NEW YORK – A team of researchers from the University of Cambridge has developed a new method that enables in situ mapping of small molecules that bind to cellular genomic DNA or chromatin-associated proteins.
Described in a Nature Biotechnology study published in January, the method, dubbed Chem-map, provides scientists a new tool to directly detect where small molecule drugs bind to chromatin, opening a window for them to further interrogate genome and chromatin function and design therapeutic targets.
"It was always very challenging to prove that the molecule actually engages with the structures in cellular chromatin," said Shankar Balasubramanian, a biochemistry professor at the University of Cambridge and the corresponding author of the study. "We have been trying to find ways of doing it for a very long time. It was not an easy problem to crack."
According to Balasubramanian, one of the motivations behind the study is that, for decades, researchers have been trying to investigate the biological function of G-quadruplexes (G4s), noncanonical secondary nucleic acid structures where guanine-rich DNA sequences fold into tetrads.
While the exact roles of G4s still need to be determined, Balasubramanian said, the scientific community generally believes that these structures are associated with gene regulation, cell fate transitions, and cancers. "The really interesting thing is that [G4s] occur in the parts of the gene structure that are involved in the regulatory regions," he noted.
However, researchers have lacked an effective way to directly study these structures and the molecules that bind to them, Balasubramanian said. Previous enrichment-based methods have tried to detect DNA binding via biophysical experiments in vitro, but typically cannot elucidate molecular binding events in the context of cellular chromatin.
"We tried such methods in the past, but just could not detect where G-quadruplex-binding molecules interact," he said, adding that one major shortcoming of previous approaches is that they typically employ noncovalent interactions, which may fall apart during DNA processing and lose the information about the binding site in situ.
To overcome this obstacle, Balasubramanian and his team developed Chem-map, which relies on small molecules to ultimately recruit transposases, which can insert indexed sequencing adapters proximal to the small-molecule-binding sites.
Mechanistically, Chem-map introduces a covalent affinity tag to the small molecule of interest in a way that doesn't interfere with its primary interaction. "The interaction doesn't have to be a permanent interaction," Balasubramanian explained. "It just has to be long enough to allow this enzyme to carry out its catalytic function proximally to the binding site."
After that, the tag recruits a transposase (Tn5) to the binding site, marking the site via proximal transposition events. Subsequently, tagmented DNA marking the small-molecule binding sites are extracted, selectively amplified, sequenced, and mapped by alignment of sequenced reads to the genome.
For their study, the Cambridge researchers demonstrated Chem-map using three different small molecule-binding modalities, including BET bromodomain protein-binding inhibitor JQ1, DNA G-quadruplex structure-binding molecules PDS and PhenDC3, and anticancer drug doxorubicin in human leukemia cells.
Overall, the researchers noted that Chem-map can be "a robust approach" for mapping the interactions of small molecules with genomic DNA and chromatin proteins in cells. "For the first time, we managed to reliably detect where quadruplex binding molecules sit," Balasubramanian said, adding that the method was also able to pick up transient interactions during the binding events.
Additionally, Chem-map revealed the chromatin binding landscape for doxorubicin, a clinically approved cancer drug that purportedly acts by targeting DNA but has not yet been directly mapped to genomic DNA in vivo. "To the best of my knowledge, this is the first study that shows where it binds," he said. "In therapeutics, the more information you have on what the drug is doing — and what it isn't doing — the better. Otherwise, you're dealing with a bit of a black box."
The method is "really elegant," said Michael Booth, a chemistry professor at University College London who was not involved in the study. "You look at it now and you think: Why has no one done that before?"
By combining different techniques to achieve completely new functionality, Booth said Chem-map offers researchers an innovative tool to directly examine small molecules’ chromatin binding events, which is "a very unexplored area that seems like it should have been explored a long time ago."
"There are plenty of small molecules that target the genome, or proteins that interact with the genome. Where do they bind? How do they react?" he added. "All these things are completely necessary for understanding biology and medicine, [and] drugs and tools that people have been using for years but don't really know how they work."
One of the potentially challenging aspects of replicating Chem-map in other labs, Booth said, is designing molecules with tags attached that will not impact their normal activities. However, beyond molecule design, he thinks the method uses fairly standard sequencing methods, and it "could easily be implemented anywhere." That said, because Chem-map is sequencing-based, it can still be relatively expensive, Booth noted.
With the Cambridge researchers’ proof of concept, Booth said one future direction for Chem-map is to design a larger portfolio of small molecules. "If you generate a bigger suite of molecules that have these tags," he said, "you can start to really try and understand the limitations and the amazing things you can identify with all the data of this method."
Balasubramanian said Chem-map could be useful in both basic and translational research to help profile drug binding to chromatin in vivo. Mirroring Booth’s point, he also thinks molecule design is an important component for future implementation.
"Depending on your molecule and your target, there is a need to optimize the molecular design," he pointed out. To that end, Balasubramanian noted that the cost of Chem-map can be influenced by the molecule synthesis process, depending on the complexity of the target. Besides that, he said the method should be "no different from any other genomic experiment."
Balasubramanian said his team will continue to improve Chem-map and release additional versions of the protocol. Additionally, he said there are potential opportunities to commercialize the technology for chromatin-targeting drug development.
"I think it has great potential," he said. "What I can say at this stage is our university looked at the [Chem-map’s] capability, and it wasn't too difficult to persuade them of [the method’s] potential, particularly in drug development and the pathway towards therapeutics."