Oligonucleotide

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 bits 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. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression (e.g. microRNA), or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

Oligonucleotides are characterized by the sequence of nucleotide residues that make up the entire molecule. The length of the oligonucleotide is usually denoted by "-mer" (from Greek meros, "part"). For example, an oligonucleotide of six nucleotides (nt) is a hexamer, while one of 25 nt would usually be called a "25-mer". Oligonucleotides readily bind, in a sequence-specific manner, to their respective complementary oligonucleotides, DNA, or RNA to form duplexes or, less often, hybrids of a higher order. 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, 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. These modifications give new properties to the oligonucleotides and make them a key element in antisense therapy.[2][3]

Synthesis
Main article: 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).[4] 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.

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.[5] Short oligonucleotide sequences also have weak intrinsic binding affinities, which contributes to their degradation in vivo.[6]

Backbone modifications

Nucleoside organothiophosphate (PS) analogs of nucleotides give oligonucleotides some beneficial properties. Key beneficial properties that PS backbones gives nucleotides are diastereomer identification of each nucleotide and the ability to easily follow reactions involving the phosphorothioate nucleotides, which is useful in oligonucleotide synthesis.[7] PS backbone modifications to oligonucleotides protects them against unwanted degradation by enzymes.[8] Modifying the nucleotide backbone is widely used because it can be achieved with relative ease and accuracy on most nucleotides.[7]

Sugar ring modifications

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.[6] They also decrease non specific protein binding, increasing the accuracy of targeting specific proteins.[6] Two of the most commonly used modifications are 2’-O-methyl and the 2’-O-methoxyethyl.[6]

Antisense oligonucleotides
Main article: Antisense therapy

Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence.[4] In the case of antisense RNA they prevent protein translation of certain messenger RNA strands by binding to them, in a process called hybridization.[9] 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.[9] RNases H is an enzyme that hydrolyzes RNA, and when used in an antisense oligonucleotide application results in 80-95% down-regulation of mRNA expression.[4]

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.[10] The antisense oligonucleotides have also been used to inhibit influenza virus replication in cell lines.[11][12]

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.[13]

Analytical techniques

Chromatography

Alkylamides can be used as chromatographic stationary phases.[14] Those phases have been investigated for the separation of oligonucleotides.[15]

Mass spectrometry

A mixture of 5-methoxysalicylic acid and spermine can be used as a matrix for oligonucleotides analysis in MALDI mass spectrometry.[16]

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.[17] One subtype of DNA microarrays can be described as substrates (nylon, glass, etc.) to which oligonucleotides have been bound at high density.[18] There are a number of applications of DNA microarrays within the life sciences.

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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
  1. Yang J, Stolee JA, Jiang H, Xiao L, Kiesman WF, Antia FD, et al. (October 2018). "Solid-Phase Synthesis of Phosphorothioate Oligonucleotides Using Sulfurization Byproducts for in Situ Capping". The Journal of Organic Chemistry. 83 (19): 11577–11585. doi:10.1021/acs.joc.8b01553. PMID 30179468.
  2. Weiss, B., ed. (1997). Antisense Oligodeoxynucleotides and Antisense RNA : Novel Pharmacological and Therapeutic Agents. Boca Raton, Florida: CRC Press
  3. Weiss B, Davidkova G, Zhou LW (1999). "Antisense RNA gene therapy for studying and modulating biological processes". Cellular and Molecular Life Sciences. 55 (3): 334–58. doi:10.1007/s000180050296. PMID 10228554.
  4. Dias N, Stein CA (March 2002). "Antisense oligonucleotides: basic concepts and mechanisms". Molecular Cancer Therapeutics. 1 (5): 347–55. PMID 12489851.
  5. Frazier KS (January 2015). "Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic pathologist's perspective". Toxicologic Pathology. 43 (1): 78–89. doi:10.1177/0192623314551840. PMID 25385330.
  6. DeVos SL, Miller TM (July 2013). "Antisense oligonucleotides: treating neurodegeneration at the level of RNA". Neurotherapeutics. 10 (3): 486–97. doi:10.1007/s13311-013-0194-5. PMC 3701770. PMID 23686823.
  7. Eckstein F (April 2000). "Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them?". Antisense & Nucleic Acid Drug Development. 10 (2): 117–21. doi:10.1089/oli.1.2000.10.117. PMID 10805163.
  8. Stein CA, Subasinghe C, Shinozuka K, Cohen JS (April 1988). "Physicochemical properties of phosphorothioate oligodeoxynucleotides". Nucleic Acids Research. 16 (8): 3209–21. doi:10.1093/nar/16.8.3209. PMC 336489. PMID 2836790.
  9. Crooke ST (April 2017). "Molecular Mechanisms of Antisense Oligonucleotides". Nucleic Acid Therapeutics. 27 (2): 70–77. doi:10.1089/nat.2016.0656. PMC 5372764. PMID 28080221.
  10. Heasman J, Kofron M, Wylie C (June 2000). "Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach". Developmental Biology. 222 (1): 124–34. doi:10.1006/dbio.2000.9720. PMID 10885751.
  11. Kumar P, Kumar B, Rajput R, Saxena L, Banerjea AC, Khanna M (November 2013). "Cross-protective effect of antisense oligonucleotide developed against the common 3' NCR of influenza A virus genome". Molecular Biotechnology. 55 (3): 203–11. doi:10.1007/s12033-013-9670-8. PMID 23729285.
  12. Kumar B, Khanna M, Kumar P, Sood V, Vyas R, Banerjea AC (May 2012). "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". Molecular Biotechnology. 51 (1): 27–36. doi:10.1007/s12033-011-9437-z. PMID 21744034.
  13. Smith RA, Miller TM, Yamanaka K, Monia BP, Condon TP, Hung G, et al. (August 2006). "Antisense oligonucleotide therapy for neurodegenerative disease". The Journal of Clinical Investigation. 116 (8): 2290–6. doi:10.1172/JCI25424. PMC 1518790. PMID 16878173.
  14. Buszewski B, Kasturi P, Gilpin RK, Gangoda ME, Jaroniec M (August 1994). "Chromatographic and related studies of alkylamide phases". Chromatographia. 39 (3–4): 155–61. doi:10.1007/BF02274494.
  15. Buszewski B, Safaei Z, Studzińska S (January 2015). "Analysis of oligonucleotides by liquid chromatography with alkylamide stationary phase". Open Chemistry. 13 (1). doi:10.1515/chem-2015-0141.
  16. Distler AM, Allison J (April 2001). "5-Methoxysalicylic acid and spermine: a new matrix for the matrix-assisted laser desorption/ionization mass spectrometry analysis of oligonucleotides". Journal of the American Society for Mass Spectrometry. 12 (4): 456–62. doi:10.1016/S1044-0305(01)00212-4. PMID 11322192.
  17. Relógio A, Schwager C, Richter A, Ansorge W, Valcárcel J (June 2002). "Optimization of oligonucleotide-based DNA microarrays". Nucleic Acids Research. 30 (11): e51. doi:10.1093/nar/30.11.e51. PMC 117213. PMID 12034852.
  18. Gong P, Harbers GM, Grainger DW (April 2006). "Multi-technique comparison of immobilized and hybridized oligonucleotide surface density on commercial amine-reactive microarray slides". Analytical Chemistry. 78 (7): 2342–51. doi:10.1021/ac051812m. 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. 10. Springer Science & Business Media. pp. 103–118. doi:10.1007/978-94-007-2172-2_3.
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