Oligo Deprotection Guide (DNA + RNA)

If your oligo is unmodified DNA

Use a standard ammonium hydroxide–based deprotection workflow unless you have a reason not to.

If you need fast turnaround / high throughput

Consider UltraFAST / AMA-based deprotection (fast, common in high-volume environments), but confirm your monomer protecting groups are compatible (especially cytosine protection).

If your oligo includes base‑labile groups, sensitive dyes, or special modifiers

Use a mild / UltraMild approach (often carbonate-in-methanol or other mild systems), and follow the modifier manufacturer’s conditions.

If your oligo is RNA or chimera (DNA/RNA)

RNA deprotection is two-stage: (1) cleavage/phosphate/base deprotection while keeping 2’ protection, then (2) 2’‑deprotection to yield functional RNA.

1) What “deprotection” actually includes

Oligo deprotection has three linked parts:

Cleavage: release oligo from solid support
Phosphate deprotection: remove cyanoethyl groups from backbone
Base/modifier deprotection: remove nucleobase protecting groups and any modifier protecting groups

You don’t “pick a deprotection method” in the abstract — you pick a method that is compatible with every component in the sequence (bases + dyes + linkers + special monomers).

2) Inventory what’s in the oligo

Before selecting conditions, list:

DNA vs RNA (or chimera)
Base protections used (especially dC and dG)
Any dyes / quenchers / hydrophobic labels
Any base‑labile or ester‑containing groups (examples include certain linkers or specialty bases)
Whether you need to retain DMT‑ON for purification

This is the fastest way to prevent the two classic failure modes:

incomplete deprotection (oligo “looks right-ish” but performs poorly)
damage to the expensive part (dye/modifier gets cooked)

The guide is explicit that sensitive components often require different deprotection schemes than “regular” oligos.

3) DNA deprotection options (how to choose)

A) Standard deprotection (baseline)

Best when:

Unmodified DNA
No base-labile dyes/modifiers

Key success factor:

Deprotect to completion (partial deprotection creates messy profiles and poor performance).

B) UltraFAST (AMA) deprotection (speed + throughput)

AMA = 1:1 ammonium hydroxide : methylamine (v/v).

Why people use it:

Deprotection can be completed in minutes, enabling same‑day release in high-volume settings.

Critical compatibility note:

UltraFAST requires acetyl-protected dC (Ac‑dC) to avoid base modification that can occur when methylamine reacts with Bz‑dC.

Practical framing:

If your shop wants fast + scalable, AMA is the default “fast lane,” but only when the chemistry is compatible.

C) Mild deprotection for ester‑sensitive / amine‑sensitive oligos

If you have components that would react badly with amines, the guide describes sodium hydroxide in aqueous alcoholic solvent as a mild alternative.

Example use case from the guide:

Situations where amine-containing reagents would create undesired chemistry (e.g., ester → amide issues).

Note:

This approach typically requires a desalting step; you can’t always just evaporate and go.

D) UltraMild deprotection (protect the fragile stuff)

UltraMild workflows are designed for:

base-labile bases / damage-repair analogs
sensitive modifiers and dyes that don’t tolerate NH₄OH or AMA

A common UltraMild strategy uses:

UltraMild monomers (e.g., Pac‑dA, Ac‑dC, iPr‑Pac‑dG)
potassium carbonate in methanol for base deprotection

Important nuance:

Deprotection time can depend on capping chemistry (some capping choices create byproducts that require longer removal).

4) RNA deprotection (what’s different and why it matters)

RNA (and chimeras) are unique because the 2’ protecting group must stay on during early steps. You only remove it after:

cleavage
cyanoethyl removal
base deprotection

A) Base deprotection for RNA

The guide highlights:

AMA at elevated temperature as a unified approach that works well for common RNA protection strategies (TOM and TBDMS), with Ac‑protected C monomers required.

It also describes an UltraMild RNA option:

NH₄OH/EtOH (3:1) at room temperature (used when oligo contains base-labile groups).

B) 2’‑deprotection (to get functional RNA)

Commonly used approaches include:

TEA·3HF-based cocktails (triethylamine trihydrofluoride), which have become widely used and are compatible with precipitation and cartridge workflows.

Practical handling warnings from the guide:

Maintain sterile / RNase‑free conditions after base deprotection.
Avoid glass for certain fluoride-based steps; use appropriate plasticware as directed.

5) Dye-containing / heavily modified oligos (how to not ruin labels)

General principle:

If a dye/modifier is present, it may dictate the deprotection method. The guide explicitly calls out dyes like TAMRA/HEX as examples that can force a different procedure than standard.

Practical “safe” posture:

When in doubt, pick the mildest conditions that still achieve complete deprotection, and verify dye stability.

A notable approach described:

For some TAMRA-containing oligos, alternative deprotection using t‑butylamine-based systems is described as an option.

6) Scale / parallel processing options: Gas-phase and on-column deprotection

When you have many oligos to process, the guide highlights methods that reduce manual labor and can be parallelized.

On-column deprotection in organic solvent (practical, scalable)

On-column deprotection in organic solvents as a practical route that captures some convenience of gas-phase methods while avoiding specialized gas handling setups. (Glen Research)

Typical concept (high-level):

Remove cyanoethyl groups
Then apply an organic-solvent deprotection reagent on-column
Then elute and proceed to downstream cleanup

(For any on-column workflow, always verify compatibility with your support chemistry + modifiers.)

7) Troubleshooting quick hits (what to check first)

If you see odd HPLC peaks / “extra” components

Incomplete deprotection can shift retention and generate confusing peak patterns; the guide shows how partial vs complete deprotection changes profiles.

If your RNA yield/purtivity is poor

Double-check that:
- base deprotection completed
- 2’ deprotection completed
- RNase-free handling was maintained

Assume chemistry damage first:

move to UltraMild conditions
confirm the modifier’s required pretreatments and stability windows