Jörg S. Hartig

Protein-dependent ribozymes report molecular interactions in real time

Most approaches to monitoring interactions between biological macromolecules require large amounts of material, rely upon the covalent modification of an interaction partner, or are not amenable to real-time detection. We have developed a generalizable assay system based on interactions between proteins and reporter ribozymes. The assay can be configured in a modular fashion to monitor the presence and concentration of a protein or of molecules that modulate protein function. We report two applications of the assay: screening for a small molecule that disrupts protein binding to its nucleic acid target and screening for protein–protein interactions. We screened a structurally diverse library of antibiotics for small molecules that modulate the activity of HIV-1 Rev-responsive ribozymes by binding to Rev. We identified an inhibitor that subsequently inhibited HIV-1 replication in cells. A simple format switch allowed reliable monitoring of domain-specific interactions between the blood-clotting factor thrombin and its protein partners. The rapid identification of interactions between proteins or of compounds that disrupt such interactions should have substantial utility for the drug-discovery process.

A comparison of DNA and RNA quadruplex structures and stabilities

Guanosine-rich sequences are prone to fold into four-stranded nucleic acid structures. Such quadruplex sequences have long been suspected to play important roles in regulatory processes within cells. Although DNA quadruplexes have been studied in great detail, four-stranded structures made up from RNA have received only minor attention, although it is known that RNA is able to form stable quadruplexes as well. Here, we compare quadruplex structures and stabilities of a variety of DNA and RNA sequences. We focus on well established DNA sequences and determine the topologies and stabilities of the corresponding RNA sequences by CD spectroscopy and CD thermal melting experiments. We find that the RNA sequences exclusively fold into the all-parallel conformation in contrast to the diverse topologies adopted by DNA quadruplexes. The thermal stabilities of the RNA structures rival those of the corresponding DNA sequences, often displaying enhanced stabilities compared to their DNA counterparts. Especially thermodynamically less stable sequences show a strong preference for potassium, with the RNA quadruplexes exhibiting much higher stabilities than the corresponding DNAs. The latter finding suggests that quadruplexes formed at critical positions in mRNAs might perturb gene expression to a larger extend than previously anticipated.

Artificial ribozyme switches containing natural riboswitch aptamer domains.

RNA Lego: The use of natural riboswitch aptamers in synthetic RNA switches (see picture) should broaden the scope of artificial RNA regulators dramatically. It is shown that thiamine pyrophosphate (TPP) aptamers can be used in engineered devices as very sensitive switches of gene expression in unmodified organisms. The approach demonstrates that intrinsic metabolites can be utilized as external effectors of cellular functions.

Predictable suppression of gene expression by 5′-UTR-based RNA quadruplexes

Four-stranded DNA and RNA quadruplexes or G4 motifs are non-B DNA conformations that are presumed to form in vivo, although only few explicit evidence has been reported. Using bioinformatics the presence of putative DNA G-quadruplexes within critical promoter regions has been demonstrated and a regulatory role in transcription has been suspected. However, in genomic DNA the presence of the complementary strand interferes with the potential to form a quadruplex motif. Contrarily RNA G4 motifs have no such limitation and consequently strong interference with gene expression is suspected. Nevertheless, experimental evidence is scarce. Here we show a well-defined structure–function relationship of synthetic quadruplex sequences in 5′-UTRs in multiple mammalian cell-lines. We establish a universal ‘translational suppressor’ effect of these motifs on gene expression at the translational level and show for the first time that specific features such as loop-length and the number of ‘GGG’-repeats further determine the suppressive impact. Moreover, a consistent and predictable repression of gene expression is observed for naturally occurring RNA G4 motifs, augmenting the functional relevance of these unusual nucleic acid structures.

A ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression

The possibility to externally control gene expression is of fundamental importance in both basic and applied life sciences. Although there are some techniques available to regulate expression in mammalian cells, they rely on the presence of ligand-sensing transcription factors, making it necessary to generate cell lines or organisms that stably express these regulatory factors. In recent years, mechanisms relying on direct RNA-ligand interactions for controlling gene expression have been both discovered in nature and engineered artificially. Among the latter, RNA switches relying on catalytically active RNA have been described. In principle, ligand-dependent triggering of mRNA self-cleavage should be a universal mechanism in order to control gene expression in a variety of organisms. Nevertheless, no examples of such aptazymes acting as RNA-based switches have been reported so far in mammalian cells. Here we present the theophylline-induced activation of an mRNA-based hammerhead ribozyme, resulting in an off-switch of gene expression. Starting from an artificial aptazyme switch reported to function in bacteria, we identified and optimized important parameters such as artificial start codons and the communicating sequence connecting ribozyme and aptamer, resulting in an RNA switch that allows for controlling transgenic expression in mammalian cells without the need to express a corresponding ligand-sensing transcription factor.

Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology

The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology.

