Epigenetics is the study of heritable silencing of genes where there is no change to the coding sequence. Interest in this area has grown significantly over the past few years particularly looking at changes induced and sustained by non-coding RNA gene silencing, histone modification and DNA methylation of cytidine in CpG islands.⁽¹⁾ At Biosearch Technologies, we offer a range of modifiers for this purpose. In particular we have amidites of 5-hydroxy-dC, 5-hydroxy-dU, 5-hydroxymethyl-dU, 5-hydroxymethyl-dC, 5-carboxy-dC, 5-formyl-dC, 5-hydroxymethyl-dC II and 5-formyl-dC III for use in the study of oxidative damage and repair, methylation and epigenetics.

Oxidised pyrimidines such as 5-hydroxy dU and 5-hydroxy dC are derived from dC via oxidative metabolic processes, UV or ionising radiation to form 5-HO-dC which spontaneously undergoes deamination to form 5-HO-dU.

Although there are repair mechanisms to convert 5-HO pyrimidines back to dC,⁽²⁾ the fact that they are observed in cellular DNA at consistent levels suggests that these repair mechanisms are inefficient,⁽³⁾ at least in certain cell types. Oligonucleotides modified with 5-hydroxy dU or 5-hydroxy dC are useful in understanding such processes.

The presence of either 5-HO-dU or 5-HO-dC can both lead to mutations resulting from their ability to mismatch with A and A/C respectively hence where the repair mechanism fails, such mutations can be permanently incorporated into the resulting gene. 5-Hydroxymethyl-dU (5-hmdU) is also a result of oxidative process or ionizing radiation but in this case dT is modified.⁽⁴⁾ It is also possible that 5-hmdU is formed by deamination of 5-hmdC but Müller and Carell recently showed that this does not contribute to the steady state levels of hmdU in mouse embryonic stem cells, but that dT is a substrate for ten eleven translocation enzymes (Tet) leading to the formation of 5-hmdU.⁽⁵⁾ Hence, hmdU is an important reagent for the study of both oxidative processes and epigenetics.

Once incorporated into an oligonucleotide, these modifiers represent the various products in the biochemical pathway of the modification of dC. In DNA, cytidine is methylated by a DNA methyl transferase catalysed reaction with S-adenosylmethionine to form 5-mdC. This is oxidised by Tet enzymes to 5-hydroxymethyl-dC which is further oxidised to 5-formyldC, which in turn is further oxidised to 5-carboxy-dC. Both 5-carboxy-dC and 5-formyl-dC can be converted back to dC via thymidine DNA glycosylase mediated base excision repair.⁽⁶⁾. The first generation hmdC phosphoramidite was fairly very well accepted but requires fairly harsh deprotection conditions. Therefore, a second generation building block (5-Hydroxymethyl-dC II) was developed that is compatible with UltraMild deprotection.⁽⁷⁾ 5-Formy-dC III has been designed to meet all of the requirements to prepare an oligo containing all of the methylated variants.⁽⁸⁾

Ref:

1. Epigenetics in human disease and prospects for epigenetic therapy, G. Egger, G. Liang, A. Aparicio and P.A. Jones. Nature, 429, 457-463, 2004.
2. Base excision repair in a network of defence and tolerance, H. Nilsen and H.E. Krokan, Carcinogenesis, 22, 987-998, 2001.
3. Endogenous oxidative damage of deoxycytidine in DNA, J.R. Wagner, H. Chia-Chieh and B.N. Ames, Proc. Nat. Acad. Sci., 89, 3380-3384, 1992.
4. Oxidative damage to DNA: formation, measurement, and biological significance, J. Cadet, M. Berger, T. Douki and J.-L. Ravanat, Rev. Physiol. Biochem. Pharmacol., 131, 1-87, 1997.
5. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA, T. Pfaffeneder, F. Spada, M. Wagner, C. Brandmayr, S.K. Laube, D. Eisen, M. Truss, J. Steinbacher, B. Hackner, O. Kotljarova, D. Schuermann, S. Michalakis, O. Kosmatchev, S. Schiesser, B. Steigenberger, N. Raddaoui, G. Kashiwazaki, U. Müller, C.G. Spruijt, M. Vermeulen, H. Leonhardt, P. Schär, M. Müller and T. Carell, Nat. Chem. Biol., 10 (7), 574-81, 2014.
6. Tet enzymes, TDG and the dynamics of DNA methylation, R.M. Kholi and Y. Zhang, Nature, 502, 472-479, 2013.
7. Efficient Synthesis of 5-Hydroxymethylcytosine Containing DNA, M. Münzel, D. Globisch, C. Trindler and T. Carell, Org. Lett., 12, 5671–5673, 2010.
8. Synthesis of a DNA promoter segment containing all four epigenetic nucleosides: 5-methyl-, 5-hydroxymethyl-, 5-formyl-, and 5-carboxy-2'-deoxycytidine, A.S. Schröder, J. Steinbacher, B. Steigenberger, F.A. Gnerlich, S. Schiesser, T. Pfaffeneder and T. Carell, Angewandte Chemie-International Edition, 53, 315-318, 2014.