Oligonucleotides are short synthetic nucleic acid polymers that generally have between 10 and 200 nucleotides. They can be single- or double-stranded, based on DNA or RNA, linked together by phosphodiester bonds. These properties are what render them essential in molecular biology, diagnosis, and therapeutics.
"Oligonucleotide" is the combination of the Greek word "oligo", little or few, and mer, part. The most commonly used way to synthesize oligonucleotides is by solid-phase chemical synthesis. This process allows exact sequence control and rapid synthesis, which allows scientists to design oligonucleotides for a specific purpose. It's not uncommon to use a chemical modification of the sugar or phosphate backbone to enhance the stability, bioavailability, and activity of oligonucleotides in living systems.
Figure 1. Schematic representation of the unmodified oligonucleotide[1].
Oligonucleotides are used in many molecular biology methods. It is believed that oligonucleotides were initially employed in lab studies only as molecular probes for the detection and measurement of particular DNA or RNA sequences. To give a simple example, oligonucleotides are routinely added as primers in PCR to copy particular DNA sequences. They are extremely specific and binding efficient, and that's why this technique works.
Further, oligonucleotides are part of sequencing methods to pinpoint nucleotide sequences in the DNA molecule. Conjugated oligonucleotides with markers, for example, conjugated to fluorescent tags have been used in DNA microarrays, Southern blotting and fluorescence in situ hybridisation (FISH) to identify complementary nucleic acid sequences with high sensitivity and specificity. Small oligonucleotides steer CRISPR/Cas9-like systems toward DNA sequences to modify the genome.
The therapeutic potential of oligonucleotides is one of the biggest focuses of recent years. Two common tactics are antisense oligonucleotides (ASOs) and small interfering RNA (siRNA).
Figure 2. Summary of trafficking and mode of action of ASOs and conjugated siRNAs[2].
ASOs are single-stranded DNA molecules made artificially to pair up with RNA sequences using Watson-Crick bases. By targeting mRNA, ASOs can:
siRNA molecules are RNA sequences with two strands, that exploit the RNA interference (RNAi) pathway to silence genes. Once bound to complementary mRNA, siRNA is processed by the RNA-induced silencing complex (RISC). There has been some success with siRNA treatments in genetic disease, viral infections and cancers.
| Comparison of ASOs and siRNA | ASOs | siRNA |
| Length | 16-21 nucleotides | 19-22 nucleotides |
| Mechanism | RNase H cleavage, splicing modulation | RNAi-mediated mRNA degradation |
| Delivery Challenges | Cellular uptake, stability | Bioavailability, immune response |
Gene editing uses oligonucleotides in a way that's largely influenced by their use in the CRISPR/Cas9 system, an era-defining gene editing system that uses short oligonucleotide sequences (also called guide RNAs or gRNAs) to modify a DNA sequence in a precise manner.
In fact, the oligonucleotide steers the Cas9 nuclease to a target sequence by complementary pairing with the DNA sequence. The Cas9 enzyme then cuts the DNA double-strand at that site, initiating the cell's non-homologous end joining (NHEJ) or homologous recombination repair (HDR) system. HDR could recombine DNA breaks, insert new nucleotides, and insert or replace genes if we had a homologous template available.
Other kinds of gene editing involve other oligonucleotides too, including oligonucleotide-directed mutagenesis (ODM), which inserts a certain nucleotide modification by causing a homologous sequence mismatch at the target sequence. The cell's DNA repair system picks up on this mistake and replaces the nucleotide in question.
Gene editing via oligonucleotides is not just for research purposes in the lab; there's a great deal of clinical potential as well.
Figure 3. Overview of CRISPR-Cas9-assisted oligonucleotide genome editing in Lactobacillus reuteri[3].
In addition to gene silencing, oligonucleotides have a variety of therapeutic roles:
a. Aptamers - these single-stranded oligonucleotides - employ specific 3D structures to bind to targets such as proteins. An example is Pegaptanib, an FDA-approved aptamer for the treatment of ocular vascular disease.
b. CpG oligonucleotides, these immunostimulatory molecules act as adjuvants in vaccines and cancer immunotherapy.
c. Oligonucleotide-based platforms have revolutionized mRNA vaccine development, as seen with the COVID-19 vaccine.
Despite their potential, oligonucleotide-based therapies face challenges, including:
Figure 4. Examples of chemical modifications used to improve the therapeutic activity of oligonucleotides[2].
The integration of oligonucleotide technologies with advances in genomics and delivery systems promises to expand their therapeutic applications, offering novel treatments for a wide array of diseases.
References
Our products and services are for research use only and cannot be used for any clinical purposes.
As a specialist in oligonucleotide therapeutics, Alfa Chemistry offers consumers a wealth of raw materials and a full range of technologies and services.
Privacy Policy | Cookie Policy | Copyright © 2025 Alfa Chemistry. All rights reserved.
