Biomedical subjects

Steffen Schubert

Publications and source records attributed to Steffen Schubert.

7 recordsLinked to original sources

PubMed2006

Strand-specific silencing of a picornavirus by RNA interference: evidence for the superiority of plus-strand specific siRNAs.

RNA interference triggered by small interfering RNAs (siRNAs) can be used to effectively contain viral spread. Here, we report on the mechanism of action of siRNAs targeting the medically important coxsackievirus B3 (CVB-3) as a typical representative of viruses with a non-segmented RNA genome in positive-strand orientation. Antiviral siRNAs can be designed to target the genomic (+)-strand, the (-)-strand that occurs as a replication intermediate, or both. In the present study, two complementary and systematic approaches are presented providing direct evidence that silencing of the viral (+)-strand is the key to inhibit CVB-3: first, we used rational siRNA design to direct silencing activity specifically against either of the two viral strands. As a second approach, we employed siRNA containing modified nucleotides to render them specific for one of the virus RNAs. Experiments with infectious coxsackievirus revealed that the inhibitory efficiency correlates exclusively with the activity of the siRNAs directed against the viral (+)-strand. Our finding that only (+)-strand specific siRNAs exert significant antiviral potency may hold true for other RNA viruses with (+)-stranded genomes as well and may therefore be helpful in the development of efficient strategies to inhibit virus propagation.

Animals↗

PubMed2005

Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions.

Contradictory reports in the literature have emphasised either the sequence of small interfering RNAs (siRNA) or the structure of their target molecules to be the major determinant of the efficiency of RNA interference (RNAi) approaches. In the present study, we analyse systematically the contributions of these parameters to siRNA activity by using deliberately designed mRNA constructs. The siRNA target sites were included in well-defined structural elements rendering them either highly accessible or completely involved in stable base-pairing. Furthermore, complementary sequence elements and various hairpins with different stem lengths and designs were used as target sites. Only one of the strands of the siRNA duplex was found to be capable of silencing via its respective target site, indicating that thermodynamic characteristics intrinsic to the siRNA strands are a basic determinant of siRNA activity. A significant obstruction of gene silencing by the same siRNA, however, was observed to be caused by structural features of the substrate RNA. Bioinformatic analysis of the mRNA structures suggests a direct correlation between the extent of gene-knockdown and the local free energy in the target region. Our findings indicate that, although a favourable siRNA sequence is a necessary prerequisite for efficient RNAi, complex target structures may limit the applicability even of carefully chosen siRNAs.

Animals↗

PubMed2005

Deletion analysis in the catalytic region of the 10-23 DNA enzyme.

In this study, the functional relevance of the core nucleotides of the RNA cleaving 10-23 DNA enzyme (DNAzyme) was investigated. Systematic deletion studies revealed that DNAzymes lacking thymine at position 8 (T8) retain catalytic activity comparable to that of the wild-type enzyme. Deletion of the adjacent cytosine at position 7 (C7) also resulted in a highly active enzyme and even the double deletion mutant C7/T8 displayed cleavage activity, although the catalytic rate under multiple turnover conditions was found to be reduced by one order of magnitude. The identification of non-essential nucleotides in the catalytic core might help to stabilize the DNAzyme against nucleolytic degradation and to overcome problems in elucidating its three-dimensional structure.

Base Sequence↗

PubMed2005

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor.

Coxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80-90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.

Animals↗

PubMed2004

Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs.

The potential of RNA interference (RNAi) to inhibit virus propagation has been well established in recent years. In several studies, however, emergence of viral escape mutants after prolonged exposure to RNAi has been observed, raising a major hurdle for a possible therapeutic application of this strategy. Here, we report the design and characterisation of a vector that allows the simultaneous expression of two short hairpin RNAs (shRNAs), thereby maintaining high silencing activity even against a viral RNA bearing mutations in one of the target sites. Two short interfering RNAs (siRNAs) against the 3D-RNA dependent RNA polymerase of coxsackievirus B3 were identified that displayed efficient inhibition of virus propagation in HeLa cells and reduced the virus titre by up to 90%. We generated two expression vectors encoding these newly identified siRNAs and evaluated their silencing efficiency against the target gene in a reporter assay. Viral escape was then simulated by introducing a point mutation into either of the target sites. This substitution led to complete abrogation of silencing by the respective vector. To bypass this blockade of silencing, an siRNA double expression vector (SiDEx) was constructed to achieve simultaneous expression of both siRNAs from one plasmid. The silencing efficiency of both siRNAs generated by SiDEx was comparable to that of the individual mono-expression vectors. In contrast to the conventional expression vectors, SiDEx displayed substantial gene regulation also of the mutated target RNA. As our approach of expressing various shRNAs from one vector is based on a simple and universally applicable cloning strategy, SiDEx may be a helpful tool to achieve sustained silencing of viruses, ultimately reducing the risk of emergence of viable mutants. An additional application of SiDEx vectors will be the simultaneous knockdown of two targeted genes for functional studies.

Coxsackievirus Infections↗

PubMed2004

Gaining target access for deoxyribozymes.

Antisense oligonucleotides and ribozymes have been used widely to regulate gene expression by targeting mRNAs in a sequence-specific manner. Long RNAs, however, are highly structured molecules. Thus, up to 90% of putative cleavage sites have been shown to be inaccessible to classical RNA based ribozymes or DNAzymes. Here, we report the use of modified nucleotides to overcome barriers raised by internal structures of the target RNA. In our attempt to cleave a broad range of picornavirus RNAs, we generated a DNAzyme against a highly conserved sequence in the 5' untranslated region (5' UTR). While this DNAzyme was highly efficient against the 5' UTR of the human rhinovirus 14, it failed to cleave the identical target sequence within the RNA of the related coxsackievirus A21 (CAV-21). After introduction of 2'-O-methyl RNA or locked nucleic acid (LNA) monomers into the substrate recognition arms, the DNAzyme degraded the previously inaccessible virus RNA at a high catalytic rate even to completion, indicating that nucleotides with high target affinity were able to compete successfully with internal structures. We then adopted this strategy to two DNAzymes that we had found to be inactive in our earlier experiments. The modified DNAzymes proved to be highly effective against their respective target structures. Our approach may be useful for other ribozyme strategies struggling with accessibility problems, especially when being restricted to unique target sites.

5' Untranslated Regions↗

PubMed2003

RNA cleaving '10-23' DNAzymes with enhanced stability and activity.

'10-23' DNAzymes can be used to cleave any target RNA in a sequence-specific manner. For applications in vivo, they have to be stabilised against nucleolytic attack by the introduction of modified nucleotides without obstructing cleavage activity. In this study, we optimise the design of a DNAzyme targeting the 5'-non-translated region of the human rhinovirus 14, a common cold virus, with regard to its kinetic properties and its stability against nucleases. We compare a large number of DNAzymes against the same target site that are stabilised by the use of a 3'-3'-inverted thymidine, phosphorothioate linkages, 2'-O-methyl RNA and locked nucleic acids, respectively. Both cleavage activity and nuclease stability were significantly enhanced by optimisation of arm length and content of modified nucleotides. Furthermore, we introduced modified nucleotides into the catalytic core to enhance stability against endonucleolytic degradation without abolishing catalytic activity. Our findings enabled us to establish a design for DNAzymes containing nucleotide modifications both in the binding arms and in the catalytic core, yielding a species with up to 10-fold enhanced activity and significantly elevated stability against nucleolytic cleavage. When transferring the design to a DNAzyme against a different target, only a slight modification was necessary to retain activity.

Binding Sites↗