oligonucleotide#Antisense oligonucleotides

{{Main|oligonucleotide 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).{{cite journal | vauthors = Dias N, Stein CA | title = Antisense oligonucleotides: basic concepts and mechanisms | journal = Molecular Cancer Therapeutics | volume = 1 | issue = 5 | pages = 347–55 | date = March 2002 | pmid = 12489851 | url = https://mct.aacrjournals.org/content/1/5/347 }} 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.{{cn|date=February 2024}}

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.{{cite journal | vauthors = Frazier KS | title = Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic pathologist's perspective | journal = Toxicologic Pathology | volume = 43 | issue = 1 | pages = 78–89 | date = January 2015 | pmid = 25385330 | doi = 10.1177/0192623314551840 | s2cid = 37981276 }} Short oligonucleotide sequences also have weak intrinsic binding affinities, which contributes to their degradation in vivo.{{cite journal | vauthors = DeVos SL, Miller TM | title = Antisense oligonucleotides: treating neurodegeneration at the level of RNA | journal = Neurotherapeutics | volume = 10 | issue = 3 | pages = 486–97 | date = July 2013 | pmid = 23686823 | pmc = 3701770 | doi = 10.1007/s13311-013-0194-5 }}

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.{{cite journal | vauthors = Eckstein F | title = Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them? | journal = Antisense & Nucleic Acid Drug Development | volume = 10 | issue = 2 | pages = 117–21 | date = April 2000 | pmid = 10805163 | doi = 10.1089/oli.1.2000.10.117 }} PS backbone modifications to oligonucleotides protects them against unwanted degradation by enzymes.{{cite journal | vauthors = Stein CA, Subasinghe C, Shinozuka K, Cohen JS | title = Physicochemical properties of phosphorothioate oligodeoxynucleotides | journal = Nucleic Acids Research | volume = 16 | issue = 8 | pages = 3209–21 | date = April 1988 | pmid = 2836790 | pmc = 336489 | doi = 10.1093/nar/16.8.3209 }} Modifying the nucleotide backbone is widely used because it can be achieved with relative ease and accuracy on most nucleotides. Fluorescent modifications on 5' and 3' end of oligonucleotides was reported to evaluate the oligonucleotides structures, dynamics and interactions with respect to environment.{{cite journal | vauthors = Michel BY, Dziuba D, Benhida R, Demchenko AP, Burger A | title = Probing of Nucleic Acid Structures, Dynamics, and Interactions With Environment-Sensitive Fluorescent Labels | language = English | journal = Frontiers in Chemistry | volume = 8 | pages = 112 | date = 2020 | pmid = 32181238 | pmc = 7059644 | doi = 10.3389/fchem.2020.00112 | bibcode = 2020FrCh....8..112M | doi-access = free }}

Another modification that is useful for medical applications of oligonucleotides is 2' sugar modifications. Modifying the 2' position sugar increases the effectiveness of oligonucleotides by enhancing the target binding capabilities of oligonucleotides, specifically in antisense oligonucleotides therapies. They also decrease non specific protein binding, increasing the accuracy of targeting specific proteins. Two of the most commonly used modifications are 2'-O-methyl and the 2'-O-methoxyethyl. Fluorescent modifications on the nucleobase was also reported.

{{main|Antisense therapy}}

Antisense oligonucleotides (ASO) are single strands of DNA or RNA that are complementary to a chosen sequence. In the case of antisense RNA they prevent protein translation of certain messenger RNA strands by binding to them, in a process called hybridization.{{cite journal | vauthors = Crooke ST | title = Molecular Mechanisms of Antisense Oligonucleotides | journal = Nucleic Acid Therapeutics | volume = 27 | issue = 2 | pages = 70–77 | date = April 2017 | pmid = 28080221 | pmc = 5372764 | doi = 10.1089/nat.2016.0656 }} Antisense oligonucleotides can be used to target a specific, complementary (coding or non-coding) RNA. If binding takes place this hybrid can be degraded by the enzyme RNase H. RNase H is an enzyme that hydrolyzes RNA, and when used in an antisense oligonucleotide application results in 80-95% down-regulation of mRNA expression.

