Antisense oligonucleotides have been used for over twenty years to inhibit gene expression levels both in vitro and in vivo. Antisense technology has opened up a new approach to the development of drug therapies, such as the production of drugs that inhibit the production of disease-causing proteins; this new technology is more selective and less toxic than traditional drugs. Antisense DNA or synthetic RNAs are oligonucleotides that are specially modified to prevent their degradation within cells and increase their affinity to the specific target mRNA.
Recent improvements in compound design and chemistry antisense have enabled this technology to become a routinely used tool in basic research, genomics, and for the validation and design of new drugs. Usually long nucleic acids between 20 and 30 bases are designed, drawn in an antisense manner with respect to the mRNA of interest; the synthetic oligonucleotide is introduced into the cell and hybridizing on the target mRNA leads to the cleavage of the mRNA by the RNAse H. The antisense oligonucleotides with a portion of DNA modified or not with phosphotiorate, can form heteroduplexes with the target RNA, degrading it and preventing the translation of the protein of interest. Gene expression levels can be monitored by Real Time PCR.
The antisense oligonucleotides introduced into the cells, if not modified, immediately undergo degradation by the cellular nucleases; currently there are two possible types of modifications:
- The S-oligos (phosphorothiorates) in which a sulfur atom replaces the non-bridging oxygen of the phosphorodiester bond. This type of oligo (phosphotiorates-PTO), however, recognize and bind to many proteins, resulting toxic or creating artifacts. For this reason it is possible to use oligo-chimeras that contain both a modification with phosphotiorate and with phosphodiester, so as to reduce the problems of toxicity and non-specificity.
- The oligos 2'-O-methyl RNA in which a methyl is added on the ribose in position two, replacing a hydroxyl group. The methyl group increases the stability of the oligo being less subject to degradation by nucleases and increasing its affinity for the target mRNA. These oligos can be further modified by introducing a methyl group in position 5 on cytidine (5'-methyl-dC). This modification combined with 2'-O-methyl further increases the stability and specificity of the oligo by increasing Tm, and also decreasing unpleasant immune responses when administered in vivo.
An alternative to antisense are i small interfering RNA (siRNA) : for more info go to the page Oligo a RNA
Standard Portfolio for Antisense Oligos (PTO / 2'-O-Methyl-RNA)