Much as LNA, tricyclo(tc)-DNA has been designed as a conformationally constrained oligonucleotide analogue. Chemically, tc-DNA deviates from natural DNA by three additional C-atoms between C(5’) and C(3’). These chemical modifications change the properties of natural oligodeoxynucleotides in the following way:
Increased RNA affinity by 2 – 4°C / modification
Increased hydrophobicity
Increased stability towards nucleolytic degradation for both, phosphate or thiophosphate internucleoside linkages
Inability to elicit RNaseH activity
Tc-oligonucleotides are manufactured in the same way as natural or LNA- oligonucleotides via solid phase phosphoramidite chemistry.
Unlike LNA, fully modified tc-oligonucleotides in the lenght range of 11-25 nucleotides can easily be prepared and produce potent antisense effects.
Due to their properties tc-oligonucleotides are particularly suited for biological and therapeutic applications where high target affinity and biostability is required and where the mechanism of action does not rely on RNaseH activity.
Duchenne muscular dystrophy (DMD) is the consequence of a X-chromosome linked genetic defect, caused by a mutation in the dystrophin gene. Dystrophin is the largest known human gene consisting of 79 exons interrupted by 78 introns. Mutations can lead to exon skipping during splicing which in turn leads to misaligned mRNA that produces unfunctional protein. The incidence rate of the disease is ca 1 out of 3500 newborn males. The majority of mutations are spontaneous and not hereditary.
Dystrophin binds to the intracellular actin filaments and links via the trans-membrane sarcoglycans to the extracellular matrix. It is essential for muscle function.
Classical mutations (e.g. in exon 52) cause exon 52 skipping during splicing which leads to a frame shifted mRNA that produces unfunctional dystrophin. Additional skipping of exon 51 induced by an antisense oligonucleotide restores the reading frame and leads to a slightly shortened but functional dystrophin variant.
