Cleavage and Deprotection of Oligonucleotides

Deprotection is typically broken into three stages:

1. Cleavage – releasing the oligo from the solid support.

2. Phosphate deprotection – removing cyanoethyl groups from the backbone.

3. Base deprotection – removing protecting groups from the bases or modifiers.

The goal is always complete deprotection without damaging sensitive modifications.

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Key Considerations

Before selecting a protocol, ask yourself:

• Does the oligo contain sensitive dyes or base-labile modifications?

• Am I working with DNA, RNA, or a hybrid structure?

• Do I need to keep the DMT group on for purification?

• How fast do I need the deprotection completed?

• Am I processing one oligo or many in parallel?

General principle: always use the mildest compatible conditions when unusual or expensive modifications are present.

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Cleavage

• Cleavage is usually combined with deprotection in a single step.

• Note: harsh basic conditions at high temperature can dissolve some silica from the support. Any resulting residue can be removed by filtration or purification.

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Key Considerations

Before selecting a protocol, ask yourself:

• Does the oligo contain sensitive dyes or base-labile modifications?

• Am I working with DNA, RNA, or a hybrid structure?

• Do I need to keep the DMT group on for purification?

• How fast do I need the deprotection completed?

• Am I processing one oligo or many in parallel?

General principle: always use the mildest compatible conditions when unusual or expensive modifications are present.

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Deprotection Strategies

Standard (Ammonium Hydroxide)

• Classic approach.

• Fresh ammonium hydroxide solution is essential for full removal of base protecting groups.

• Typical conditions:

  • iBu-dG: 55 °C for 16 h or 65 °C for 8 h

  • dmf-dG, Ac-dG: 55 °C for 4 h or 65 °C for 2 h

  • iPr-Pac-dG: 55 °C for 30 min

UltraFast (Ammonium Hydroxide/Methylamine, AMA)

• Mix equal volumes of ammonium hydroxide and 40% aqueous methylamine.

• Cleavage and deprotection in minutes rather than hours.

• Example: 65 °C for 5–10 min.

• Requires Ac-dC instead of Bz-dC to prevent unwanted side reactions.

Mild and UltraMild

• For sensitive modifications (e.g., fluorescent dyes, base-labile groups).

• Methods include:

  • Potassium carbonate in methanol (0.05 M, RT, 4 h)

  • t-Butylamine/Water (1:3) at 60 °C for 6 h

  • “Ultra-UltraMild” methods using special supports and gentle reagents.

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RNA-Specific Notes

• RNA deprotection must preserve 2′-protecting groups during cleavage and base deprotection.

• After base deprotection, a separate 2′-deprotection step is required (commonly TEA·3HF or TBAF-based).

• UltraMild RNA monomers are often recommended for compatibility with dyes and modifications.

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Dye-Containing Oligos

• Dyes and quenchers are often base-sensitive and expensive.

• Always match the deprotection method to the most fragile component.

• Incompatible conditions can lead to degraded or non-functional probes.

• When in doubt, choose slower and milder conditions.

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Summary

Cleavage and deprotection are critical for producing functional oligos. Each synthesis should be reviewed in advance to determine:

• Which deprotection method is compatible with all components.

• Whether pretreatments are required (e.g., for amino modifiers).

• The balance between speed and safety of the final product.

Best practice: start from the most sensitive component in your oligo and select conditions that protect it while still ensuring complete deprotection.

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Quick Reference Tables

Table 1: Standard DNA Deprotection

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Table 2: UltraFast (AMA, NH4OH + Methylamine)

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Table 3: Mild & UltraMild Approaches

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Table 4: RNA-Specific Conditions

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Troubleshooting – Common Problems & Fixes

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✅ Tip: When in doubt, start with the mildest compatible protocol. It’s easier to repeat deprotection than to recover from degraded oligos.