The use of Morpholino antisense oligonucleotides for gene knockdowns in vertebrates, which is now a standard technique in developmental biology and is used to study altered gene expression and gene function, was first developed by Janet Heasman using Xenopus.{{cite journal | vauthors = Heasman J, Kofron M, Wylie C | title = Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach | journal = Developmental Biology | volume = 222 | issue = 1 | pages = 124–34 | date = June 2000 | pmid = 10885751 | doi = 10.1006/dbio.2000.9720 | doi-access = free }} FDA-approved Morpholino drugs include eteplirsen and golodirsen. The antisense oligonucleotides have also been used to inhibit influenza virus replication in cell lines.{{cite journal | vauthors = Kumar P, Kumar B, Rajput R, Saxena L, Banerjea AC, Khanna M | title = Cross-protective effect of antisense oligonucleotide developed against the common 3' NCR of influenza A virus genome | journal = Molecular Biotechnology | volume = 55 | issue = 3 | pages = 203–11 | date = November 2013 | pmid = 23729285 | doi = 10.1007/s12033-013-9670-8 | s2cid = 24496875 }}{{cite journal | vauthors = Kumar B, Khanna M, Kumar P, Sood V, Vyas R, Banerjea AC | title = Nucleic acid-mediated cleavage of M1 gene of influenza A virus is significantly augmented by antisense molecules targeted to hybridize close to the cleavage site | journal = Molecular Biotechnology | volume = 51 | issue = 1 | pages = 27–36 | date = May 2012 | pmid = 21744034 | doi = 10.1007/s12033-011-9437-z | s2cid = 45686564 }}

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. Many genetic diseases including Huntington's disease, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) have been linked to DNA alterations that result in incorrect RNA sequences and result in mistranslated proteins that have a toxic physiological effect.{{cite journal | vauthors = Smith RA, Miller TM, Yamanaka K, Monia BP, Condon TP, Hung G, Lobsiger CS, Ward CM, McAlonis-Downes M, Wei H, Wancewicz EV, Bennett CF, Cleveland DW | display-authors = 6 | title = Antisense oligonucleotide therapy for neurodegenerative disease | journal = The Journal of Clinical Investigation | volume = 116 | issue = 8 | pages = 2290–6 | date = August 2006 | pmid = 16878173 | doi = 10.1172/JCI25424 | doi-access = free | pmc = 1518790 }}

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.{{Cite journal |last1=Ming |first1=Xin |last2=Alam |first2=Md Rowshon |last3=Fisher |first3=Michael |last4=Yan |first4=Yongjun |last5=Chen |first5=Xiaoyuan |last6=Juliano |first6=Rudolph L. |date=2010-06-15 |title=Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor |url=http://dx.doi.org/10.1093/nar/gkq534 |journal=Nucleic Acids Research |volume=38 |issue=19 |pages=6567–6576 |doi=10.1093/nar/gkq534 |pmid=20551131 |issn=1362-4962|pmc=2965246 }} 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.{{Cite journal |last1=Hawner |first1=Manuel |last2=Ducho |first2=Christian |date=2020-12-16 |title=Cellular Targeting of Oligonucleotides by Conjugation with Small Molecules |journal=Molecules |volume=25 |issue=24 |pages=5963 |doi=10.3390/molecules25245963 |pmid=33339365 |pmc=7766908 |issn=1420-3049|doi-access=free }} 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.{{Cite journal |last=Crooke |first=S. T. |date=2017 |title=Cellular uptake and trafficking of antisense oligonucleotides |url=https://pubmed.ncbi.nlm.nih.gov/28244996/ |journal=Nat. Biotechnol. |volume=35 |issue=3 |pages=230–237|doi=10.1038/nbt.3779 |pmid=28244996 |s2cid=1049452 }}

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. This has been achieved with small molecule-ON conjugates for example bearing an N-acetyl galactosamine which targets receptors of hepatocytes.{{Cite journal |last1=Prakash |first1=Thazha P. |last2=Graham |first2=Mark J. |last3=Yu |first3=Jinghua |last4=Carty |first4=Rick |last5=Low |first5=Audrey |last6=Chappell |first6=Alfred |last7=Schmidt |first7=Karsten |last8=Zhao |first8=Chenguang |last9=Aghajan |first9=Mariam |last10=Murray |first10=Heather F. |last11=Riney |first11=Stan |last12=Booten |first12=Sheri L. |last13=Murray |first13=Susan F. |last14=Gaus |first14=Hans |last15=Crosby |first15=Jeff |date=July 2014 |title=Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice |journal=Nucleic Acids Research |volume=42 |issue=13 |pages=8796–8807 |doi=10.1093/nar/gku531 |issn=1362-4962 |pmc=4117763 |pmid=24992960}} 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). Another broadly used and heavily investigated entity for targeted delivery and increased cell uptake of oligonucleotides are antibodies.

