Antisense Oligonucleotides (AONS)
Control of gene expression is a central theme of gene expression; since the pioneering work of Zamenick et. Al where is shown that a complimentary oligonuicleotide near the ribosomal binding site of a mRNA, can bind and arrest translation. Since a series of modifications around the native phosphodiester bond have been developed; these modified oligos are nuclease resistant and some exhibit better stability.
This is a summary:
- Phosphorothioate oligos
- 2’O methyl Oligos
- 2’-5’ linked Oligos
- Morpholino Oligos
- Locked Nucleic Acids (LNA) Oligos
- Bridged Nucleic Acids (BNA) oligos
- Phosphorothioates S-Oligos
Due to the abundant presence of DNAse’s in the cellular environment the degradation of natural oligos turned to be a challenge.
Introducing a sulfur atom in place of one of the non-bridging oxygen confers nuclease resistance to the oligomer. S-Oligos are about 20 bases in length; whereas it is possible to substitute every position of the oligomer with sulfur atoms, this diminishes its solubility; a suggested strategy is to substitute only the 3 outermost positions of the oligo( that is the first 3 and the last 3 positions) this way solubility is still good and nuclease protection is built in.
Antisense Oligonucleotides (AONs)
Bio-Synthesis has over 23 years of experience in oligo research and applications and our staff can assist you with the optimal design for your antisense research.
Key Features in AONs
Fundamentally AONs should possess the following features:
- AONs must have good stability extra and intracellularly, that is must be nuclease resistant
- AON’s must have good penetrating ability, that is the ability to translocate across the cell membrane.
- AONs must have good selectivity and strong binding towards the intended target.
Nuclease Stability
The internucleotide linkages, ie the phosphodiester link is made nuclease resistant by a variety of modifications, ie:
- Oxygen replaced by sulfur(S oligos), morpholino, ethyl/methylphosphonates
- Locked Nucleic acids (LNAs*) where the carbon 2 is linked directly to carbon 4 of the base
- Bridged Nucleic acids (BNAs*) six-member bridged structure with an N-O linkage. This novel nucleic acid analogue can be synthesized and incorporated into oligonucleotides. when compare BNA with PNA, BNA has better base-pair stacking and specificity.
Membrane Translocation
Uptake of the AONs can be accomplished by:
- Linking of “Trojan Peptides”, these are 10-20 positively charged peptides that can be attached to the amino terminus and facilitate transport of the AON’s
- A variety of transfection agents( generally cationic reagents that permeabilize the cell membrane)
- The use of C and U propyne analogs (pdU and pdC)
Binding Affinity and Specificity
This involves modifiying the exocyclic bases, involved in Watson-Crick base pairing.
The most versatile approach is by use of bridged nucleic acid. BNA analogs have been shown to bind tighter than normal bases than PNA, thereby allowing shorter probe size and lesser nucleotide modifications.
There are of course the S oligos modifications, 2’-Ome-RNA,C5 methylated pyrimidines and other base modified analogs which confer higher binding/affinity than native oligos.