Synthetic oligonucleotides are custom-made, short strands of nucleic acids, either DNA or RNA, that are created in a laboratory setting. These molecules are distinct from naturally occurring genetic material because their sequences are designed and built to precise specifications. They typically consist of 10 to 50 nucleotides. This synthetic nature allows scientists to develop highly specific tools and therapeutic agents for a wide array of biological and medical applications.
How Synthetic Oligonucleotides Are Created
Synthetic oligonucleotides are created using a chemical process called solid-phase synthesis, often utilizing phosphoramidite chemistry. This automated method involves building the oligonucleotide chain on a solid support material, such as controlled pore glass or polystyrene. Each addition of a nucleotide involves a series of chemical reactions, ensuring the precise sequence is formed.
The process begins by attaching the first nucleotide to the solid support. Then, a cycle of four main steps—deprotection, coupling, capping, and oxidation—is repeated for each additional nucleotide. This stepwise addition allows for the creation of exact sequences, which are then cleaved from the solid support, deprotected, and purified once the desired length is achieved. This automated synthesis enables the rapid and cost-effective production.
Diverse Applications in Biology and Medicine
Synthetic oligonucleotides have transformed various fields, from fundamental biological research to medical diagnostics and therapeutics. Their ability to bind to specific genetic sequences makes them highly versatile tools. These molecules are widely used in molecular biology and diagnostics, but their potential in therapeutic applications is particularly significant.
In therapeutics, oligonucleotides treat genetic disorders and other diseases. Antisense oligonucleotides (ASOs), for instance, can bind to messenger RNA (mRNA) molecules, blocking the production of disease-related proteins. An example is nusinersen (Spinraza), an ASO used to treat spinal muscular atrophy by modifying gene expression. Small interfering RNA (siRNA) molecules are another type of oligonucleotide therapeutic that triggers the degradation of specific mRNA molecules, leading to gene silencing. These RNA-based interventions can address diseases including some inborn errors of metabolism and rare genetic disorders.
Oligonucleotides also play a role in medical diagnostics. They are used as primers in the Polymerase Chain Reaction (PCR), a technique that amplifies small amounts of DNA for detecting specific mutations or pathogens, such as in COVID-19 testing. Also, oligonucleotides serve as probes for detecting complementary DNA or RNA through molecular hybridization, which is useful in DNA sequencing and gene expression analysis. Their precision allows for the identification of specific genetic markers associated with various conditions.
Beyond diagnostics and therapeutics, synthetic oligonucleotides are tools in fundamental biological research. They are employed in gene editing technologies like CRISPR-Cas systems, where specific RNA oligonucleotides guide the Cas enzyme to target and modify particular DNA sequences. Oligonucleotides are also used for gene synthesis, enabling researchers to create artificial genes or modify existing ones to study gene function. Furthermore, they act as probes for studying the intricate roles of genes within cells.
Variations and Enhancements of Synthetic Oligonucleotides
Synthetic oligonucleotides can be either DNA or RNA, each serving different purposes. DNA oligonucleotides are commonly used as primers for DNA amplification and sequencing, while RNA oligonucleotides, such as siRNA, are often employed for gene silencing. The choice between synthetic DNA and RNA depends on the specific biological target and desired function.
To overcome limitations of natural oligonucleotides, such as instability and poor cellular uptake, scientists introduce various chemical modifications. Backbone modifications, like phosphorothioate linkages, can increase the oligonucleotide’s stability against degradation by enzymes in the body, which is particularly beneficial for therapeutic applications.
Other enhancements include fluorescent labels for detection in research assays, allowing scientists to track their location or activity within cells or tissues. Affinity tags can also be incorporated for purification. Modifications can also improve the delivery of oligonucleotides to target cells or organs, for instance, by adding cholesterol to enhance cell membrane penetration or N-acetylgalactosamine (GalNAc) for liver-specific targeting. These structural and chemical alterations are made to improve the oligonucleotide’s stability, binding affinity, specificity, and overall performance for its intended application.