Antisense agents are valuable tools to inhibit the expression of a target gene in a sequence‐specific manner, and may be used for functional genomics, target validation and therapeutic purposes. Three types of anti‐mRNA strategies can be distinguished. Firstly, the use of single stranded antisense‐oligonucleotides; secondly, the triggering of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes; and thirdly, RNA interference induced by small interfering RNA molecules. Despite the seemingly simple idea to reduce translation by oligonucleotides complementary to an mRNA, several problems have to be overcome for successful application. Accessible sites of the target RNA for oligonucleotide binding have to be identified, antisense agents have to be protected against nucleolytic attack, and their cellular uptake and correct intracellular localization have to be achieved. Major disadvantages of commonly used phosphorothioate DNA oligonucleotides are their low affinity towards target RNA molecules and their toxic side‐effects. Some of these problems have been solved in ‘second generation’ nucleotides with alkyl modifications at the 2′ position of the ribose. In recent years valuable progress has been achieved through the development of novel chemically modified nucleotides with improved properties such as enhanced serum stability, higher target affinity and low toxicity. In addition, RNA‐cleaving ribozymes and deoxyribozymes, and the use of 21‐mer double‐stranded RNA molecules for RNA interference applications in mammalian cells offer highly efficient strategies to suppress the expression of a specific gene.

The design of antisense oligonucleotides containing locked nucleic acids (LNA) was optimized and compared to intensively studied DNA oligonucleotides, phosphorothioates and 2'-O-methyl gapmers. In contradiction to the literature, a stretch of seven or eight DNA monomers in the center of a chimeric DNA/LNA oligonucleotide is necessary for full activation of RNase H to cleave the target RNA. For 2'-O-methyl gapmers a stretch of six DNA monomers is sufficient to recruit RNase H. Compared to the 18mer DNA the oligonucleotides containing LNA have an increased melting temperature of 1.5-4 degrees C per LNA depending on the positions of the modified residues. 2'-O-methyl nucleotides increase the T(m) by only 2'-O-methyl > DNA > phosphorothioate. Three LNAs at each end of the oligonucleotide are sufficient to stabilize the oligonucleotide in human serum 10-fold compared to an unmodified oligodeoxynucleotide (from t(1/2) = approximately 1.5 h to t(1/2) = approximately 15 h). These chimeric LNA/DNA oligonucleotides are more stable than isosequential phosphorothioates and 2'-O-methyl gapmers, which have half-lives of 10 and 12 h, respectively.

Guanine-rich sequences can adopt intramolecular four-stranded structures, called G-quadruplexes. These motifs have been intensively investigated on the DNA level, but their overall biological relevance remains elusive. Only recently has research concerning the function of G-quadruplexes in RNAs commenced. Here, we demonstrate for the first time, that an RNA G-quadruplex structure inhibits translation in vivo in eukaryotic cells. We investigated the function of a highly conserved, thermodynamically stable RNA G-quadruplex in the 5′-UTR of the mRNA of the human Zic-1 zinc-finger protein. Using dual luciferase reporter assay, we demonstrate that the Zic-1 RNA G-quadruplex represses protein synthesis inside eukaryotic cells. Quantitative RT-PCR assays confirmed that the reduction of protein synthesis is due to regulation of the translation process and not a consequence of reduced transcription. Western blot analysis revealed that expression of Zic-1 is strongly reduced by a 73 nucleotides-long fragment of the UTR containing the G-quadruplex motif. These structures might add to the more recently discovered elements in untranslated regions of mRNAs that regulate their translation.

Locked nucleic acids (LNAs) and double‐stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA–DNA–LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2′‐O‐methyl RNA–DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA–DNA–LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co‐transfection study showed that siRNA is the most potent inhibitor of VR1–green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC₅₀ of 0.06 nM. An LNA gapmer was found to be the most efficient single‐stranded antisense oligonucleotide, with an IC₅₀ of 0.4 nM being 175‐fold lower than that of commonly used phosphorothioates (IC₅₀ ∼70 nM). In contrast, the efficiency of a 2′‐O‐methyl‐modified oligonucleotide (IC₅₀ ∼220 nM) was 3‐fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA–DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.

‘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.