Real-time monitoring strategies for optimization of in vitro transcription and quality control of RNA.

RNA-based therapeutics and vaccines are opening up new avenues for modern medicine. To produce these useful RNA-based reagents, in vitro transcription (IVT) is an important reaction that primarily determines the yield and quality of the product. Therefore, IVT condition should be well optimized to achieve high yield and purity of transcribed RNAs. To this end, real-time monitoring of RNA production during IVT, which allows for fine tuning of the condition, would be required. Currently, light-up RNA aptamer and fluorescent dye pairs are considered as useful strategies to monitor IVT in real time. Fluorophore-labeled antisense probe-based methods can also be used for real-time IVT monitoring. In addition, a high-performance liquid chromatography (HPLC)-based method that can monitor IVT reagent consumption has been developed as a powerful tool to monitor IVT reaction in near real-time. This mini-review briefly introduces some strategies and examples for real-time IVT monitoring and discusses pros and cons of IVT monitoring methods.

Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme.

Circular RNA (circRNA) has various advantages over linear mRNA that is gaining success as a new vaccine and therapeutic agent. Thus, circRNA and its engineering methods have attracted attention recently. In this study, we developed a new in vitro circRNA engineering method by end-to-end self-targeting and splicing (STS) reaction using Tetrahymena group I intron ribozyme. We found that only the P1 helix structure of the group I intron was enough to generate circRNA by STS reaction. The efficacy of circRNA generation by STS reaction was comparable to the method using a permuted intron-exon (PIE) reaction. However, an end-to-end STS reaction does not introduce any extraneous fragments, such as an intronic scar that can be generated by PIE reaction and might trigger unwanted innate immune responses in cells, into circRNA sequences. Moreover, generated circRNA was efficiently purified by ion pair-reversed phase high-pressure liquid chromatography and used for cell-based analysis. Of note, efficient protein expression and stability with least innate immune induction by the circRNA with coxsackievirus B3 IRES were observed in cells. In conclusion, our new in vitro circRNA strategy can effectively generate highly useful circRNAs in vitro as an alternative circRNA engineering method.

Targeted suicide gene therapy for liver cancer based on ribozyme-mediated RNA replacement through post-transcriptional regulation.

Hepatocellular carcinoma (HCC) has high fatality rate and limited therapeutic options. Here, we propose a new anti-HCC approach with high cancer-selectivity and efficient anticancer effects, based on adenovirus-mediated Tetrahymena group I trans-splicing ribozymes specifically inducing targeted suicide gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To confer potent anti-HCC effects and minimize hepatotoxicity, we constructed post-transcriptionally enhanced ribozyme constructs coupled with splicing donor and acceptor site and woodchuck hepatitis virus post-transcriptional regulatory element under the control of microRNA-122a (miR-122a). Adenovirus encoding post-transcriptionally enhanced ribozyme improved trans-splicing reaction and decreased human TERT (hTERT) RNA level, efficiently and selectively retarding hTERT-positive liver cancers. Adenovirus encoding miR-122a-regulated ribozyme caused selective liver cancer cytotoxicity, the efficiency of which depended on ribozyme expression level relative to miR-122a level. Systemic administration of adenovirus encoding the post-transcriptionally enhanced and miR-regulated ribozyme caused efficient anti-cancer effects at a single dose of low titers and least hepatotoxicity in intrahepatic multifocal HCC mouse xenografts. Minimal liver toxicity, tissue distribution, and clearance pattern of the recombinant adenovirus were observed in normal animals administered either systemically or via the hepatic artery. Post-transcriptionally regulated RNA replacement strategy mediated by a cancer-specific ribozyme provides a clinically relevant, safe, and efficient strategy for HCC treatment.

Group I Intron-Based Therapeutics Through Trans-Splicing Reaction.

