Oligonucleotide
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What is an oligonucleotide?
An oligonucleotide is a short chain of nucleotides that consists of either single- or double-stranded DNA or RNA. They are typically 20 nucleotides or fewer in length, but can consist of as many as 100 or even 200 nucleotides.
Oligonucleotides were originally used in lab research as molecular probes to detect complementary DNA or RNA sequences. Today, they are especially important in drug development because of their ability to influence gene expression once bound to a target sequence.
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What are oligonucleotides made of?
Oligonucleotides are short chains of nucleotides that can be single- or double-stranded DNA or RNA. Nucleotides, the building blocks of oligonucleotides and nucleic acids, consist of a nitrogen-containing base, a sugar, and a phosphate group.
DNA contains the nucleotides adenine, guanine, cytosine, and thymine, while RNA uses uracil instead of thymine. The sequence of these nucleotides forms the genetic code.
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How are oligonucleotides different from regular DNA or RNA?
An oligonucleotide is a short chain of nucleotides, typically containing up to 20 units, but they can consist of as many as 100 or even 200 nucleotides. They can be single- or double-stranded DNA or RNA.
Oligonucleotides are often synthesized for use as molecular probes to detect complementary DNA or RNA sequences. They also occur naturally as microRNA and breakdown products of longer nucleotide sequences. Because of their complementarity and sequence specificity, oligonucleotides are relevant and versatile for various molecular biology techniques. They also possess a high degree of chemical stability, making them convenient for research, diagnostics, and therapeutic applications.
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How do scientists create oligonucleotides in the lab?
Scientists synthesize oligonucleotides in the lab using automated processes refined from methods developed in the 1970s and early 1980s by biochemists Har Gobind Khorana, Robert L. Letsinger, and Marvin H. Caruthers. Letsinger's work, which replaced phosphodiester with phosphotriester intermediates, was essential to the automated synthesis of gene fragments.
Oligonucleotides are used in research as primers for polymerase chain reaction (PCR) to clone and sequence genes. They are also used in quantitative PCR (qPCR) for rapid detection of infectious agents such as SARS-CoV-2. In medicine, they are used to diagnose diseases like Alzheimer disease and cystic fibrosis and to treat conditions such as cytomegalovirus retinitis and Duchenne muscular dystrophy.
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What are some uses of oligonucleotides in genetics research?
Oligonucleotides are useful in genetics research due to their versatility in molecular biology techniques. They can be designed to bind to target messenger RNA (mRNA) molecules and alter gene expression, making them important in drug development.
Oligonucleotides are essential in PCR (polymerase chain reaction), a method used to exponentially amplify specific DNA sequences. In vitro mutagenesis, including oligonucleotide-directed mutagenesis, utilizes synthetic oligonucleotides to create specific mutations in cloned DNA for studying gene function. They also have uses in microarrays, which facilitate direct comparisons of molecular activity between different samples, advancing disease detection and treatment.
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How can oligonucleotides help diagnose or treat genetic diseases?
Oligonucleotides play a crucial role in diagnosing and treating genetic diseases due to their ability to interact with specific DNA or RNA sequences. They can detect mutations associated with conditions like Alzheimer disease, cystic fibrosis, and sickle cell anemia.
Antisense oligonucleotides (ASOs) can bind to messenger RNA (mRNA) to modify gene expression. For example, nusinersen is used to treat spinal muscular atrophy, and fomivirsen is used to treat cytomegalovirus retinitis in AIDS patients. Oligonucleotide-directed mutagenesis facilitates the study of gene function by creating specific mutations in cloned DNA, which can then be introduced into cells to observe the effects.
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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.
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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.
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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.