A new study by researchers at the University of Chicago is a proof of concept that shows how DNA nanodevices can target specific cell types in living organisms, and how they might be used in the future for biomedical purposes.

DNA is an appealing candidate for a programmable medicine delivery device because of its known structural and binding properties. DNA-based nanodevices have shown promise as carriers of encapsulated cargo, however, directing these nanodevices to specific organs or cells where diseases may be localized has remained challenging, limiting their therapeutic potential.

Researchers have partially overcome this challenge by leveraging the transparency and powerful genetics of a microscopic roundworm – the nematode C. elegans. The team utilized C. elegans cell surface receptors that specifically bind to engineered DNA nanodevices, enabling them to home in to the exact location of interest. In addition to implications for diseases confined to individual organs, this innovation could represent a pathway for wider use of DNA nanodevices in more complex model organisms, as well as for therapeutics.

“If we could send cargo in, exactly where we want it, and release it, that would have enormous potential for drug delivery or environment sensing. That could be the future of this technology,” said Kasturi Chakraborty, PhD, the lead author on the paper, which was published on July 28 in eLife. The project was the product of ten years of work and collaborations between the labs of Yamuna Krishnan, PhD, Professor of Chemistry, and Paschalis Kratsios, PhD, Assistant Professor Neurobiology.

The researchers first tested if they could direct a DNA nanodevice to granules in C. elegans intestinal epithelial cells called lysosome-related organelles, or LROs, which play a role in things like pigmentation and immunity. They found a receptor, SID-2, that could bind small interfering RNA (siRNA), and designed their nanodevice using both DNA and RNA. The hybrid nanodevice (a 50 base pair long, double stranded RNA with a DNA scaffold) could be internalized, sent into intestinal epithelial cells and localized to LROs after binding SID-2.

Unfortunately, the SID-2 receptor present in LROs is not expressed in all cell types, including neurons. Therefore, simply feeding C. elegans the DNA nanodevices, as was done to target the LROs, would not be sufficient to reach neurons. The DNA nanodevice could still work, but it needed a new receptor target to latch onto. Their targeting technique was inspired by previous work in immune cells and the Golgi network, “but the antibodies we used there were too big to get inside the cells we were interested in. We needed something much smaller,” Chakraborty said.

To sidestep this additional obstacle, through a long and rigorous screening process, third author Sunaina Surana developed an antibody called 9E that binds DNA and can be fused to proteins expressed in neurons.

To test the ability of 9E to direct the DNA nanodevice in C. elegans neurons, the Krishnan lab joined forces with the Kratsios lab in the Department of Neurobiology. The researchers genetically fused 9E to synaptobrevin (SNB-1), a membrane protein found on synaptic vesicles. Neurons expressing the SNB-1-9E fusion protein successfully attracted and bound the DNA nanodevices. “We knew it could work,” Chakraborty said, “but we didn’t know how to get to the specific neurons we wanted to target. That’s where Dr. Kratsios came in.”

But the researchers were not yet satisfied. Instead, they sought to push even further, towards targeting the DNA nanodevices to specific neurons. Kratsios’s lab is studying neuronal development in C. elegans and has developed molecular tools that provide genetic access to specific neuronal populations and even specific regions of synapses. By leveraging these tools, the precision of the DNA nanodevice targeting could push the envelope on generic tissue level-targeting and achieve cellular and sub-cellular level accuracy.

Finally, to show that the nanodevices could not only reach their target but remain functional when they got there, second author Anees Palapuravan used pHlava-9E, which combined the capabilities of a DNA-based pH reporter but could engage 9E. When pHlava-9E was directed to neuronal cell membrane, it displayed the fluorescent signal expected of neutral pH environment like the cell membrane, thus indicating that the nanodevice remained functional.

Very few chemical technologies can be targeted to specific cells in a multicellular organism, and this work positions DNA nanodevices among fluorescent proteins and natural pathogens as the only other extraneously-introduced technology to possess both cell- and organelle-level specificity.

“The interest in what’s happening inside cells has increased a lot, and we need new technologies to shine light on what’s going on there. We as scientists often take cells out of their environment and then study them,” Chakraborty said. “But having technologies to study them in their local environment inside an animal would be awesome, and this kind of work in C. elegans is one step towards that. Getting the technology to the cells, and to specific locations inside the cells, is really exciting to me.”

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