Recent advances in imaging technology have made it possible to visualise intracellular dynamics, which offers a greater understanding of several key biological principles for accelerating therapeutic development. Fluorescent labeling is one such technique that’s used to discover intracellular proteins, their dynamics, and dysfunction. Each internal in addition to external probes with fluorescent dyes are used for this purpose, although external probes can higher visualize intracellular proteins as in comparison with the interior probes. Nonetheless, their application is restricted by non-specific binding to intracellular components, leading to a low goal specific signaling and better background noise.

Recently, a fluorescent dye-labeled immunosensor referred to as Quenchbody (Q-body) has been successfully used to detect antigens in solutions or on the cell surface. A Q-body is basically an antibody fragment with the power to bind a particular antigen.

Against this backdrop, researchers from Japan and Singapore led by Prof. Hiroshi Ueda from Tokyo Institute of Technology (Tokyo Tech), Japan recently reported the applicability of Q-bodies for imaging of intracellular proteins in live cells. Their findings are actually published in Chemical Science.

For the reason that Q-body works as a site-specific and antigen-dependent imaging tool, we hypothesized that it should display antigen-dependent switchable fluorescence on interacting with the goal protein, enabling precise visualization of intracellular dynamics. We demonstrated this by synthesizing a Q-body for p53, a tumor suppressor biomarker protein that plays a very important role in DNA repair, cell division, and cell death.”

Prof. Hiroshi Ueda, Tokyo Institute of Technology

The team synthesized a “double” fluorescence dye-labeled Q-body called “C11_Fab Q-body,” which displayed higher sensitivity and goal specificity compared to traditional probes in human cancer cells expressing p53. For the reason that expression of p53 increases in cancer cells, they electroporated the Q-body in several human cancer cell lines to validate their hypothesis.

In comparison with a conventional probe that displayed continuous fluorescence signals even within the absence of p53, the Q-body probe displayed fluorescence signals in “fixed” cells (cells with denatured proteins to halt decomposition) expressing p53. Furthermore, the Q-body probe could visualize each wild (control) and mutant type p53 in fixed cell samples.

Further, the team observed fluorescence signals with 8-fold higher intensity in live human colon cancer cell lines with p53 expression as in comparison with the negatives. Interestingly, the Q-body was stable in the long run, displaying fluorescence intensity changes with experimentally induced changes in p53 levels.

Flow cytometry revealed higher immunofluorescence with Q-body in cells expressing p53. Moreover, on sorting, the ratio and fluorescence signal of those cells was significantly higher (see Fig. 1) in comparison with the others (with or without Q body).

What are the implications of those findings? Prof. Ueda answers, “The prevailing techniques are unable to offer precise imaging of less abundant intracellular targets with high specificity and sensitivity. On this context, our study demonstrates the potential of Q-bodies in live cell imaging for higher visualization of dynamical intracellular changes, and provides an approach for intracellular antigen-specific sorting of live cells using a Q-body.”

Going ahead, we will expect the event of many more Q-bodies for visualizing several other intracellular biomarkers, opening doors to improved cell-based therapeutic development and cancer research.

Source:

Tokyo Institute of Technology

Journal reference:

Dai, Y., et al. (2022) Intra Q-body: an antibody-based fluorogenic probe for intracellular proteins that enables live cell imaging and sorting. Chemical Science. doi.org/10.1039/d2sc02355e.