In 1982, the Cech group discovered that an intron structure in an rRNA precursor of Tetrahymena thermophila is sufficient to complete splicing without assistance from proteins. This was the first moment that scientists recognized RNAs can have catalytic activities derived from their own unique three-dimensional structures and thus play more various roles in biological processes than thought before. Several additional catalytic RNAs, called ribozymes, were subsequently identified in nature followed by intense studies to reveal their mechanisms of action and to engineer them for use in fields such as molecular cell biology, therapeutics, imaging, etc. Naturally occurring RNA-targeting ribozymes can be broadly classified into two categories by their abilities: Self-cleavage and self-splicing. Since ribozymes use base-pairing to recognize cleavage sites, identification of the catalytic center of naturally occurring ribozymes enables to engineer from "self" to "trans" acting ones which has accelerated to design and use ribozyme as valuable tools in gene therapy fields. Especially, group I intron-based trans-splicing ribozyme has unique property to use as a gene therapeutic agent. It can destroy and simultaneously repair (and/or reprogram) target RNAs to yield the desired therapeutic RNAs, maintaining endogenous spatial and temporal gene regulation of target RNAs. There have been progressive improvements in trans-splicing ribozymes and successful applications of these elements in gene therapy and molecular imaging approaches for various pathogenic conditions. In this chapter, current status of trans-splicing ribozyme therapeutics, focusing on Tetrahymena group I intron-based ribozymes, and their future prospects will be discussed.

Therapeutic applications of group I intron-based trans-splicing ribozymes.

Since the breakthrough discovery of catalytic RNAs (ribozymes) in the early 1980s, valuable ribozyme-based gene therapies have been developed for incurable diseases ranging from genetic disorders to viral infections and cancers. Ribozymes can be engineered and used to downregulate or repair pathogenic genes via RNA cleavage mediated by trans-cleaving ribozymes or repair and reprograming mediated by trans-splicing ribozymes, respectively. Uniquely, trans-splicing ribozymes can edit target RNAs via simultaneous destruction and repair (and/or reprograming) to yield the desired therapeutic RNAs, thus selectively inducing therapeutic gene activity in cells expressing the target RNAs. In contrast to traditional gene therapy approaches, such as simple addition of therapeutic transgenes or inhibition of disease-causing genes, the selective repair and/or reprograming abilities of trans-splicing ribozymes in target RNA-expressing cells facilitates the maintenance of endogenous spatial and temporal gene regulation and reduction of disease-associated transcript expression. In molecular imaging technologies, trans-splicing ribozymes can be used to reprogram specific RNAs in living cells and organisms by the 3'-tagging of reporter RNAs. The past two decades have seen progressive improvements in trans-splicing ribozymes and the successful application of these elements in gene therapy and molecular imaging approaches for various pathogenic conditions, such as genetic, infectious, and malignant disease. This review provides an overview of the current status of trans-splicing ribozyme therapeutics, focusing on Tetrahymena group I intron-based ribozymes, and their future prospects. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Development of RNA aptamer that inhibits methyltransferase activity of dengue virus.

OBJECTIVES: To develop an RNA aptamer specific for the methyltransferase (MTase) of dengue virus (DENV) which is essential for viral genome replication and translation acting directly on N-7 and 2'-O-methylation of the type-I cap structure of the viral RNA. RESULTS: We identified 2'-fluoro-modified RNA aptamers that can specifically bind DENV serotype 2 (DENV2) MTase using systematic evolution of ligands by exponential enrichment technology. We truncated the chosen aptamer into a 45-mer RNA sequence that can bind DENV2 MTase with K d  ~ 28 nM and inhibit N-7 methylation activity of the protein. Moreover, the 45-mer truncated aptamer could not only bind with an K d  ~ 15.6 nM but also inhibit methylation activity of DENV serotype 3 (DENV3) MTase. The 45-mer aptamer competitively impeded binding of both DENV2 and DENV3 genomic RNA to MTase of each serotype. CONCLUSION: The selected 45-mer truncated RNA aptamer specifically and avidly bound DENV MTase and competitively inhibited its methylation activity, and thus could be useful for the development of anti-DENV agents.

