Oligonucleotide synthesis refers to the artificial construction of short sequences of nucleic acids—typically DNA or RNA—that range from 10 to 200 nucleotides in length. These synthetic strands serve as essential building blocks across a multitude of molecular biology applications, including PCR primers, qPCR probes, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), aptamers, CRISPR guide RNAs, and synthetic genes. Oligonucleotides are programmable, highly specific, and chemically tunable, which makes them a versatile tool for use in diagnostics, gene editing, therapeutics, and synthetic biology.
Figure 1. (a) Common designs and chemical modifications of ASOs and siRNAs. (b) Chemical diversity of artificial nucleic acids for therapeutic applications[1].
Alfa Chemistry provides a comprehensive platform for both standard and customized oligonucleotide synthesis, enabling researchers to access high-fidelity oligos for cutting-edge biological innovations.
The cornerstone of modern oligonucleotide synthesis is solid-phase phosphoramidite chemistry, a method introduced by Beaucage and Caruthers in the 1980s. This highly efficient and automated technique follows a four-step cyclic process:
A. Deprotection – Acidic removal of the 5' DMT (dimethoxytrityl) group to expose a free hydroxyl group.
B. Coupling – Activation of the incoming 3'-phosphoramidite nucleotide using tetrazole derivatives, followed by its attachment to the growing chain.
C. Capping – Acetylation of unreacted hydroxyl groups to prevent errors.
D. Oxidation – Conversion of the unstable trivalent phosphite linkage to a stable pentavalent phosphate.
This strategy enables stepwise elongation of oligonucleotides from the 3' to 5' direction while immobilized on a solid support, typically controlled pore glass (CPG) or polystyrene beads. Alfa Chemistry utilizes state-of-the-art synthesizers to achieve high coupling efficiency, low truncation rates, and precise length control, with error rates typically as low as 1 in 600 bases.
Figure 2. The main four-step phosphoramidite chemistry is widely used in commercial oligonucleotide synthesizers. The starting nucleoside is attached to the specific substrate via its 3' hydroxyl group. The synthesis cycle includes four steps: deprotection, base coupling, capping and oxidation. The synthesis proceeds in the 3' to 5' direction[2].
Column-based synthesis remains the gold standard for high-purity oligonucleotide production, especially when target specificity and low error rates are paramount. Each oligo is synthesized in a discrete microcolumn where reagents are sequentially pumped through in a highly controlled, programmable fashion. This method supports scalable production, from nanomole to micromole quantities, and is ideal for applications demanding rigorous quality assurance, such as therapeutic ASOs and diagnostic probes.
While traditional systems operate with throughput limitations (96–768 oligos per run), improvements in automation and reagent delivery have reduced costs to $0.05–$0.15 per nucleotide.
Figure 3. Automated oligonucleotide synthesis. (a) The synthesis process includes designing the target sequence, delivering chemical reagents via an inkjet printer, and performing oligonucleotide synthesis in a microreactor chip. (b) Individual microreactors are filled with silica beads to increase the surface area for subsequent synthesis. These beads are fixed in the microreactor by a sintering process. (c) Oligonucleotide synthesis on silica beads[3].
Alfa Chemistry offers extensive customization, including backbone modifications (phosphorothioate, 2'-O-methyl, LNA), fluorescent labels, and terminal functionalization.
Microarray-based (or chip-based) synthesis emerged in the 1990s to address the need for massively parallel, cost-effective oligonucleotide production. This platform allows the synthesis of tens of thousands to millions of oligos on a single chip, with per-nucleotide costs dropping to as low as $0.00001.
Several microarray strategies have been developed:
| Method | Mechanism |
| Photolithographic (PPG) | Photodegradable 5' protection groups; mask-based UV exposure |
| Digital Micromirror Device (DMD) | Maskless UV patterning using programmable micromirrors |
| Photogenerated Acid (PGA) | Light-induced acid deprotection |
| Electrochemical Activation | Site-selective DMT removal via redox reactions |
| Inkjet Printing | Direct deposition of phosphoramidite monomers |
These methods are particularly suited for applications like DNA microarrays, resequencing, and gene synthesis via oligo assembly.
Alfa Chemistry collaborates with platform developers to offer custom oligo pools for synthetic genome design, functional screening, and library generation.
Current phosphoramidite chemistry limits practical oligo length to approximately 200 bases due to cumulative coupling inefficiencies and error accumulation. Beyond this, high-fidelity gene assembly relies on overlapping short oligonucleotides followed by enzymatic stitching.
Recent innovations include:
Figure 4. Fabrication process of the patterned SiO2-COC slide, phosphoramidite chemistry, and fluorescence image of an in situ oligonucleotide array on SiO2-COC slide[4].
Alfa Chemistry remains committed to integrating both established and next-generation methodologies to provide oligonucleotides tailored for complex genomic applications.
Oligonucleotides play a central role across numerous disciplines:
Alfa Chemistry provides a diverse library of oligonucleotide synthesis building blocks to enable researchers to design and synthesize their own custom oligonucleotides. The key intermediate building blocks with their tightly controlled purities and physicochemical properties, for seamless implementation in automated SPOS workflows. The use of these pre-functionalized synthetic units allows for a dramatic reduction in synthetic complexity and synthesis yields and also allows for modular design of complex oligonucleotides.
1. How long can synthetic oligonucleotides be?
Most chemically synthesized oligos are under 200 bases. For sequences beyond this, overlapping oligos are assembled enzymatically into longer constructs.
2. What is the difference between phosphorothioate and natural phosphodiester backbones?
Phosphorothioate modifications increase nuclease resistance, enhancing oligo stability in biological environments, especially for therapeutic use.
3. Can I order oligos with 5' or 3' modifications?
Yes. Alfa Chemistry supports various terminal modifications such as fluorescent dyes, biotin, amino linkers, and phosphate groups.
4. What's the turnaround time for custom oligonucleotide synthesis?
Typically 3–7 business days, depending on length, purity (desalted, HPLC, PAGE), and modifications.
5. How are chip-synthesized oligos purified and recovered?
Most microarray-synthesized oligos are recovered via cleavage from the surface followed by amplification or enzymatic assembly. However, they often exhibit lower individual purity than column-synthesized oligos.
6. What quality control methods are used?
Alfa Chemistry employs MALDI-TOF, capillary electrophoresis, and UV spectroscopy to confirm sequence integrity and purity.
7. Are enzymatic methods still used for oligonucleotide synthesis?
Primarily for amplification (e.g., PCR) rather than de novo synthesis, though emerging enzymatic synthesis platforms are under active development.
References
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