Color is exciting and fun—just look at Andy Warhol’s striking silkscreen paintings of Marilyn Monroe. Now, a new method uses bright red and green fluorescence to detect global levels of both methylated and hydroxymethylated cytosine (5mCs and 5hmCs) modifications of genomic DNA in the same straightforward assay, which could lead to easy and inexpensive clinical tests.
Elmar Weinhold and Yuval Ebenstein’s labs (RWTH Aachen University and Tel Aviv University) wanted to use a pop of color to make this measurement easy enough for personnel working in high-volume clinical labs to run, while also ensuring accuracy and sensitivity. The team had already worked out how to bring hues to 5hmC, so they turned their attention to labeling 5mC. But it’s not easy to work with an inert methyl group, so they instead devised a way to color unmodified cytosines, and that info can be used to calculate the amount of methylation. Here’s the method:
- They produced a double mutant of a CpG-specific DNA methyltransferase M.Mpel called M.Mpel (dm) that has a larger-than-usual active site to accommodate unnatural cofactors
- M.Mpel (dm) along with unnatural cofactors enables the transfer of azides to DNA specifically at CpGs, and adds a vibrant fluorophore with azide-alkyne cycloaddition
- Place samples on a multi-sample slide and analyze the fluorescence with a slide scanner and imaging software
The team used the method to label colon samples from patients with colon cancer and healthy controls—the same samples they had already labeled previously for 5hmC. The results:
- As reported before, 5hmC levels declined in cancer samples
- The new data showed that 5mC levels remained about the same in patient samples on average
Next up, they combined the 5hmC and CpG labeling on the same samples at the same time on chronic lymphocytic leukemia (CLL) and control samples, both whole blood and peripheral blood mononuclear cells (PBMCs). They labeled for 5hmC (red fluorescence), then labeled unmodified CpGs (green fluorescence).
- With single-molecule microscopy, they added a blue fluorophore for the DNA, and saw blue strings with red and green dots
- Then, they conducted the high-throughput version with many labeled samples per slide analyzed with a slide scanner and saw that both modifications were lower in patients’ blood and cells
The method was easy, fast and sensitive, and delivered the same results with tissues and blood, at least for CLL, which means that a simple blood test using the method could be sufficient.
Bring some color to your world at ChemBioChem, July 2023.