Fluorescent antibody-based probe works in living cells

The research team's fluorescent antibody-based, genetically encoded probe works in living cells, and is low-cost.

A Frankenbody labeling mitochondria is shown.
A Frankenbody labeling mitochondria is shown.
Ning Zhao/Colorado State University

Antibodiesthe biomolecules that immune systems deploy to find, tag, and destroy invading pathogenswork by binding to specific targets, called epitopes, on the surfaces of antigens. Scientists have long harnessed this selective tagging mechanism in natural antibodies to engineer antibody-based probes that let them purify and study different types of proteins within cells. One technique to do so, epitope tagging, involves fusing an epitope to a protein of interest and using fluorescently labeled antibodies to make those proteins visible, but it only works in fixed, dead cells.

Recognizing these limitations, a team of researchers from Colorado State University (CSU; Fort Collins, CO) and the Tokyo Institute of Technology (Tokyo Tech; Tokyo, Japan) has developed an antibody-based, genetically encoded probe that works in living cells.

According to first author Ning Zhao, a postdoctoral researcher who designed most of the experiments, their new antibody-based probe is affectionately called a "frankenbody." Like stitching new limbs on a body, the scientists have taken the binding regions of a normal antibody, the "sticky parts," and grafted them to a different scaffold that remains stable in live cells, but retains the specificity of the antibody.

"We're interested in intracellular antibodies because you can use them as imaging reagents in a live cell," says Tim Stasevich, an assistant professor in the Department of Biochemistry and Molecular Biology at CSU, who partially led the work along with Tokyo Tech professor Hiroshi Kimura. "You don't need a tag, like a green fluorescent protein, because instead you have this fluorescent antibody that will bind to your protein that you want to visualize."

The new probe would be a useful complement to the green fluorescent protein (GFP), which involves genetically fusing a light-up green tag to a protein of interest. However, the GFP is limited by its relatively large size and the time it takes to fluoresce. With the CSU researchers' new probe, however, the tag is smaller and becomes fluorescent faster, so the "birth" of a protein of interest can be captured in real time.

With the goal of making their tool immediately useful, the scientists designed their probe to work with the classic HA tag. HA is a widely used small linear epitope tag that's derived from a portion of the human influenza virus protein hemagglutinin.

"For the longest time, people have been looking at HA-tagged proteins in fixed, dead cells," Stasevich says. "Now, we can image the dynamics of those proteins in live cells."

Stasevich's lab is particularly interested in studying RNA translation, and they plan to use their new system to more easily design new RNA imaging experiments.

The HA tag is a chain of just nine amino acids and the probe is genetically encoded on a plasmid that can be easily transferred into a cell, Zhao explains. This is in contrast to traditional antibodies, which can cost a lab several hundreds of dollars per order, suffer from lot-to-lot variability, and are difficult to get into cells. The new probe from Stasevich's team therefore provides a low-cost solution for protein and RNA translation imaging.

In their paper that describes the work, the scientists demonstrated some applications, including single-protein tracking, single-RNA translation imaging, and amplified fluorescence imaging in zebrafish embryos. All of these experiments are more challenging when using traditional fluorescent protein tags.

Based on this development, the research team has several new imaging reagents in the works, Stasevich says.

Full details of the work appear in the journal Nature Communications.

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