Epigenetic modifications

With the explosion in interest in epigenetics in recent years, the need has arise for oligonucleotides containing epigenetic cytosine modifications (Figure 1).

Cytosine base modifications for use in epigenetic research

Figure 1 | Cytosine base modifications for use in epigenetic research

Synthesis and deprotection of oligonucleotides with modified cytosine

Oligonucleotides containing modified cytosines for epigenetic research are synthesized using conventional phosphoramidite solid-phase oligonucleotide synthesis, but replacing the standard cytosine phosphoramidite with modified cytosine phosphoramidites (Figure 2).

Structures of nucleoside phosphoramidite used to synthesize oligos containing epigenetic cytosine analogues

Figure 2 | Structures of nucleoside phosphoramidite used to synthesize oligos containing epigenetic cytosine analogues

Some changes to standard synthesis and deprotection conditions are required for these modified bases. The epigenetic bases (apart from 5-Methylcytosine) have functional groups at the 5-position that must be protected, which dictates the deprotection conditons for oligos containing these modifications, and compatibility with other modifications.

5-Methylcytosine

The 5-methylcytosine phosphoramidite monomer (Figure 2a) requires no modification to standard conditions for oligo synthesis and deprotection.

5-Hydroxymethylcytosine

Two 5-hydroxymethylcytosine phosphoramidite monomers are available. The standard 5-hydroxymethylcytosine phosphoramidite monomer (Figure 2b), in which the 5-hydroxymethyl group is protected as a cyanoethyl ether, can be deprotected using ammonium hydroxide, the standard reagent for oligonucleotide deprotection; however, harsh deprotection conditions must be used (65 °C for several days, or 75 °C for 17 h), which may be incompatible with other bases. This monomer cannot be deprotected using either sodium hydroxide or potassium carbonate. The alternative 5-hydroxymethylcytosine phosphoramidite monomer (Figure 2c) is compatible with sodium hydroxide and potassium carbonate deprotection, but urea derivatives are formed when standard ammonium hydroxide deprotection is used.

5-Formylcytosine

In the standard formylcytosine phosphoramidite monomer (Figure 2d) the aldehyde is is masked as a protected 1,2-diol. After oligonucleotide synthesis and deprotection (either using ammonium hydroxide, sodium hydroxide or potassium carbonate), the 1,2-diol must be converted to the aldehyde by oxidation with aqueous sodium periodate in a separate step (Figure 3). After the oxidation reaction is complete (in 30 minutes at 4 °C), excess periodate is removed by gel filtration or quenching with ethylene glycol, before HPLC purification.

Scheme showing the deprotection and oxidation of the 5-formylcytosine base after oligo synthesis

Figure 3 | Scheme showing the deprotection and oxidation of the 5-formylcytosine base after oligo synthesis

This standard 5-formylcytosine phosphoramidite has the advantage that it can be used with both standard (ammonium hydroxide) mild (aqueous base) deprotection conditions, which makes it potentially compatible with a wide range of other modifications; the disadvantage is that a separate oxidation step is required after oligo synthesis, which may be incompatible with some other modified bases.

An alternative 5-formylcytosine phosphoramidite (Figure 2e) is in development, in which the aldehyde is not protected, but the 4-amino (amidine) group is protected with as a benzoyl amide (Figure 2). This alternative 5-formylcytosine phosphoramidite monomer cannot be deprotected with potassium carbonate.

5-Carboxycytosine

In the 5-carboxycytosine phosphoramidite monomer (Figure 2f) the acid is protected as an ethyl ester, and the 4-amino (amidine) group is protected as a benzoyl amide. Standard deprotection with ammonium hydroxide yields the amide as a side product, along with the desired carboxylic acid. Deprotection with sodium hydroxide avoids this problem, although the use of sodium hydroxide in oligonucleotide deprotection prevents the use of dimethylformamidine-protected phosphoramidites in the rest of the oligonucleotide. The 5-carboxycytosine phosphoramidite monomer cannot be deprotected with potassium carbonate.

For further information see the Glen Research epigenetic base report.