Oligonucleotide Synthesis

  • Oligonucleotide synthesis is the chemical process of creating short strands of nucleic acids—typically DNA or RNA—with a defined sequence of nucleotides. This synthetic capability has been foundational to the development of modern molecular biology, genomics, diagnostics, and therapeutic technologies. Most oligonucleotides used in laboratories and clinical applications today are produced using automated solid-phase synthesis, which allows for high precision, scalability, and customization.
  • The most widely used method for oligonucleotide synthesis is phosphoramidite chemistry, developed in the 1980s. This approach is based on a stepwise addition of protected nucleoside phosphoramidite building blocks to a growing oligonucleotide chain attached to a solid support, such as controlled pore glass (CPG) or polystyrene beads. The synthesis proceeds from the 3′ to the 5′ end of the oligonucleotide and consists of four major steps in each cycle: deprotection, coupling, capping, and oxidation.
  • In the deprotection step, the 5′-dimethoxytrityl (DMT) protecting group is removed from the terminal nucleotide to expose the hydroxyl group. Next, in the coupling step, the activated phosphoramidite of the next nucleotide (protected at the base and phosphate groups) is added, forming a phosphite triester bond. Any unreacted terminal hydroxyl groups are then capped to prevent incomplete sequences from elongating. Finally, oxidation is performed to convert the unstable phosphite linkage into a stable phosphate diester. These cycles repeat until the desired sequence is fully assembled.
  • After the full-length synthesis, the oligonucleotide is still attached to the solid support and contains multiple protecting groups. A cleavage and deprotection step—commonly using ammonium hydroxide or other basic conditions—removes the protecting groups from the bases and backbone and detaches the oligonucleotide from the solid support. The resulting crude product is a mixture containing the full-length oligonucleotide along with shorter sequences and side products.
  • Purification is critical to ensure the integrity and performance of synthesized oligonucleotides. Depending on the intended application, various methods are employed, including desalting, reverse-phase high-performance liquid chromatography (RP-HPLC), anion-exchange chromatography, or polyacrylamide gel electrophoresis (PAGE). For high-purity needs, such as clinical or gene-editing applications, more stringent purification is used to separate full-length products from truncated ones.
  • Modern oligonucleotide synthesis also allows for the introduction of chemical modifications to enhance the functionality, stability, or delivery of the molecule. These include fluorescent dyes, biotin tags, thiol groups, or modified backbones such as phosphorothioates and locked nucleic acids (LNAs). Such modifications are essential in therapeutic oligonucleotides like antisense molecules, siRNAs, aptamers, and guide RNAs used in CRISPR systems.
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