mRNA fusion constructs serve in a general cell‐based assay to profile oligonucleotide activity

A cellular assay has been developed to allow measurement of the inhibitory activity of large numbers of oligonucleotides at the protein level. The assay is centred on an mRNA fusion transcript construct comprising of a full‐length reporter gene with a target region of interest inserted into the 3′‐untranslated region. Luciferase and fluorescent reporter genes were used in the constructs. The insert can be from multiple sources (uncharacterised ESTs, partial or full‐length genes, genes from alternate species, etc.). Large numbers of oligonucleotides were screened for antisense activity against a number of such constructs bearing different reporters, in different cell lines and the inhibitory profiles obtained were compared with those observed through screening the oligonucleotides against the corresponding endogenous genes assayed at the mRNA level. A high degree of similarity in the profiles was obtained indicating that the fusion constructs are suitable surrogates for the endogenous messages for characterisation of antisense oligonucleotides (ASOs). Furthermore, the results support the hypothesis that the secondary structure of mRNAs are divided into domains, the nature of which is determined by primary nucleotide sequence. Oligonucleotides whose activity is dependent on the local structure of their target mRNAs (e.g. ASOs, short interfering RNAs) can be characterised via such fusion RNA construct

Second Generation of Antisense Oligonucleotides: From Nuclease Resistance to Biological Efficacy in Animals

From efforts to improve the biophysical properties of antisense oligonucleotides by incorporating backbone- or sugar-modified nucleoside analogs, 2'-O-methoxyethyl ribonucleosides 8b were identified as building blocks for a second generation of antisense oligonucleotides. Compounds containing these modifications were demonstrated to combine the benefit of a high binding affinity to the RNA complement with a large increase in nuclease resistance, allowing the use of regular phosphodiester linkages. Chimeric oligonucleotides with 2'-O-methoxyethyl ribonucleosides, 8b, in the wings and a central DNA-phosphorothioate window were shown to efficiently downregulate C-'raf' kinase and PKC-α messenger-RNA in tumor cell lines resulting in a profound inhibition of cell proliferation. The same compounds were able to effectively reduce the growth of tumors in animal models at low concentrations indicating the potential utility of these second generation antisense oligonucleotides for therapeutic applications

Development of Artificial Ribonucleases Using Macrocyclic Lanthanide Complexes

The sequence-specific recognition and cleavage of ribonucleic acids (RNA) represents a fundamental challenge. Metal complexes are among the best synthetic catalysts for the cleavage of RNA. Covalent attachment of suitable macrocyclic lanthanide complexes to oligonucleotides allows the cleavage of complementary RNAs in a sequence-specific manner. The preparation, properties and use of such artificial ribonucleases are highlighted. In particular, the design and preparation of constructs cleaving RNA with multiple turn-over is described

mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity

A cellular assay has been developed to allow measurement of the inhibitory activity of large numbers of oligonucleotides at the protein level. The assay is centred on an mRNA fusion transcript construct comprising of a full-length reporter gene with a target region of interest inserted into the 3′-untranslated region. Luciferase and fluorescent reporter genes were used in the constructs. The insert can be from multiple sources (uncharacterised ESTs, partial or full-length genes, genes from alternate species, etc.). Large numbers of oligonucleotides were screened for antisense activity against a number of such constructs bearing different reporters, in different cell lines and the inhibitory profiles obtained were compared with those observed through screening the oligonucleotides against the corresponding endogenous genes assayed at the mRNA level. A high degree of similarity in the profiles was obtained indicating that the fusion constructs are suitable surrogates for the endogenous messages for characterisation of antisense oligonucleotides (ASOs). Furthermore, the results support the hypothesis that the secondary structure of mRNAs are divided into domains, the nature of which is determined by primary nucleotide sequence. Oligonucleotides whose activity is dependent on the local structure of their target mRNAs (e.g. ASOs, short interfering RNAs) can be characterised via such fusion RNA constructs