After uptake into target cells antisense oligonucleotides bind to corresponding complementary mRNA, whereby the translation and also the biosynthesis of the target protein is specifically inhibited.
The complex of antisense DNA oligonucleotide and the bound RNA strand is recognised by RNase H, which enzymatically cleaves the RNA. As a result, the antisense DNA oligonucleotide is released and thus again available for binding complementary RNA.
Some critical aspects are the cellular uptake, as the negatively charged DNA can only poorly pass through cell membranes. Here, various lipophilic molecules (see modifications) have proven to be very helpful.
Furthermore, unprotected, short DNA oligonucleotides are rapidly degraded in cells. In order to prevent or at least slow down the degradation, in addition to phosphothioates various 2 ´-modifications are successfully used.
Phosphothioat-Oligonucleotide (Phosphorothioat, PTO)
The phosphorothioate modification of the DNA backbone is one of the first and still widely used modification for antisense oligonucleotides (antisense oligos of the first generation). In the phosphate backbone, one of the two oxygen atoms which is not involved in the internucleotide bridge, is replaced by a sulfur atom (Fig.). Due to the four different substituents, the central phosphorus atom is a chiral center which effects diastereomer formation.
In oligonucleotides, all (Full-PTO) or just single phosphates (Part-PTO) can be replaced by phosphorothioate. Often it seems to be sufficient to incorporate two to four PTOs at the end of the oligos to achieve the desired stability against degradation.
Phosphorothioates are presented in the order system of biomers.net by asterisks (*). If all or just single phosphodiester should be replaced by phosphorothioates, a * have to be inserted into the sequence at the respective position.
- A normal sequence without phosphorothioates:
- The identical sequence, but as a "full-PTO-oligo" (all inter-nucleotidic phosphodiester were changed to PTO):
a * g * c * t * g * g * a * c * t * g * g * g * a * c * t * g * g * a * g * t * g * a
- The sequence with single PTOs, in this case three PTO at both ends:
a * g * c * gtgacgtggacgtgag * t * g * a
- The sequence with a PTO-bridge at the 3 'end:
agcgtgacgtggacgtgagtg * a
If a PTO-Oligo should be modified at one end, for example with a fluorescent dye, it is often useful, to "stabilize" also the phosphate bridge between the oligo and the modification (synthesizing as a PTO).
At a 5'-modification, in this case a * is also added to the 5'-end of the oligos (at a 3'-modification according to the 3'-end) and the desired modification is selected in the menu:
- * a * g * c * t * g * g * a * c * t * g * g * g * a * c * t * g * g * a * g * t * g * a, 5'-modification: biotin: PTO between biotin and oligo
By definition it is a ribose if an oxygen is linked at 2´-carbon of the sugar. biomers.net offers here a selection of different modifications:
Antisense oligonucleotides having a modification at the 2'-hydroxyl group (2´-O-methyl, 2´-O-methoxyethyl), often show an increased stability to enzymatic degradation. This also facilitates an improved distribution of the oligos in the cell.1 Furthermore, it was observed that 2´-modified oligonucleotides (2´-O-methyl, 2´-O-methoxyethyl, 2´-O-propyl, 2´-O-fluoro) bind with greater affinity to complementary RNA.2
Chimeric oligonucleotides can be made from various types of building blocks. In contrast to naturally occurring molecules, synthetic oligos can simultaneously consist of DNA and RNA or PNA. It is also possible that these oligos are mixed from right- and left-handed components (see L-DNA, L-RNA, PNA). Furthermore, mixtures are possible with reverse building blocks, whereby molecules can be generated that terminate at both ends with 3 'or 5'.
- An Antisense Oligonucleotide Primer. Hogrefe RI; Antisense and Nucleic Acid Drug Development (1999), 9(4): 351-357.
Antisense Oligonucleotide Modifications
An ineffectual uptake of oligonucleotide conjugates to the cell often represent the greatest difficulty for a successful development of new oligonucleotide-based therapeutics.
In terms of antisense oligonucleotides, a wide variety of modifications are available. Matched to the researcher´s particular method, various internal or terminal changes of an oligonucleotide are possible. Small lipophilic molecules which can be attached to an oligonucleotide may facilitate the passage of the highly negatively charged DNA or RNA through the cell membrane and thus the uptake into the cell.
Small lipophilic molecules which can be attached to oligonucleotide, allow the passage through the cell membrane and thus the uptake into the cell of the highly negatively charged DNA or RNA. The attachment of cholesterol or tocopherol to DNA or RNA molecules may improve the stability and the distribution of the conjugates in the cell1 (Lipophilic Modifications).
Modifications which may also inhibit the degradation of the oligos by nucleases are inverted-ends. A further nucleotide is attached "upside down" to the 3'-terminal nucleotide so that the oligonucleotide has a 5'-terminus on both sides. Besides protection against digestion, this modification efficiently blocks elongation during PCR.