An oligo synthesizer is a machine that builds short strands of DNA or RNA, known as oligonucleotides. These instruments are fundamental in modern biological research and biotechnology by creating custom genetic sequences. The ability to precisely control the order of building blocks in these strands has made oligo synthesizers indispensable tools in various scientific and medical advancements.
Understanding Oligonucleotides and Their Importance
Oligonucleotides, often called oligos, are short, synthetic single strands of DNA or RNA. Their name comes from the Greek word “olígoi,” meaning “few” or “small,” combined with “nucleotide,” the basic building block of nucleic acids. These molecules range from 10 to 200 nucleotides or more in length.
Custom-designed oligonucleotides are fundamental tools in biological sciences, allowing researchers to precisely target and manipulate genetic information. They are used to understand how genes work, identify genetic diseases, and develop new treatments. The ability to create specific sequences makes them versatile for exploring and altering biological systems.
How Oligo Synthesizers Work
Oligo synthesizers operate on the principle of solid-phase synthesis, where the growing oligonucleotide chain is attached to a solid support material, such as controlled-pore glass beads. This process involves the stepwise addition of individual nucleotide building blocks, called phosphoramidites, to the chain. The synthesis proceeds in a 3′ to 5′ direction, adding one base at a time until the desired sequence is complete.
Each addition of a nucleotide involves a cycle of four chemical steps. The first step, called detritylation, removes a protective chemical group from the 5′ end of the growing chain, preparing it for the next nucleotide. In the coupling step, the desired phosphoramidite building block is activated and forms a bond with the unblocked 5′-hydroxyl group on the growing chain. This reaction creates a phosphite triester linkage between the newly added nucleotide and the existing chain.
Following coupling, a capping step blocks any unreacted 5′-hydroxyl groups that failed to couple. This step prevents the formation of “failure sequences.” Finally, in the oxidation step, the unstable phosphite triester linkage is converted into a more stable phosphate triester bond, strengthening the connection between the nucleotides. After all desired bases have been added, the completed oligonucleotide is cleaved from the solid support and undergoes further processing.
Types of Oligo Synthesizers and Overcoming Challenges
The evolution of oligo synthesizers has led to various designs. Historically, column-based systems were used for individual syntheses, with each oligonucleotide synthesized in its own separate column. Modern advancements include array-based or multi-well plate formats, allowing for higher throughput and the parallel synthesis of many oligonucleotide sequences. Some machines can synthesize 96 different oligos in a few hours using a standard 96-well microtiter plate format.
Despite these advancements, challenges remain in oligonucleotide synthesis, particularly in achieving high coupling efficiency and synthesizing complex sequences. Coupling efficiency refers to the percentage of growing chains that successfully add a new nucleotide in each cycle. Even a small decrease in efficiency can significantly reduce the yield of full-length oligonucleotides as the desired length increases. For instance, a 100-mer oligonucleotide with 98% coupling efficiency would theoretically yield only about 13% full-length product. Factors like moisture can lower coupling efficiency, so anhydrous conditions are maintained.
Synthesizing complex sequences, such as those with secondary structures, repetitive regions, or extreme GC content, also presents difficulties. These features can interfere with the chemical reactions, leading to lower yields or errors. To address these limitations, researchers are developing new methods, including enzymatic synthesis approaches. These emerging techniques, which use enzymes like terminal transferase (TdT) or template-dependent polymerases, aim to achieve higher coupling efficiencies and synthesize much longer oligonucleotides with less hazardous waste.
Applications of Synthesized Oligonucleotides
Custom-synthesized oligonucleotides are used in various scientific and medical fields for many applications. In research, they are commonly used as primers in the polymerase chain reaction (PCR), a technique that amplifies specific DNA segments for further study. Oligonucleotides also serve as probes in techniques like DNA microarrays and fluorescence in situ hybridization (FISH) to identify specific DNA or RNA sequences, aiding in gene expression studies and mutation detection.
Beyond research, these molecules are important tools in diagnostics, helping to detect genetic diseases and identify pathogens. For example, allele-specific oligonucleotides (ASOs) are used to detect genetic variations. In therapeutics, synthesized oligonucleotides are used as direct therapeutic agents. Examples include antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), which can regulate gene expression and are being explored for treating various conditions, including Duchenne muscular dystrophy and spinal muscular atrophy. Oligonucleotides also play a role in gene editing technologies like CRISPR-Cas9, where they serve as guide RNAs to direct the enzyme to specific locations in the genome for targeted modifications.