A general design strategy for protein-responsive riboswitches in mammalian cells

RNAs are ideal for the design of gene switches that can monitor and program cellular behavior because of their high modularity and predictable structure-function relationship. We have assembled an expression platform with an embedded modular ribozyme scaffold that correlates self-cleavage activity of designer ribozymes with transgene translation in bacteria and mammalian cells. A design approach devised to screen ribozyme libraries in bacteria and validate variants with functional tertiary stem-loop structures in mammalian cells resulted in a designer ribozyme with a protein-binding nutR-boxB stem II and a selected matching stem I. In a mammalian expression context, this designer ribozyme exhibited dose-dependent translation control by the N-peptide, had rapid induction kinetics and could be combined with classic small molecule–responsive transcription control modalities to construct complex, programmable genetic circuits.

Reporter ribozymes for real-time analysis of domain-specific interactions in biomolecules: HIV-1 reverse transcriptase and the primer-template complex.

The major task in the postgenome-era is to decipher the function of thousands of new proteins, their involvement in regulatory networks and to check their suitability as pharmaceutical drug targets. For this reason, novel methods are required that facilitate rapid and reliable identification of molecular interactions of complex biological systems compatible with high-throughput screening protocols. Knowledge about the interaction partners, binding affinity, and interacting domains represent the basis for identification of the biological function and discovery of novel inhibitors, modulators, and drug leads of a given protein.[1] Although there are several powerful methods available for detection and quantification of molecular interactions,[2±7] they are often not generally applicable or compatible with high-throughput screening protocols, in real time. Hence, the development of novel, broadly applicable methods, independent of target protein function, is of fundamental importance. We are interested in using ribozymes for developing functional assays that allow the analysis of interactions of biologically relevant molecules in real-time.We have reported a novel system for rapid and reliable measurement of the catalytic activity of the hammerhead ribozyme (HHR) by using substrate oligonucleotides labeled with two fluorescent dyes.[8] The spatial proximity of the two dyes results in fluorescence quenching of the donor fluorophore by fluorescence resonance energy transfer (FRET). Ribozyme cleavage activity can be then monitored by a time-dependent increase of fluorescence in real-time. By using these reporter ribozymes we have identified novel inhibitors of the hammerhead ribozyme[9] and of the HIV-1 Rev protein,[10] which were also able to inhibit the biological function of the target molecule in vivo. Herein, we report the rational design of a reporter ribozyme, which is specifically regulated by HIV-1 reverse transcriptase (HIV-1 RT). We demonstrate that the HIV-1 RT dependent reporter ribozyme is not only capable of selectively detecting the presence of HIV-1 RT but also of sensing the domain-specific interaction of other HIV-1 RT binders such as the primer±template complex. For the construction of the reporter ribozyme we have chosen a strategy similar to that used for previous systems that are regulated by small organic molecules, by inserting an aptamer sequence into stem II of the HHR. Proper folding of stem III is essential for cleavage activity of the HHR.[11±14] We have chosen an aptamer which was selected by Tuerk et al. from a combinatorial RNA-library and which binds HIV-1 RT with an affinity of 25 pm.[15] The crystal structure of the RNA±protein complex shows that the anti-HIV-1 RTaptamer in the complex with HIV-1 RT forms a pseudoknot structure, in which the 5’and 3’-ends of the aptamers are spatially separated.[16] We have deliberately chosen an aptamer with a pseudoknot structure because this motif is often used as a regulatory element in nature. For example, formation of a pseudoknot induces a frameshift in some viral mRNA sequences.[17] In some eukaryotic transcripts, a pseudoknot structure in the 5’untranslated region leads to activation of a regulatory protein, which then locally controls translation of the transcript.[18] Owing to these known structural and regulatory features of pseudoknot motifs the anti-HIV-1 RT aptamer seemed to be well suited as a regulatory element of a hammerhead ribozyme. The aptamer was inserted into stem II of the HHR, as shown in Figure 1a, resulting in a fusion construct FK-1 with competing folds of the ribozyme and the pseudoknot structures. The simultaneous folding of both domains is impossible in this design, because in the absence of HIV-1 RT the inserted aptamer sequence folds into a hairpin loop structure (see Figure 1a, left). As shown in Figure 1b, the reporter ribozyme FK-1 is active in the absence of HIV-1 RT due to the folding of the hairpin loop, forming stem II in FK-1. The presence of an unpaired loop was proven by digestion with ribonucleases specific for single-stranded RNA (Figure 2). In the presence of HIV-1 RT, the catalytic activity of FK-1 is inhibited (Figure 1b) due to the induction of the pseudoknot fold by the protein. This leads to disruption of stem II and, hence, to the inhibition of cleavage activity. To further characterize and verify the influence of the stability of stem II on the capability of structural changes of FK-1, two variants of FK-1 were generated, one with weakened and one with stabilized stem II structures. Deletion of the GC base pair highlighted in gray in Figure la yields construct FK-2 with a destabilized stem II (Figure 1c). The complete absence of catalytic activity of FK-2 (Figure 1d) indicates that the lack of the stabilizing GC base pair diminishes formation of the catalytically active conformation in favor of the pseudoknot fold. Indeed, nuclease digest reactions of FK-2 shown in Figure 2 resulted in cleavage patterns, which are in accordance with the kinetic data in Figure 1d, thus supporting the exclusive formation of the pseudoknot. For further validation of this hypothesis we constructed a third version of the reporter ribozyme, which is only capable of forming the catalytically active fold, but not the pseudoknot structure. Starting from FK-1, an additional GC base pair was inserted, which leads to stabilization of stem II in the construct FK-3 (Figure 1e). Figure 1 f shows, as expected, that FK-3 is catalytically active, with the cleavage activity remaining unchanged even in the presence of HIV-1 RT. Owing to the increased stability of stem II, the protein is no longer able to induce the folding of the pseudoknot. Indeed, the nuclease COMMUNICATIONS

Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

Significance Riboswitches are short RNA sequences for ligand-dependent modulation of gene expression in cis. This study demonstrates that an artificial riboswitch, a ligand-dependent self-cleaving ribozyme (aptazyme), can knockdown expression of an adeno- (DNA) virus early and a measles (RNA) virus structural gene, impacting biological outcomes, i.e. inhibiting viral genome replication and infectivity, respectively. It is the first report of riboswitches for replication control of human-pathogenic viruses and of their function in fully cytoplasmic (virus) systems. For future applications, aptazymes can be customized in other viruses facilitating analyses of viral gene functions or as a safety switch in oncolytic viruses. Because of their small size and RNA-intrinsic activity, we propose aptazymes as an alternative for inducible promoters in eukaryotic gene expression control. Aptazymes are small, ligand-dependent self-cleaving ribozymes that function independently of transcription factors and can be customized for induction by various small molecules. Here, we introduce these artificial riboswitches for regulation of DNA and RNA viruses. We hypothesize that they represent universally applicable tools for studying viral gene functions and for applications as a safety switch for oncolytic and live vaccine viruses. Our study shows that the insertion of artificial aptazymes into the adenoviral immediate early gene E1A enables small-molecule–triggered, dose-dependent inhibition of gene expression. Aptazyme-mediated shutdown of E1A expression translates into inhibition of adenoviral genome replication, infectious particle production, and cytotoxicity/oncolysis. These results provide proof of concept for the aptazyme approach for effective control of biological outcomes in eukaryotic systems, specifically in virus infections. Importantly, we also demonstrate aptazyme-dependent regulation of measles virus fusion protein expression, translating into potent reduction of progeny infectivity and virus spread. This not only establishes functionality of aptazymes in fully cytoplasmic genetic systems, but also implicates general feasibility of this strategy for application in viruses with either DNA or RNA genomes. Our study implies that gene regulation by artificial riboswitches may be an appealing alternative to Tet- and other protein-dependent gene regulation systems, based on their small size, RNA-intrinsic mode of action, and flexibility of the inducing molecule. Future applications range from gene analysis in basic research to medicine, for example as a safety switch for new generations of efficiency-enhanced oncolytic viruses.

Artificial Riboswitches : Synthetic mRNA-based Regulators of Gene Expression

For half a century, bacterial regulation of gene expression has been known to be dominated by proteins that interact with metabolites, which results in altered transcription initiation. lthough the expression of the majority of genes is controlled by protein-based mechanisms, the discovery of RNA-based feedback devices that enable regulation of expression without the need for engaged proteins came as a surprise. Breaker and co-workers initially discovered that the use of such mechanisms, termed riboswitches, is widespread in bacteria. For excellent reviews that highlight naturally occurring riboswitches, we refer to the recent literature. Riboswitches are typically located in the 5’-untranslated region (5’-UTR) of bacterial mRNA, and consist mainly of a first domain (called aptamer domain) that specifically senses a metabolite, and a second domain (the expression platform) that facilitates control over transcription termination or translation initiation by a structural rearrangement (see Scheme 1). With respect to the revolutionary findings of Breaker and coworkers, it is very intriguing that researchers have successfully constructed similar, artificial systems even several years before naturally occurring riboswitches were discovered. The generation of such man-made, RNA-based regulators was possible by using aptamer technology for the recognition of ligands by RNAs. Aptamers are in-vitro-selected nucleic acid sequences that specifically bind to a ligand of choice. Such artificial, RNA-based switches enable the control of gene expression, uncoupled from the intrinsic metabolism. Although natural riboACHTUNGTRENNUNGswitches are mainly found in bacteria, artificial systems have been constructed for eukaryotic organisms as well. Such tailormade regulatory devices should prove of value as tools in biotechnology as well as synthetic biology applications. Here, we give an overview of the different concepts that are based on the insertion of ligand-sensing elements into mRNAs, thereby enabling the regulation of expression of the respective message. Due to space restrictions we will neither discuss artificial trans-acting mechanisms such as small-molecule-regulated, RNA-based transcriptional activators, nor ligand-controlled antisense constructs for the regulation of gene expression.