User:Mpuar/Oligonucleotides

Synthesis
The mechanism of action of antisense oligonucleotides within a cell can be split into three phases: prehybridization, hybridization, and post hybridization. In prehybridization antisense oligonucleotides enter and distribute within the cell and concentrate at the target RNA site. Then the antisense oligonucleotides have to locate the correct region of RNA that they will hybridize with. They hybridization process involves interactions with proteins, such as Ago2 as well as other intracellular materials (Crooke 2017). The individual steps of the hybridization process are still unclear, and a topic of continued research (Crooke 2017). It is known that double stranded siRNA and single strand siRNA organize the antisense oligonucleotides, and help in the binding process between the antisense oligonucleotides and the target RNA. Target RNA structures are the main factors that determine hybridization, while proteins bound to the RNA have an effect only in rare cases. Post hybridization processes include a number of chemical mechanisms that degrade, disable, or modify the target RNA to cause the desired pharmacological effect of the therapy. The details of the post hybridization mechanisms are also a research interest of scientists in the field (Crooke 2017).

Chemical analysis

 * Chromatography

Alkylamides can be used as chromatographic stationary phases. Those phases have been investigated for the separation of oligonucleotides.


 * Mass spectrometry

A mixture of 5-methoxysalicylic acid and spermine can be used as a matrix for oligonucleotides analysis in MALDI mass spectrometry.

Creating chemically stable short oligonucleotides was the earliest challenge in developing antisense oligonucleotide therapies. Naturally occurring oligonucleotides are easily degraded by nucleases, an enzyme that cleaves nucleotides and is ample in every cell type. Short oligonucleotides also have weak intrinsic binding affinities, which contributes to their easy degradation (DeVos 2013). It was found that nucleoside phosphorothioates analogs of nucleotides give them some beneficial properties such as diastereomer identification of each nucleotide, reactions involving phosphorothioate nucleotides can be easily followed, and the modification can be performed on almost all nucleosides (Eckstein 1985). The phosphorothioate backbone modifications are the most widely used method to protect antisense oligonucleotides from degradation. As seen in Figure 1, there is a large scope of modifications that can be made to the backbone beyond simple phosphorothioation. The modification also interacts with the enzyme RNaseH, which promotes the cleavage of RNA at specific targets. This property is very useful when applying antisense oligonucleotides to modify disease genes. Furthermore phosphorothioate modifications help cells intake the antisense oligonucleotides more effectively by aiding the attachment of antisense oligonucleotides to plasma proteins.