Antisense oligonucleotides have been used for over twenty years to inhibit gene expression levels both in vitro and in vivo. Antisense technology has opened a new approach to the development of drug therapeutics. Antisense drugs are designed to inhibit the production of disease-causing proteins and offer the potential to be highly selective in their action and less toxic than traditional drugs.
Antisense oligonucleotides, either as synthetic DNA or RNA, are oligonucleotides which have been modified in various ways. The purpose of this modification is to prevent oligonucleotide degradation within cells, and to increase affinity to the specific target mRNA.
Recent improvements in design and chemistry of antisense compounds have enabled this technology to become a routinely used tool in basic research, genomics, target validation and drug discovery. It is becoming increasingly popular to confirm phenotypes after use of RNAi by gene silencing antisense DNA oligos. A nucleic acid sequence, usually 18-25 bases long, is designed in antisense orientation to the mRNA of interest; the sequence is made as a synthetic oligonucleotide and is introduced into the cell or organism. Hybridization of the antisense oligo to the target mRNA results in RNase H cleavage of the message which prevents protein translation and thereby blocks gene expression. Antisense oligonucleotides containing a native DNA or phosphorothioate-modified DNA segment of at least six bases long will bind the target mRNA and form an RNA/DNA heteroduplex, which is a substrate for endogenous cellular RNases H.1+2. The decrease in mRNA levels can be measured using real-time PCR.
While unmodified oligodeoxynucleotides can display some antisense activity, they are subject to rapid degradation by nucleases and are therefore of limited utility. The simplest and most widely used nuclease-resistant chemistry available for antisense applications is the phosphorothioate (S-oligos) modification. In phosphorothioates, a sulfur atom replaces a non-bridging oxygen in the oligo phosphate backbone.
S-oligos can show greater non-specific protein binding than unmodified phosphodiester oligos, which can cause toxicity or other artifacts when present at high concentrations. These problems can be reduced or eliminated using chimeric designs. A phosphorothioate/phosphodiester chimera generally has one to four S-modified internucleoside linkages on both the 5'- and 3'-ends with a central core of unmodified DNA.
Current chimeric antisense oligo design combines the use of DNA with 2'-O-Methyl RNA bases and the replacement of dC with 5-Methyl-dC, especially in the context of a CpG motif. 2'-O-Methyl RNA increases both nuclease stability and affinity (Tm) of the antisense oligo to the target mRNA. 2'-O-Methyl RNA bases, however, do not activate RNase H cleavage. The preferred antisense design incorporates 2'-O-modified RNA in chimeric antisense oligos that retain an RNase H activating domain of DNA (or phosphorothioate DNA). Substitution of 5-Methyl dC for dC will slightly increase the Tm of the antisense oligo. Use of 5-Methyl dC in CpG motifs can also reduce the chance of adverse immune responses in vivo. metabion recommends that all antisense oligos receive HPLC purification and that oligos undergo a Na+ salt exchange before use in cells or live animals to ensure that salts used in purification are removed. Performing double purification by HPLC and gel filtration metabion's antisense oligos are ready-to-use for in vivo applications.
RNA interference (RNAi) is a new technique that is considered a major technology breakthrough. So exciting is it that it has attracted attention in the regular media as well as the scientific press. The big deal is that we can now design experiments that can target a specific gene and "knock it down" or "silence" it. This means that scientists can then study what happens when a gene is inactivated (e.g. as might happen in cancer or many other disease states).
This is done by designing small pieces of RNA (known as "short interfering RNAs" or "siRNAs") based on some known information about the gene's mRNA sequence. Since short sequences of nucleic acids are known as oligos these are often referred to as RNAi oligos.
These RNA's are then transfected into cells and the effects of "gene silencing" are analyzed.
The key to the success of the technique is designing the best RNAi oligos from what we know of the gene sequence. The gene may be several thousand bases in length and the oligos are only 21-23 long so there are many options.
metabion delivers highly purified antisense oligos mainly as
- Phosphorothioates (PTOs): increased cell uptake, nuclease resistance,
elicitation of RNAse H activity (click here for pricing) - 2'-O-Methyl RNA: resistance to a wide variety of ribo- and deoxyribonucleases, formation of more stable hybrids with complementary RNA strands than equivalent DNA and RNA sequences
- RNA/RNAi