oligonucleotide
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oligonucleotide, a short chain of nucleotides (nitrogen-containing units linked to a sugar and a phosphate group) that consist of either single- or double-stranded DNA or RNA.
Characteristics
Oligonucleotides originally were used almost exclusively in laboratory research, being synthesized and used generally as molecular probes to detect complementary DNA or RNA sequences. Today they are especially important in the realm of drug development, owing to their ability to influence gene expression once bound to a target sequence; often such agents are antisense oligonucleotides (ASOs), single- or double-stranded sequences designed to selectively bind to and alter the function of target messenger RNA (mRNA) molecules. Oligonucleotides also occur naturally, in the form of microRNA and as breakdown products of longer nucleotide sequences.
In general, oligonucleotides are 20 nucleotides or fewer in length. However, they can consist of as many as 100 or even 200 nucleotides. Their length, which is denoted by the suffix -mer (e.g., 20-mer), is dictated largely by the length of the desired target sequence and by the application for which they are used. Oligonucleotides attach to their respective complementary DNA or RNA targets in a sequence-specific manner, and, because they have the properties of complementarity and sequence specificity and can be manipulated easily, they are versatile and relevant for a wide variety of molecular biology techniques. Oligonucleotides also possess a high degree of chemical stability and are therefore convenient not only for research applications but also for use as diagnostic tools and as therapeutic agents.
Applications in research and medicine
An example of the use of oligonucleotides in research is in the form of primers for the polymerase chain reaction (PCR), which was developed in 1983 by Kary B. Mullis, an American biochemist who won the Nobel Prize for Chemistry in 1993 for his invention. PCR, which is used for cloning and sequencing genes, relies on primers that typically are 18–22 nucleotides in length and are designed to match a target sequence. Oligonucleotides are likewise used for quantitative PCR (qPCR), an application of which is the rapid detection of infectious agents, such as SARS-CoV-2 (the cause of COVID-19). Other research applications of oligonucleotides include in situ hybridization and microarrays, which analyze gene expression and are used to detect single nucleotide polymorphisms.
In addition to the detection of infectious agents, oligonucleotides are used in the diagnosis of certain types of disease, particularly in testing for genetic conditions. Examples include the detection of mutations that cause or are involved in Alzheimer disease, cystic fibrosis, epilepsy, sickle cell anemia, and spinocerebellar ataxia.
In therapeutics, fomivirsen, which in 1998 became the first ASO to be approved for use in human patients, is used in the treatment of cytomegalovirus retinitis in individuals with AIDS. Eteplirsen, another ASO, is used to treat Duchenne muscular dystrophy. Other agents have also been approved for certain inherited conditions; for example, nusinersen, which was approved in 2016, is used to treat spinal muscular atrophy, a hereditary neuromuscular disorder. Market growth for oligonucleotide therapeutics has been significant; by 2024 more than 100 such agents were under development.
Historical developments in oligonucleotide synthesis
The first functional synthetic oligonucleotide was produced in the 1970s by Indian-born American biochemist Har Gobind Khorana. Khorana, using directed chemical synthesis, generated a functional oligonucleotide and thereby the first synthetic gene. American biochemist Robert L. Letsinger, who had developed some of the first methods for solid phase synthesis of peptides, subsequently refined Khorana’s approach, replacing phosphodiester with phosphotriester intermediates, which provided greater protection of phosphate from undesirable reactions. This led to his development of the so-called phosphite-triester method, which provided a foundation for the phosphoramidite method introduced in the early 1980s by Marvin H. Caruthers, who had studied under Letsinger and Khorana during his graduate and postgraduate studies, respectively. Letsinger’s work was paramount to laying the groundwork for efficient automated synthesis of gene fragments.
Modern oligonucleotide synthesis is largely automated. Areas of ongoing refinement include finding ways to increase the production of modified therapeutic oligonucleotides and to increase the synthesis of oligonucleotides used in PCR, qPCR, and genetic testing.