The instructions for making all the proteins the body needs are encoded in DNA and found in every cell. But every cell is different and the proteins they need to produce at a given time change based on environmental cues. This information can be gleaned from messenger molecules known as RNA, which ferry protein-making instructions from DNA to the cellular factories where proteins are assembled.

Accessing a cell’s full complement of RNA, known as its “transcriptome,” is, therefore, a powerful research tool. However, existing techniques are unable to target individual cells without removing them from their natural tissue environments.    

A multidisciplinary Penn team, featuring researchers from the Perelman School of Medicine and the Departments of Chemistry and Biology in the School of Arts & Sciences, has now demonstrated a process to do just that. Their new technique is called TIVA, short for transcriptome in vivo analysis.

The team used TIVA to physically isolate the RNA of a single cell within living tissue in mice and human cells by “tagging” or capturing the RNA with a custom-built molecule.

With the ability to target a single cell inside intact tissue, rather than one isolated in a suspension, this method provides a unique opportunity to assess how cells really work in the body. It can also provide insight into how that function may go awry in various diseases and eventually help in testing new drugs.  

“Our data showed that the tissue microenvironment shapes the RNA landscape of individual cells,” says Jim Eberwine, professor of pharmacology at Penn Medicine and co-director of the Penn Genome Frontiers Institute.

The TIVA tag is a Swiss Army Knife-type of molecule, designed to contain the multiple chemical tools it needs to accomplish its task of capturing RNA. These tools include a means of entering cells, a fluorescent marker that indicates they are in place, a way for researchers to collect them after they complete their mission, and most important, a site that binds to RNA that can be activated at the appropriate time and place.

This aspect was critical to the researchers’ goal of targeting a single cell that was still embedded in live tissue. Unable to introduce the TIVA tag to the target cell without damaging its neighbors, the researchers designed this binding site with a removable chemical cage; the tag can’t begin capturing RNA until a laser with a specific wavelength breaks the cage open. That way, the researchers could have the molecule enter all the cells of a tissue sample, but only activate the ones in the specific cell they wanted to study.

“By coming in with blue light,” says Ivan Dmochowski, associate professor of chemistry, “the molecule falls apart in such a way that the [binding site] is revealed and can start binding to RNA.”

The team used the TIVA tag to profile gene expression in single neurons from intact brain tissue, and compared it to other cells in different growing conditions. The researchers say they were surprised to discover that cells in suspension expressed more genes as compared to cells in intact tissue, as if the isolated cells were making more RNA to be ready for anything in the absence of getting any meaningful chemical signals from surrounding cells.

Their findings reinforce the notion that “real biology” can’t be extrapolated from cells grown in unnatural conditions.