Each synthesis starts on a solid support (e.g., CPG) carrying the 3′‑end nucleoside. The nucleoside bears a 5′‑DMT group (acid‑labile) and base‑protecting groups stable to acid but removable later. For RNA, 2′‑OH is protected (e.g., TBDMS or TOM) throughout synthesis.

• Support choices: CPG, controlled‑morphology supports for flow/scale
• Linkers: cleavable linkers tuned to DNA/RNA and downstream deprotection
• Custom loads to balance yield vs. sterics for long/modified sequences

A mild acid removes the 5′‑DMT protecting group, exposing the 5′‑OH so the next nucleotide can couple. Online monitoring of the released DMT cation provides a quick measure of coupling efficiency and synthesis progress.

The incoming nucleoside phosphoramidite is activated by a tetrazole‑type catalyst and reacts with the exposed 5′‑OH to form a phosphite triester linkage. High coupling efficiency is critical for long or complex sequences.

• Base: A, C, G, T/U; analogs (LNA, cEt, 2′‑F/OMe, etc.) as needed
• Add‑ons: internal labels, spacers, linkers via compatible amidites
• Double/additional coupling to boost yield at difficult sites

Unreacted 5′‑OH groups are acetylated so they cannot enter subsequent cycles. This prevents “n−1” deletions from propagating and simplifies purification.

The phosphite triester is oxidized (e.g., iodine) to the natural phosphate linkage (PO). For phosphorothioate (PS) backbones, a sulfurizing reagent replaces one non‑bridging oxygen with sulfur, enhancing nuclease resistance and pharmacokinetics for ASOs/siRNA.

• PO vs. PS or mixed PO/PS patterns (full or partial)
• Compatible with LNA/cEt and other therapeutic motifs

The DMT–Coupling–Capping–Oxidation steps repeat until the full sequence is assembled. Trityl monitoring and test cleaves assess efficiency, especially for long or GC‑rich designs.

After chain assembly, the oligo is cleaved from the support and deprotected to remove base‑ and phosphate‑protecting groups. Conditions depend on chemistry (DNA vs RNA) and labels.

• DNA: ammonia/AMA variants; heat/time tuned to protect labels
• RNA: base deprotection then 2′‑protecting group removal (e.g., TBDMS/TOM)
• Sensitive labels (e.g., some dyes) use milder protocols

Choice depends on application and modification set. We recommend HPLC (RP or IEX) for probes, long or modified oligos, and regulated programs.

• Desalt/Cartridge: quick cleanup for routine PCR
• RP‑HPLC: hydrophobic separation; great for dyes/modified oligos
• IEX‑HPLC: charge‑based; useful for PS and length variants
• PAGE: high resolution for short/critical applications

We install functional groups during synthesis or via post‑synthetic conjugation (amine, thiol, azide, alkyne, DBCO, maleimide, etc.) for dyes, quenchers, biotin, lipids, PEG, peptides, and GalNAc.

• 5′/internal/3′ placements; spacers for accessibility
• Orthogonal click chemistries (CuAAC, SPAAC, TCO–tetrazine)
• Duplex assembly (siRNA) with stoichiometry verification

RNA uses 2′‑protecting groups (e.g., TBDMS/TOM) and may integrate 2′‑OMe, 2′‑F, LNA/cEt, or terminal caps for stability. Conditions balance complete deprotection with label integrity and low depurination.

• Identity: Mass spectrometry (MS)
• Purity: Analytical HPLC (RP/IEX), optional PAGE/CE
• Quantitation: UV/OD260 with extinction coefficients
• GMP: batch records, method validation, endotoxin/bioburden (as applicable)

For large‑scale and regulated programs, we harmonize raw material specs, change control, equipment qualification, and cleaning validation. Process performance is characterized during tech transfer and monitored across lots.