Inhibition of Japanese encephalitis virus (JEV) replication by specific RNA aptamer against JEV methyltransferase.

Japanese encephalitis virus (JEV) is the most common etiological agent of epidemic viral encephalitis. JEV encodes a single methyltransferase (MTase) domain located at the N-terminal region of the viral nonstructural protein NS5. JEV MTase is essential for viral replication and specifically catalyzes methylation of the viral RNA cap, which occurs exclusively in the cytoplasm. Therefore, JEV MTase is a potential target for antiviral therapy. Here, we identified specific and avid RNA aptamer (Kd âˆ¼ 12 nM) with modified 2'-O-methyl pyrimidines against JEV MTase. The RNA aptamer efficiently inhibited viral cap methylation activity of MTase and interfered with JEV production in cells. Moreover, we generated a 24-mer truncated aptamer that could specifically bind to JEV MTase with high affinity (Kd ∼16 nM). The 24-mer aptamer efficiently inhibited JEV production and replication in cells. Therefore, MTase-specific RNA aptamer might be useful as an anti-JEV agent.

Therapeutic Applications of Aptamer-Based Riboswitches.

Aptamers bind to their targets with high affinity and specificity through structure-based complementarity, instead of sequence complementarity that is used by most of the oligonucleotide-based therapeutics. This property has been exploited in using aptamers as multifunctional therapeutic units, by attaching them to therapeutic drugs, nanoparticles, or imaging agents, or as direct molecular decoys for inducing loss-of-function or gain-of-function of targets. One of the most interesting fields of aptamer application is their development as molecular sensors to regulate artificial riboswitches. Naturally, the riboswitches sense small-molecule metabolites and respond by regulating the expression of the corresponding metabolic genes. Riboswitches are cis-acting RNA structures that consist of the sensing (aptamer) and the regulating (expression platform) domains. In principle, diverse riboswitches can be engineered and applied to control different steps of gene expression in bacterial species as well as eukaryotes, by simply replacing aptamers against various endogenous and/or exogenous targets. Although these engineered aptamer-based riboswitches are recently gaining attention, it is clear that aptamer-based riboswitches have a potential for next-generation therapeutics against various diseases because of their controllability, specificity, and modularity in regulating gene expression through various cellular processes, including transcription, splicing, stability, RNA interference, and translation. In this review, we provide a summary of the recently developed and engineered aptamer-based riboswitches focusing on their therapeutic availability and further discuss their clinical potential.

In vitro selection of RNA aptamers that selectively bind danofloxacin.

Danofloxacin is a synthetic fluoroquinolone with broad spectrum antibacterial activity that is used for the treatment of respiratory diseases in animal husbandry. However, danofloxacin has many adverse reactions and is toxic to humans. Especially, it detrimentally affects muscle, central nerve system, peripheral nerve system, liver, and skin in those who ingest foods in which danofloxacin has accumulated. Prescreening and determination of the level of danofloxacin in foods or food products is necessary for human health. Aptamers are composing of oligonucleotides that specifically interact with target molecules. They are emerging as detection/diagnostic ligands. Here, we used the SELEX in vitro selection technology to identify specific and high-affinity RNA aptamers with 2'-fluoro-2'-deoxyribonucleotide modified pyrimidine nucleotides against danofloxacin. Selected RNA aptamers bound specifically to danofloxacin, but not to tetracycline. Truncation of RNA aptamer up to 36 mer did not comprise specificity and affinity. The truncated RNA aptamer specifically bound to target chemical, allowing the discrimination of danofloxacin from other fluoroquinolones. The isolated specific aptamer could be a potential agent used for the rapid and cost-effective detection and sensing of danofloxacin, replacing instrumental methods including the more expensive and time-consuming methods of high performance liquid chromatography and liquid chromatography/mass spectrometry.

