- Oligonucleotides are short sequences of nucleotides—typically composed of DNA or RNA—that are synthetically produced and designed to have specific sequences. These molecules usually range from 2 to about 100 nucleotides in length, though most commonly used oligonucleotides fall between 15 to 50 bases. They are foundational tools in modern molecular biology, biotechnology, and medicine, with applications in diagnostics, therapeutics, genomics, and synthetic biology.
- The structure of an oligonucleotide is similar to that of natural nucleic acids: it consists of a sugar-phosphate backbone with attached nitrogenous bases (adenine, cytosine, guanine, thymine in DNA, and uracil instead of thymine in RNA). The sequence of bases is custom-designed to be complementary to specific target sequences, enabling them to hybridize with high specificity. This specificity underpins their use as primers in polymerase chain reaction (PCR), probes in nucleic acid hybridization assays, and guides in gene-editing technologies such as CRISPR-Cas9.
- Oligonucleotides are synthesized chemically through solid-phase synthesis methods, most commonly using phosphoramidite chemistry. This approach allows precise control over the sequence, length, and chemical modifications, enabling the production of high-purity oligonucleotides on demand. After synthesis, these molecules can be further modified with fluorescent dyes, affinity tags like biotin, or backbone alterations such as phosphorothioate linkages or locked nucleic acids (LNAs) to improve stability and functionality, especially for use in biological systems.
- One of the most significant biomedical applications of oligonucleotides is in antisense and RNA interference (RNAi) therapeutics. In these strategies, synthetic oligonucleotides are used to bind complementary RNA sequences and block their translation into proteins or trigger their degradation, thereby modulating gene expression. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are now being developed or approved for treating a variety of genetic and viral diseases, including spinal muscular atrophy, Duchenne muscular dystrophy, and hepatitis B.
- In molecular diagnostics, oligonucleotides serve as probes that detect the presence of specific DNA or RNA sequences associated with pathogens, mutations, or biomarkers. Their high specificity makes them ideal for applications like qPCR, microarrays, and next-generation sequencing (NGS). Moreover, oligonucleotides are critical components in synthetic biology, where they are used to design genetic circuits, encode artificial genes, or build nanostructures through techniques like DNA origami.
- Despite their versatility, the stability and delivery of oligonucleotides in biological systems remain challenges. Unmodified oligonucleotides are susceptible to degradation by nucleases in serum and cells. To overcome this, chemical modifications to the sugar, phosphate backbone, or bases are introduced to enhance resistance to enzymatic degradation, improve cellular uptake, and prolong half-life.
