Nanoneedles help locate methylated mRNA in living cells

A new technique that relies on nanoneedles to fish for RNA in live cells offers a new way to measure intriguing but poorly understood chemical tags that pop on and off messenger RNA (J. Am. Chem. Soc. 2022, DOI: 10.1021/jacs.2c01036). These modifications—methyl groups that attach to RNA bases in various ways—affect gene expression and have links to development, cancer progression, and other cellular processes.

Researchers have a relatively solid understanding of how methylation on DNA regulates genes, but are just beginning to uncover the details of this process in RNA—in part because tools for studying RNA methylation in single cells or for probing how multiple types of methyl tags might work together have been lacking. “There are really no such tools out there,” says Chuan He, an RNA biologist at the University of Chicago who was not involved in the work. The new technique “allows you to point [a] needle at different parts of the cell and analyze the modifications.”

Researchers have identified at least four types of RNA methylations , with the most common being N⁶-methyladenosine (m⁶A). Currently, scientists identify m⁶A and other RNA methylations with liquid chromatography combined with mass spectrometry (LC-MS) or with high-throughput RNA sequencing. But the former can’t identify the genes on which the marks sit, and the latter is costly and time consuming. Also, both methods require prior isolation and purification of the RNA. The new technique is faster and easier, requiring no sequencing or complex chemical analysis, says Peng Shi, a biomedical engineer at City University of Hong Kong who co-led the work. And it can be conducted in intact, live cells.

The method rests on an RNA sampling method that Shi and his colleagues developed in 2015, in which an array of nanodiamond needles pierce a living cell membrane, snatch up genetic material, and sneak back out without damaging the cell. They attached m⁶A-binding antibodies onto the needles to pull out mRNA with that methylation from thousands of cells at once. The nanoneedles were attached to a specially fabricated chip on which the researchers amplified a specific gene of interest. The nanodiamond needles are small enough to target specific regions in a cell—say, a dendrite of a neuron—allowing researchers to assess the spatial distribution of RNA methylations within a cell, Shi says.

To determine whether the RNA of a gene might be tagged by two different types of methyl group, the researchers added another layer of detection. After capturing RNA with m⁶A modifications, they applied a second antibody that can pick out a less common methylation called N¹-methyladenosine (m¹A) on those strands. Then they used fluorescent tags to locate any m¹A sites and determine whether both m¹A and m⁶A methylations occur on the same gene.

“One can see some really interesting applications” of the method, He says—for example, tracking the distribution of RNA methylation within a neuron or a developing embryo. Shi says his team now plans to investigate spatial and temporal profiles of different types of RNA methylation in genes to see how these markers relate to malignancies and metastasis in cancer.