Oligonucleotide
Oligonucleotides are short
Oligonucleotides are characterized by the
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. These modifications give new properties to the oligonucleotides and make them a key element in antisense therapy.[4][5]
Synthesis
Oligonucleotides are chemically synthesized using building blocks, protected phosphoramidites of natural or chemically modified nucleosides or, to a lesser extent, of non-nucleosidic compounds. The oligonucleotide chain assembly proceeds in the 3' to 5' direction by following a routine procedure referred to as a "synthetic cycle". Completion of a single synthetic cycle results in the addition of one nucleotide residue to the growing chain. A less than 100% yield of each synthetic step and the occurrence of side reactions set practical limits of the efficiency of the process. In general, oligonucleotide sequences are usually short (13–25 nucleotides long).[6] The maximum length of synthetic oligonucleotides hardly exceeds 200 nucleotide residues. HPLC and other methods can be used to isolate products with the desired sequence.[citation needed]
Chemical modifications
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.[7] Short oligonucleotide sequences also have weak intrinsic binding affinities, which contributes to their degradation in vivo.[8]
Backbone modifications
Nucleoside organothiophosphate (PS) analogs of nucleotides give oligonucleotides some beneficial properties. Key beneficial properties that PS backbones give nucleotides are diastereomer identification of each nucleotide and the ability to easily follow reactions involving the phosphorothioate nucleotides, which is useful in oligonucleotide synthesis.[9] PS backbone modifications to oligonucleotides protects them against unwanted degradation by enzymes.[10] Modifying the nucleotide backbone is widely used because it can be achieved with relative ease and accuracy on most nucleotides.[9] Fluorescent modifications on 5' and 3' end of oligonucleotides was reported to evaluate the oligonucleotides structures, dynamics and interactions with respect to environment.[11]
Sugar ring modifications
Another modification that is useful for medical applications of oligonucleotides is
Antisense oligonucleotides
Antisense oligonucleotides (ASO) are single strands of DNA or RNA that are complementary to a chosen sequence.
The use of
Neurodegenerative diseases that are a result of a single mutant protein are good targets for antisense oligonucleotide therapies because of their ability to target and modify very specific sequences of RNA with high selectivity.
Cell internalisation
Cell uptake/internalisation still represents the biggest hurdle towards successful oligonucleotide (ON) therapeutics. A straightforward uptake, like for most small-molecule drugs, is hindered by the polyanionic backbone and the molecular size of ONs. The exact mechanisms of uptake and intracellular trafficking towards the place of action are still largely unclear. Moreover, small differences in ON structure/modification (vide supra) and difference in cell type leads to huge differences in uptake. It is believed that cell uptake occurs on different pathways after adsorption of ONs on the cell surface. 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. After passing the cell membrane, ON therapeutics are encapsulated in early endosomes which are transported towards late endosomes which are ultimately fused with lysosomes containing degrading enzymes at low pH.[18] To exert its therapeutic function, the ON needs to escape the endosome prior to its degradation. Currently there is no universal method to overcome the problems of delivery, cell uptake and endosomal escape, but there exist several approaches which are tailored to specific cells and their receptors.[19]
A conjugation of ON therapeutics to an entity responsible for cell recognition/uptake not only increases the uptake (vide supra) but is also believed to decrease the complexity of the cell uptake as mainly one (ideally known) mechanism is then involved.[18] This has been achieved with small molecule-ON conjugates for example bearing an N-acetyl galactosamine which targets receptors of hepatocytes.[20] These conjugates are an excellent example for obtaining an increased cell uptake paired with targeted delivery as the corresponding receptors are overexpressed on the target cells leading to a targeted therapeutic (compare antibody-drug conjugates which exploit overexpressed receptors on cancer cells).[19] Another broadly used and heavily investigated entity for targeted delivery and increased cell uptake of oligonucleotides are antibodies.
Analytical techniques
Chromatography
Alkylamides can be used as chromatographic stationary phases.[21] Those phases have been investigated for the separation of oligonucleotides.[22] Ion-pair reverse-phase high-performance liquid chromatography is used to separate and analyse the oligonucleotides after automated synthesis.[23]
Mass spectrometry
A mixture of
DNA microarray
DNA microarrays are a useful analytical application of oligonucleotides. Compared to standard cDNA microarrays, oligonucleotide based microarrays have more controlled specificity over hybridization, and the ability to measure the presence and prevalence of alternatively spliced or polyadenylated sequences.[26] One subtype of DNA microarrays can be described as substrates (nylon, glass, etc.) to which oligonucleotides have been bound at high density.[27] There are a number of applications of DNA microarrays within the life sciences.[citation needed]
See also
- Aptamers, oligonucleotides with important biological applications
- Morpholinos, oligos with non-natural backbones, which do not activate RNase-H but can reduce gene expression or modify RNA splicing
- Polymorphism, the appearance in a population of the same gene in multiple forms because of mutations; can often be tested with ASO probes
- CpG Oligodeoxynucleotide, an ODN with immunostimulatory properties
- Polypurine reverse-Hoogsteen hairpins, PPRHs, oligonucleotides that can bind either DNA or RNA and decrease gene expression.
References
- S2CID 52157806.
- PMID 25380780.
- ^ PMID 28696921.
- ^ Weiss, B., ed. (1997). Antisense Oligodeoxynucleotides and Antisense RNA : Novel Pharmacological and Therapeutic Agents. Boca Raton, Florida: CRC Press
- S2CID 9448271.
- ^ PMID 12489851.
- S2CID 37981276.
- ^ PMID 23686823.
- ^ PMID 10805163.
- PMID 2836790.
- ^ PMID 32181238.
- ^ PMID 28080221.
- PMID 10885751.
- S2CID 24496875.
- S2CID 45686564.
- PMID 16878173.
- PMID 20551131.
- ^ PMID 33339365.
- ^ S2CID 1049452.
- PMID 24992960.
- S2CID 97825477.
- .
- PMID 12134814.)
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: CS1 maint: numeric names: authors list (link - S2CID 18280663.
- S2CID 2093309.
- PMID 12034852.
- PMID 16579618.
Further reading
- Spingler B (January 2012). "Chapter 3. Metal-Ion-Promoted Conformational Changes of Oligonucleotides". In Sigel A, Sigel H, Sigel RK (eds.). Interplay between metal ions and nucleic acids. Vol. 10. Springer Science & Business Media. pp. 103–118. )
External links
- RNAi Atlas: a database of RNAi libraries and their target analysis results
- physorg.com | Genetic source of muscular dystrophy neutralized