DNA markers and disease: NMR and MS clues

Marked out

Modifications to DNA associated with various autoimmune diseases as well as cancer can be identified thanks to work by a UK team. The researchers have developed a way to enrich the rare gene segments that contain the modified base hydroxymethylcytosine and to identify individual hydroxymethylcytosine molecules in DNA. Such changes act as markers telling the cell which genes it should read and which it shouldn't. As such, they are key to a better understanding of diseases involving DNA as well as offering a new approach to the development of therapies. The team used NMR spectroscopy and mass spectrometry to follow reactions in the laboratory.

The standard four bases - adenine, guanine, cytosine and thymine - that comprise the genetic code within DNA in every cell are ubiquitous but different tissues transcribe different sections of DNA. DNA has been likened to a computer operating system with RNA, which builds the proteins it encodes being the software running on the system rather than DNA being some kind of blueprint itself. Epigenetic factors, markers, are utilised by different cells in different ways and at different times in development to initiate transcription of specific genes as necessary. These markers might include a modification of the base cytosine in which different side groups are attached, such as a methyl or hydroxymethyl group. Researchers are aware that dense methylation of regulatory gene segments switches off the corresponding genes. For instance, during embryonic development, the pattern of methylation initiates the cell differentiation that causes the seemingly identical cells to begin to form different tissues. Moreover, changes in these methylation patterns can cause health problems as autoimmune responses are triggered or cell replication is no longer held in check so that tumours grow and cancer developments.

Epigenetic detection

Sequencing techniques that are able to specifically detect such epigenetic bases are important to understanding foetal development and disease. Hagan Bayley at the University of Oxford University and colleagues, Wen-Wu Li, Lingzhi Gong, explain that until now, the identification of hydroxymethylcytosine has been complex, expensive, and error-prone task. They have now developed a chemical modification that allows for the differentiation of hydroxymethylcytosine and methylcytosine through sequencing in nanopores.

The new result builds on technology developed by Oxford Nanopore, a company founded by Bayley in 2005. The process involves threading a strand of DNA through a membrane-embedded protein nanopore without the need for an enzymatic amplification step and can be used to obtain the sequence of individual strands. The current flowing through the pore correlates with the specific base in the pore at a given time.

Chemical processing

The team explains that they have now developed a chemical reaction between hydroxymethylcytosine, bisulfite, and a cysteine-containing peptide that does not affect any other base, modified or otherwise. They used high-performance liquid chromatography (HPLC) to isolate the products and proton NMR spectroscopy as well as UV-Vis absorption spectroscopy, and negative and positive ion mode electrospray ionization mass spectrometry to identify the further modified bases.

Their experiments showed that the reaction modifications can boost the resolution possible by improving the difference in current measured when the reacted hydroxymethylcytosine is in the pore. Moreover, that team has demonstrated that it is also possible to attach a fluorescent marker to the modified site, or a molecular "eye" that can be used to attach the rare hydroxymethylcytosine-containing DNA fragments to "hooks" that allow the fragments to be enriched over unmodified fragments, enabling rapid sequence analysis. The team suggests that the same approach might be used to modify other epigenetic marker bases.

"The next step is to enrich the hyrodxymethylcytosine-containing DNA in embronic stem cells and central nerve cells using our method, and develop methods of detecting other rare epigenetic bases," team member Li told SpectroscopyNOW. "Utimately, we hope to sequence those enriched DNA segments using the nanopore approach."