An oligonucleotide is a short, synthetic strand of DNA or RNA, typically between 12 and 30 building blocks (called nucleotides) long. These small molecules are designed to recognize and bind to specific genetic sequences inside cells, which makes them powerful tools in both medicine and laboratory diagnostics. As of April 2025, the FDA has approved 22 oligonucleotide-based drugs, and the therapeutic market is projected to reach nearly $7 billion in 2026.
Basic Structure and How It Works
Each nucleotide in an oligonucleotide contains three parts: a sugar, a phosphate group, and one of four chemical bases (adenine, thymine, guanine, or cytosine in DNA; uracil replaces thymine in RNA). The bases on one strand pair with complementary bases on another strand, a process called Watson-Crick base pairing. A pairs with T (or U in RNA), and G pairs with C. This pairing is what gives oligonucleotides their precision: a 20-nucleotide sequence can be designed to match one specific stretch of genetic code out of the entire genome.
A typical single-stranded DNA oligonucleotide of 20 nucleotides has a molecular weight of about 7,000 daltons and carries 19 negative electrical charges along its backbone. That’s tiny compared to most proteins, but large enough to carry a meaningful amount of genetic information. The negative charge, however, creates challenges for getting these molecules into cells, which is why chemical modifications and delivery systems play such a large role in oligonucleotide science.
Three Major Types of Therapeutic Oligonucleotides
Antisense Oligonucleotides (ASOs)
ASOs are single-stranded molecules, usually 12 to 30 nucleotides long, that bind directly to a complementary messenger RNA inside a cell. Once bound, they can silence a gene in two main ways. The first is physical blocking: the ASO sits on the RNA and prevents the cell’s machinery from reading it. The second involves recruiting a natural enzyme called RNase H, which recognizes the DNA-RNA pair and cuts the RNA apart, destroying the message before it can be turned into protein. Some ASOs are also designed to alter how RNA is spliced, skipping over defective sections of a gene to produce a functional protein.
Small Interfering RNAs (siRNAs)
Unlike ASOs, siRNAs are double-stranded RNA molecules, usually 19 to 22 base pairs long. They work through a different cellular pathway called RNA interference. Once inside a cell, the double strand gets loaded into a protein complex called RISC. The complex discards one strand (the passenger) and keeps the other (the guide strand), which then directs RISC to find and destroy the matching RNA target. The first siRNA drug, patisiran, received FDA approval in 2018 for a hereditary condition that causes harmful protein buildup.
Aptamers
Aptamers take a completely different approach. Instead of targeting genetic sequences, these single-stranded DNA or RNA oligonucleotides fold into complex three-dimensional shapes that can grab onto proteins and small molecules, much like antibodies. They’re sometimes called “chemical antibodies” because they bind their targets with high specificity based on the target’s physical shape rather than its genetic sequence. Pegaptanib, an aptamer approved in 2004, was designed to bind a growth factor involved in abnormal blood vessel formation in the eye.
Chemical Modifications That Make Them Work
Natural DNA and RNA strands are fragile inside the body. Enzymes called nucleases chew them apart within minutes. To make oligonucleotide drugs survive long enough to reach their targets, chemists have developed several key modifications to the basic structure.
The most common is the phosphorothioate (PS) backbone, where one oxygen atom in the phosphate linkage is replaced with sulfur. This simple swap dramatically increases resistance to enzymatic breakdown. The very first approved oligonucleotide drug, fomivirsen (approved in 1998 for a viral eye infection), was a fully PS-modified 21-nucleotide strand. Nearly every oligonucleotide drug since has used PS modifications in some form.
A second generation of modifications targets the sugar portion of each nucleotide. Adding a small chemical group at a specific position on the sugar ring improves both stability and binding strength. The two most successful versions, known as MOE and 2′-fluoro modifications, appear in many approved drugs. Nusinersen, an 18-nucleotide drug approved for spinal muscular atrophy, uses a combination of PS backbone and MOE sugar modifications throughout its entire length.
Locked nucleic acids (LNA) go even further by chemically locking the sugar into a rigid shape, which greatly increases how tightly the oligonucleotide grips its RNA target. Recent research has shown that combining LNA modifications with PS backbones can improve both cellular uptake and gene-silencing activity beyond what either modification achieves alone. Another distinct chemistry, phosphorodiamidate morpholino (PMO), replaces both the sugar and the phosphate with an entirely different uncharged backbone. Eteplirsen, a 30-nucleotide PMO drug approved in 2016 for Duchenne muscular dystrophy, carries no negative charge at all.
Getting Oligonucleotides Into the Right Cells
Designing an oligonucleotide that binds the right target is only half the challenge. The other half is delivering it to the correct tissue. Two strategies have emerged as the most successful so far, both focused on liver delivery.
Lipid nanoparticles (LNPs) are tiny fat-based capsules that protect the oligonucleotide in the bloodstream and help it enter liver cells. Patisiran, the first approved siRNA drug, uses this approach. LNPs were also the delivery technology behind the mRNA COVID-19 vaccines, which brought the concept into mainstream awareness.
A newer and increasingly dominant approach uses a sugar molecule called GalNAc (N-acetylgalactosamine) attached directly to the oligonucleotide. Liver cells display a receptor on their surface that naturally binds GalNAc, pulling the conjugated drug inside. This targeted delivery increases drug levels in liver cells by six to seven times compared to unconjugated oligonucleotides at the same dose. GalNAc conjugation has now largely replaced LNPs for liver-targeted oligonucleotide therapies because it’s simpler: the drug can be injected under the skin rather than infused intravenously.
Delivery to organs beyond the liver remains one of the field’s biggest unsolved problems. Researchers are exploring conjugation with antibodies, vitamins, and other targeting molecules to direct oligonucleotides to muscle, brain, and other tissues.
Oligonucleotides in Diagnostics
Outside of therapeutics, oligonucleotides are essential tools in medical testing. Every PCR test, including the ones used widely during the COVID-19 pandemic, relies on short oligonucleotide primers. These primers are designed to match sequences flanking a region of interest in a pathogen’s DNA, allowing the test to selectively copy and detect that specific genetic material.
Oligonucleotides also serve as probes in diagnostic assays. A probe is a short, labeled single strand designed to bind a complementary target sequence. In one common format called TaqMan, the probe carries two fluorescent dyes. When the probe binds its target and gets broken apart during the copying process, the dyes separate, producing a measurable light signal. This is what allows real-time PCR machines to quantify exactly how much of a target pathogen is present in a sample. DNA probes built on the same hybridization principle are used in genetic testing to identify mutations, detect chromosomal abnormalities, and screen for inherited conditions.
How Oligonucleotides Are Made
Oligonucleotide synthesis is a fully automated chemical process performed on a solid support, similar to an assembly line. The standard method uses a four-step cycle that adds one nucleotide at a time to a growing chain. The key ingredient in each cycle is a specially prepared building block called a phosphoramidite, a chemistry first developed in 1981 that remains the industry standard. Each cycle attaches a new nucleotide to the exposed end of the chain, and after the final nucleotide is added, the finished strand is released from the solid support and purified.
Modern synthesizers can produce oligonucleotides up to about 100 nucleotides in length with high accuracy, though most therapeutic and diagnostic applications use sequences well under that limit. The process is fast enough that custom oligonucleotides for research use can be ordered online and delivered within days.