genus Clostridium | J Microbiol Methods | 17658189 |
| group II intron | This article provides insight into modes of DNA target site recognition used by mobile group II introns. | Zhuang, F | A. M. Lambowitz | 2009 | EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting | RNA | 19155322 |
| group II intron | This article used an efficient mobile group II intron with an IEP lacking the En domain for the efficient recognition of DNA target sites at different locations in the Sinorhizobium meliloti chromosome. | Garcia-Rodriguez, F. M | N. Toro | 2014 | Use of the computer-retargeted group II intron RmInt1 of Sinorhizobium meliloti for gene targeting | RNA Biol | 24646865 |
| RNase P | This article uses a sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli for inhibiting HCV replication and demonstrates the utility of this ribozyme for antiviral applications. | Mao, X | G. Li | 2014 | Inhibition of hepatitis C virus by an M1GS ribozyme derived from the catalytic RNA subunit of Escherichia coli RNase P | Virology journal | 24885776 |
| RNase P | This study is the first to use a sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications. | Trang, P | F. Liu | 2000 | Effective inhibition of human cytomegalovirus gene expression and replication by a ribozyme derived from the catalytic RNA subunit of RNase P from Escherichia coli | Proc Natl Acad Sci U S A | 10811889 |
| hammerhead ribozyme | This article reported the use of hammerhead ribozyme for the generation of gRNAs both in vitro and in zebrafish embryos. | Lee, R. T | P. W. Ingham | 2016 | Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis | PLoS One | 27832146 |
| hammerhead ribozyme | This article developed tetracycline-dependent ribozymes, which can switch on gene expression up to 8.7-fold upon addition of tetracycline. | Beilstein, K | B. Suess | 2015 | Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes | ACS Synth Biol | 25265236 |
| hammerhead ribozyme | This article develops an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. | Gao, Y | Y. Zhao | 2014 | Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing | J Integr Plant Biol | 24373158 |
| group II intron | This article suggests retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria. | Karberg, M | A. M. Lambowitz | 2001 | Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria | Nat Biotechnol | 11731786 |
| group II intron | This article shows mobile group II introns have been used to develop a novel class of gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. | Zhong, J | A. M. Lambowitz | 2003 | Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker | Nucleic Acids Res | 12626707 |
Ribocentre