In vitro selection of RNA aptamer specific to Salmonella typhimurium.

Salmonella is a major foodborne pathogen that causes a variety of human diseases. Development of ligands directly and specifically binding to the Salmonella will be crucial for the rapid detection of, and thus for efficient protection from, the virulent bacteria. In this study, we identified a RNA aptamer-based ligand that can specifically recognize Salmonella Typhimurium through SELEX technology. To this end, we isolated and characterized an RNase-resistant RNA aptamer that bound to the OmpC protein of Salmonella Typhimurium with high specificity and affinity (Kd ~ 20 nM). Of note, the selected aptamer was found to specifically bind to Salmonella Typhimurium, but neither to Gram-positive bacteria (Staphylococcus aureus) nor to other Gram-negative bacteria (Escherichia coli O157:H7). This was evinced by aptamer-immobilized ELISA and aptamer-linked precipitation experiments. This Salmonella species-specific aptamer could be useful as a diagnostic ligand against pathogen-caused foodborne sickness.

Rapid detection of food pathogens using RNA aptamers-immobilized slide.

The purpose of this study was to develop a simple and rapid detection system for foodborne bacteria, which consisted of an optical microscope and its slide chip with artificial antibodies, or RNA aptamers. From an RNA pool, three each RNA aptamers were built by the method of SELEX (systematic evolution of ligands by exponential enrichment) for components of cell wall, LPS (lipopolysaccharide) from E. coli O157:H7, teichoic acid from Staphylococcus aureus and a cell membrane protein of OmpC from Salmonella typhimurium, respectively. These aptamers were hybridized with thiol-conjugated 16 dT-linker molecules in order to be immobilized on silver surface which was, in advance, fabricated on glass slide, using a spin-coating method. To confirm that each aptamers retained its specific binding activities to their antigenic live bacteria, microscopic view of bound cells immobilized on silver film were observed. Furthermore, we observed the fluorescence-emitting bacteria-aptamer complex immobilized on silver film after adding RNA aptamers hybridized with fluorophore, FAM-conjugated 16 dT-linker molecules. As a result, the RNA aptamers-immobilized slide system developed in this study was a useful new tool to rapidly monitor individual food pathogens.

An RNA aptamer that binds carcinoembryonic antigen inhibits hepatic metastasis of colon cancer cells in mice.

BACKGROUND & AIMS: Carcinoembryonic antigen (CEA) is expressed by many types of cancer cells; its overexpression induces cell adhesion, increases resistance to anoikis, and promotes hepatic metastasis of colon cancer cells. The amino acid sequence PELPK in its hinge region, between the N and A1 domains, is required for migration of cancer cells to the liver. We sought to identify ligands of this domain for use in diagnosis and therapy. METHODS: We screened for RNA aptamers against the domain of CEA required for metastasis using systematic evolution of ligands by exponential enrichment. The specificity and affinity of the aptamer for CEA protein were characterized by mobility shift, uptake, and surface plasmon resonance assays. We analyzed the effects of the aptamer on metastatic properties of cells, as well as metastasis of colon cancer cells in mice. RESULTS: Using systematic evolution of ligands by exponential enrichment, we identified an RNA aptamer that bound to the PELPK sequence in CEA with high affinity and specificity. The isolated aptamer bound specifically to CEA-positive cells and inhibited interactions between CEA and heterogeneous nuclear ribonucleoprotein M4. The aptamer inhibited homotypic aggregation, migration, and invasion by CEA-positive cancer cells, but did not affect adhesion of endothelial cells. The aptamer induced colon cancer cell anoikis by interrupting the interaction between death receptor 5 and CEA. The aptamer prevented metastasis of human colon cancer cells to the livers of mice. CONCLUSIONS: An RNA aptamer that binds to the PELPK sequence in CEA inhibits its interactions with heterogeneous nuclear ribonucleoprotein M4 and death receptor 5, migration and invasion by colon cancer cells, and hepatic metastasis of colon cancer cells in mice. It promoted cancer cell anoikis and might be used to identify CEA-positive tumors in patients or be developed as an anti-cancer reagent.

