Everything RNA

This article is a practical, end-to-end guide to chemical RNA oligo synthesis (solid‑phase phosphoramidite workflows), with an emphasis on 2′‑O‑TBDMS RNA. It is written for trained users operating automated synthesizers and standard downstream cleanup/purification workflows.

Safety note: Many steps involve corrosives/toxic reagents (strong acids, alkylamines, fluoride reagents such as TEA·3HF, etc.). Only trained personnel should perform these procedures using appropriate PPE and local safety/SDS requirements.

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Quick Start Recommendations (Read This First)

1) Coupling strategy 

Start with double coupling early, then increase to 3× and 4× coupling later as the strand gets longer.

Why: even small drops in stepwise yield compound fast with length (Sproat reports typical stepwise coupling yields for TBDMS RNA around ~98.5–99% under good conditions).
A 60‑mer at 99% stepwise yield is only ~55% full‑length in theory—so you don’t save by under‑coupling; you pay for it in purity and yield.

Suggested coupling schedule:

• 1–25 nt: 2× coupling (double couple every cycle)

• 26–50 nt: 2× coupling, then begin 3× coupling for “difficult” positions (G, or known troublesome motifs)

• 51–80 nt: 3× coupling for most cycles; optionally keep 2× for easy cycles if performance is excellent

• 80+ nt: 3× coupling baseline, plus 4× coupling for the last ~10–20 additions (the portion most sensitive to cumulative failures)

Mechanically, “3×” or “4×” coupling means you repeat the coupling step multiple times before oxidation/capping—a standard approach used to increase effective coupling efficiency.

For longest RNA oligos, try alternatively protected RNA phosphoramidites (e.g., TOM, TC)

2) Use a strong activator

For TBDMS RNA, avoid weak/old activators and use strong activators (examples include ETT or BMT).

Glen Research also highlights that activator choice matters, a BTT (often excellent for RNA) and DCI (frequently favored for longer oligos and scale).

3) Ruthlessly control water

Water kills yield and increases side products. It cannot be over-emphasized the importance of very dry solvents; a commonly cited target is <10 ppm water, verified by Karl Fischer if possible.

4) Deprotection + desilylation quality is often the real limiter

Incomplete desilylation can show up as “N+” peaks (often mistaken for artifacts from double coupling).

A practical desilylation workflow

1. Dissolve dried oligo in DMSO, warming to fully dissolve (commonly around 65 °C). Use a fresh desilylation cocktail (NMP or DMSO + TEA + TEA·3HF). Use polypropylene tubes/containers where specified.

2. Add the specified volume of cocktail to the oligo solution, then heat ( ~65 °C for ~150 minutes, with a warning not to overrun past ~160 minutes).

3. Quench with RNA quenching/loading buffer (quench addition immediately after the timed reaction).

4. Proceed directly to SPE/desalting.

5) After desilylation: quench and clean up fast

Move quickly: pre neutral pH can harm RNA, and the sample should be loaded onto SPE/desalting workflows promptly after quench.

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What Makes RNA Synthesis Different From DNA?

RNA synthesis is harder primarily because the 2′‑OH mu synthesis. The commonly used 2′‑O‑silyl protecting groups (e.g., TBDMS) add steric bulk, which can reduce coupling efficiency and require longer/multiple couplings.

RNA synthesis uses the same general phosphoramidite cycle, but conditions and reagents must be tuned to preserve yield and avoid degradation.

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Cleavage & Base Deprotection: Avoid “Self‑Inflicted” RNA Damage

Harsh base deprotection—especially heating in aqueous ammonium hydroxide—can cause severe RNA degradation.

Faster/milder approaches (e.g., alkylamine-based strategies or methylamine-based methods) can reduce exposure but can be potentially damaging, so CE protecting group choices matter (e.g., N‑acetyl protection for cytidine will be required for certain fast protocols).

Practical takeaway:

• Choose deprotection conditions protecting groups, modifications, and length.

• Favor milder/shorter deprotection when possible to reduce strand scission.

 

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Purification: Choosing the Right Method for Length and Use Case

A) Leave DMT “ON” for long RNAs when it helps purification

For RNAs longer than ~25 nt, it can be advantageous to leave the 5′‑DMT group ON to exploit its hydrophobicity during purification.

B) Common purification options

• Reverse‑phase HPLC (RP‑HPLC) (especially effective with longer RNAs)

• Anion‑exchange HPLC

• SPE/desalting cartridges (fast cleanup to remove salts/reagents after desilylation)

• PAGE (when very high resolution is required, typically for shorter/medium RNAs or specialized applications)

C) Salt form matters (especially for MS and secondary structures)

Sodium salts are not suitable for ESI‑MS. Convert to ammonium salt forms for MS.
For sequences with ≥4 consecutive guanines, sodium or potassium salts can form quadruplexes; using appropriate ion‑exchange conditions (e.g., lithium-based systems) can help manage these.

If MS looks “wrong,” check:

• Salt form (avoid sodium; consider ammonium exchange)

• Ionization settings/method suitability for longer RNAs (your ionization tuning can be a major factor).

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Troubleshooting Guide (Fast Pattern Recognition)

Symptom: “N+” peaks, unexpected heavier species

Likely causes:

• Incomplete desilylation (common)

• Misinterpreted artifacts from multiple coupling (over coupling-- check mass)

Fixes:

• Temperature/solvent system

• Consider extended desilylation time where validated (longer desilylation improving outcomes)

Symptom: Lots of N‑1 / truncations

Likely causes:

• Coupling efficiency dropping late in synthesis

• Water contamination

• Activator choice not suitable

Fixes:

• Move from 2× → 3× → 4× coupling late in sequence, and/or extend coupling time 

• Improve dryness control (<30 ppm water target)

• Use strong RNA activators (ETT/BTT/BMT)

Symptom: Degradation / smeared chromatograms / unexpectedly low recovery

Likely causes:

• Overly harsh base deprotection (heated aqueous NH4OH is a known offender)

• Too much time in damaging conditions post‑desilylation (handling delays)

Fixes:

• Switch to milder/faster deprotection protocols compatible with your protecting groups

• Quench and load onto cleanup promptly after desilylation