Alkylamides can be used as chromatographic stationary phases.{{cite journal | vauthors = Buszewski B, Kasturi P, Gilpin RK, Gangoda ME, Jaroniec M | title = Chromatographic and related studies of alkylamide phases. | journal = Chromatographia | date = August 1994 | volume = 39 | issue = 3–4 | pages = 155–61 | doi = 10.1007/BF02274494 | s2cid = 97825477 }} Those phases have been investigated for the separation of oligonucleotides.{{cite journal | vauthors = Buszewski B, Safaei Z, Studzińska S | title = Analysis of oligonucleotides by liquid chromatography with alkylamide stationary phase. | journal = Open Chemistry | date = January 2015 | volume = 13 | issue = 1 | doi = 10.1515/chem-2015-0141 | doi-access = free }} Ion-pair reverse-phase high-performance liquid chromatography is used to separate and analyse the oligonucleotides after automated synthesis.{{Cite journal|date=2002-06-07|title=Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction|url=https://www.sciencedirect.com/science/article/abs/pii/S0021967302003060|journal=Journal of Chromatography A|language=en|volume=958|issue=1–2|pages=167–182|doi=10.1016/S0021-9673(02)00306-0|pmid=12134814 |issn=0021-9673|last1=Gilar |first1=M. |last2=Fountain |first2=K. J. |last3=Budman |first3=Y. |last4=Neue |first4=U. D. |last5=Yardley |first5=K. R. |last6=Rainville |first6=P. D. |last7=Russell Rj |first7=2nd |last8=Gebler |first8=J. C. |url-access=subscription }}

A mixture of 5-methoxysalicylic acid and spermine can be used as a matrix for oligonucleotides analysis in MALDI mass spectrometry.{{cite journal | vauthors = Distler AM, Allison J | title = 5-Methoxysalicylic acid and spermine: a new matrix for the matrix-assisted laser desorption/ionization mass spectrometry analysis of oligonucleotides | journal = Journal of the American Society for Mass Spectrometry | volume = 12 | issue = 4 | pages = 456–62 | date = April 2001 | pmid = 11322192 | doi = 10.1016/S1044-0305(01)00212-4 | bibcode = 2001JASMS..12..456D | s2cid = 18280663 }} ElectroSpray Ionization Mass Spectrometry (ESI-MS) is also a powerful tool to characterize the mass of oligonucleotides.{{cite journal | vauthors = Shah S, Friedman SH | title = An ESI-MS method for characterization of native and modified oligonucleotides used for RNA interference and other biological applications | journal = Nature Protocols | volume = 3 | issue = 3 | pages = 351–6 | date = March 2008 | pmid = 18323805 | doi = 10.1038/nprot.2007.535 | s2cid = 2093309 }}

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.{{cite journal | vauthors = Relógio A, Schwager C, Richter A, Ansorge W, Valcárcel J | title = Optimization of oligonucleotide-based DNA microarrays | journal = Nucleic Acids Research | volume = 30 | issue = 11 | pages = 51e–51 | date = June 2002 | pmid = 12034852 | pmc = 117213 | doi = 10.1093/nar/30.11.e51 }} One subtype of DNA microarrays can be described as substrates (nylon, glass, etc.) to which oligonucleotides have been bound at high density.{{cite journal | vauthors = Gong P, Harbers GM, Grainger DW | title = Multi-technique comparison of immobilized and hybridized oligonucleotide surface density on commercial amine-reactive microarray slides | journal = Analytical Chemistry | volume = 78 | issue = 7 | pages = 2342–51 | date = April 2006 | pmid = 16579618 | doi = 10.1021/ac051812m }} There are a number of applications of DNA microarrays within the life sciences.{{cn|date=February 2024}}

• 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.

{{Reflist}}

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• {{cite book | vauthors = Spingler B | veditors = Sigel A, Sigel H, Sigel RK | title = Interplay between metal ions and nucleic acids. | publisher = Springer Science & Business Media | date = January 2012 |volume=10 |doi=10.1007/978-94-007-2172-2_3 |pages=103–118|chapter=Chapter 3. Metal-Ion-Promoted Conformational Changes of Oligonucleotides | journal = Metal Ions in Life Sciences | pmid = 22210336 }}

{{refend}}

• [https://web.archive.org/web/20150210230607/http://rnaiatlas.ethz.ch/ RNAi Atlas]: a database of RNAi libraries and their target analysis results
• [http://www.physorg.com/news166974009.html physorg.com | Genetic source of muscular dystrophy neutralized]

{{Nucleic acids}}

• [https://www.sciencedirect.com/science/article/abs/pii/S0022354924001527?fr=RR-9&ref=pdf_download&rr=882ea24ccf6b15b8 A Review on Commercial Oligonucleotide Drug Products]

Category:Nucleic acids