Small pores aren’t just highly sought after in the cosmetics industry. They can really lend a hand in smoothing out the rougher epigenetics experimental challenges researchers face nowadays.
Just ask Dr. Meni Wanunu and Marija Drndic at the University of Pennsylvania, along with colleagues from New England Biolabs, Boston University, and Temple University, who put solid-state nanopores to work recently and obtained some glowing DNA methylation profiling results.
In addition to immunoprecipitation approaches for enriching 5hmC marks, researchers have been working hard to introduce robust techniques for discerning 5-methylcystosine (5mC) and 5-hydroxymethylcytosine (5hmC) at the single base level. (If you want to get a quick 5hmC fix, check out our 5hmC Analysis Guide)
Poring into 5hmC with Current
The research team tackled the 5hmC-mC conundrum with their nanopore set-up. Two electrolyte chambers were separated by a thin silicon nitride membrane through which a tiny tunnel was punched. Adding some juice drove ions from one chamber into the other through the tunnel, producing a current.
When long stretches of double-stranded DNA passed through the tunnel—which Wanunu says resembled “spaghetti being sucked into the mouth”—the drop in current reflected the DNA’s structure. By making identical-sequence strands of DNA with cytosine (C), mC, or hmC, the investigators demonstrated, both experimentally and with molecular dynamics simulations:
- DNA’s physical properties depended on the cytosine modification polarity which, in turn, produced characteristic current measurements.
- More polar hmC-DNA was more flexible than unmodified DNA. This correlation between cytosine polarity and DNA flexibility “may have epigenetic consequences,” says Wanunu.
- Few hundred molecules were all that was necessary to tell apart mC-DNA from hmC-DNA.
- C-DNA or mC-DNA with varying proportions of hmC produced different currents from which the amount of hmC could be calculated.
The investigators suggest nanopores may be the way to directly map hmC and mC patterns in DNA straight out of living cells. Get the details from Journal of the American Chemical Society December 2010