In vitro selection of Escherichia coli O157:H7-specific RNA aptamer.

Escherichia coli (E. coli) O157:H7 is a major foodborne pathogen that causes life-threatening symptoms in humans worldwide. To rapidly and properly identify the pathogen and avoid its toxic effects, ligands which can directly and specifically bind to the virulent E. coli O157:H7 serotype should be identified. In this study, a RNA aptamer-based ligand which can specifically distinguish the pathogen E. coli O157:H7 from others was developed by a subtractive cell-SELEX method. To this end, an RNA library was first incubated with the E. coli K12 strain, and the RNAs binding to the strain were discarded. The precluded RNAs were then used for the selection of O157:H7-specific aptamers. After 6 rounds of the subtractive cell-SELEX process, the selected aptamer was found to specifically bind to the O157:H7 serotype, but not to the K12 strain. This was evidenced by aptamer-immobilized ELISA, real-time PCR analysis, or an aptamer-linked precipitation experiment. Importantly, the isolated RNA aptamer that distinguishes between the virulent serotype and the nonpathogenic strain specifically bound to an O157:H7-specific lipopolysaccharide which includes the O antigen. This novel O157:H7-specific aptamer could be of potential application as a diagnostic ligand against the pathogen-related food borne illness.

Identification of RNA aptamer specific to mutant KRAS protein.

Mutations in the KRAS gene are required for early occurrence and maintenance of tumorigenesis and are the most frequently found in many types of human malignant diseases. Therefore, approaches targeting RAS function have been proposed for cancer therapy. However, no selective and specific inhibitors of KRAS have yet been developed as anticancer agents. In this study, by employing counter-systematic evolution of ligands by exponential enrichment technique, we identified and characterized an RNA aptamer that specifically bound to mutant KRAS protein with a point mutation in codon 12 of the KRAS gene. Real-time polymerase chain reaction analysis, surface plasmon resonance measurements, and competitive precipitation experiments showed that the selected aptamer contained activities of specific and high-affinity binding to the mutant KRAS (K(D) approximately 4.04 nM) but much less binding to the wild type (K(D) approximately 227 nM). This RNA aptamer could be useful as a ligand for specific therapeutics and diagnostics against mutant KRAS-mediated cancers.

Selection of RNA aptamers specific to active prostate-specific antigen.

A counter-SELEX procedure with recombinant purified active prostate specific antigen (PSA) was used to identify specific RNA aptamers against the active PSA. We developed two different kinds of counter-SELEX methods; one includes pre-clearance step with inactive proPSA protein, and the other with tagged GST protein. After 9 iterative selection cycles, several identical RNA aptamers can be identified from both counter-SELEX methods. Real-time PCR analysis and gel retardation experiment showed that the aptamers have a specific binding activity against the active PSA, but not for GST or proPSA. These aptamers could be of potential use as specific diagnostic, imaging and/or therapeutic agents against prostate cancer.

RNA aptamer-based sensitive detection of SARS coronavirus nucleocapsid protein.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of a newly emerged disease SARS. The SARS-CoV nucleocapsid (N) protein is one of the most abundant structural proteins and serves as a diagnostic marker for accurate and sensitive detection of the virus. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant N protein, we selected a high-affinity RNA aptamer capable of binding to N protein with a dissociation constant of 1.65 nM. Electrophoretic mobility shift assays and RNA competition experiments showed that the selected aptamer recognized selectively the C-terminal region of N protein with high specificity. Using a chemiluminescence immunosorbent assay and a nanoarray aptamer chip with the selected aptamer as an antigen-capturing agent, we could sensitively detect N protein at a concentration as low as 2 pg/ml. These aptamer-antibody hybrid immunoassays may be useful for rapid, sensitive detection of SARS-CoV N protein.