Antisense/ASO, siRNA & miRNA Design and Protocols

Antisense/ASO, siRNA & miRNA Design / Protocol

Success or failure of an anti-sense experiment fundamentally depends on first selecting the right target sequence within the particular mRNA of interest. The anti-sense oligo, along with its appropriate chemical modifications, is then designed around that sequence. The following should be taken into consideration when selecting the mRNA target sequence:

Anti-sense oligos typically should be about 20 bases long; such oligos are easy to synthesize, form stable DNA-RNA duplexes, and are long enough to be unique, at least in the human genome. Uniqueness is important; it is critical that the anti-sense oligo not bind, even partially, to a non-target mRNA. If as few as 6-7 base pairs are formed between the anti-sense oligo and non-target mRNA, that likely would be sufficient to initiate RNase H activity, leading to cleavage of the wrong target.

Whatever sequence within the mRNA is chosen, it must be accessible to binding by an oligo. Thus, the secondary and tertiary structure of the RNA must be taken into consideration to minimize the possibility that the chosen sequence is inaccessible to binding. The use of a robust RNA folding program (e.g., Sfold or mfold) to assist with this aspect of anti-sense oligo design is highly recommended.

Generally speaking, RNA sequences involved in binding to proteins, ribosomes or spliceosomes tend to be accessible for binding to oligos. In particular, targeting either the methionine (AUG) initiation codon (to block translation) or splice sites (to block splicing) are often good strategies. However, note that, in the latter case, the anti-sense oligo must be able to enter the nucleus.

Because unmethylated CG motifs are common in bacterial, but not eukaryotic, DNA, their presence in an anti-sense oligo may trigger an immune response in in vivo experiments if the organism's immune system interprets it as a bacterial infection. CG-mediated immune response is particularly strong when the CG sequence is embedded as part of a purine-purine-C-G-pyrimidine-pyrimidine sequence. One way to avoid this problem is to be careful to choose oligos that either lack CG, or at least lack the above flanking sequences around a CG. If elimination of CG is not possible, then a good alternative is to replace the C in CG with 5-methyl-C, which does not stimulate the immune system or deleteriously affect hybridization.

Anti-sense oligos containing either single GGGG runs or repeated GG or GGG runs in close proximity can form intra-strand tetraplexes (single structures of four strands). G tetraplexes often have high affinity for proteins, which can result in potent, non-antisense biological effects that may interfere with an anti-sense experiment, particularly when such effects mimic anti-sense activity. Whenever possible, such G motifs should be avoided. When elimination of such motifs is unavoidable, then a good alternative is to replace one or more of the Gs with 7-deaza-G or 6-thio-G, which block G-tetraplex formation.

Several studies have conclusively shown that the activity of an anti-sense oligo against its mRNA target is sequence-motif content-dependent. A major study of over 1000 phosphorothiolated anti-sense oligos showed that the presence of motifs CCAC, TCCC, ACTC, GCCA, and CTCT positively correlated with anti-sense activity, while GGGG, ACTG, TAA, CCGG, and AAA negatively correlated with anti-sense activity.

References

(1) Sazani, P., Kole, R. Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing. J. Clin. Invest. (2003), 112: 481-486.
(2) Juliano, R., Alam, Md.R., Dixit, V., Kang, H. Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. (2008), 36: 4158-4171.
(3) Chan, J.H., Lim, S., Wong, W.S. Antisense oligonucleotides: from design to therapeutic applications. Clin. Exp. Pharmacol. Physiol. (2006), 33: 533-540.
(4) Kurreck, J. Antisense technologies. Improvement through novel chemical modifications. Eur. J. Biochem. (2003), 270: 1628-1644.
(5) Crooke, S.T. Progress in antisense technology. Annu. Rev. Med. (2004), 55: 61-95.