Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics.
Commonly made in the laboratory by solid-phase chemical synthesis,[1] these small fragments of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes.
This basic property serves as a foundation for the use of oligonucleotides as probes for detecting specific sequences of DNA or RNA.
Examples of procedures that use oligonucleotides include DNA microarrays, Southern blots, ASO analysis,[3] fluorescent in situ hybridization (FISH), PCR, and the synthesis of artificial genes.
Oligonucleotides are composed of 2'-deoxyribonucleotides (oligodeoxyribonucleotides), which can be modified at the backbone or on the 2' sugar position to achieve different pharmacological effects.
The oligonucleotide chain assembly proceeds in the 3' to 5' direction by following a routine procedure referred to as a "synthetic cycle".
A less than 100% yield of each synthetic step and the occurrence of side reactions set practical limits of the efficiency of the process.
[citation needed] Creating chemically stable short oligonucleotides was the earliest challenge in developing ASO therapies.
Naturally occurring oligonucleotides are easily degraded by nucleases, an enzyme that cleaves nucleotides and is ample in every cell type.
Notably, studies show that most tissue culture cells readily take up ASOs (phosphorothiote linkage) in a non-productive way, meaning that no antisense effect is observed.
In contrast to that conjugation of ASO with ligands recognised by G-coupled receptors leads to an increased productive uptake.
[17] Next to that classification (non-productive vs. productive), cell internalisation mostly proceeds in an energy-dependant way (receptor mediated endocytosis) but energy-independent passive diffusion (gymnosis) may not be ruled out.
[18] This has been achieved with small molecule-ON conjugates for example bearing an N-acetyl galactosamine which targets receptors of hepatocytes.