Recent advances in imaging technology have made it possible to visualize intracellular dynamics, which offers a better understanding of several key biological principles to accelerate therapeutic development. Fluorescent labeling is one such technique which is used to identify intracellular proteins, their dynamics and their dysfunction. Both internal and external probes with fluorescent dyes are used for this purpose, although external probes can visualize intracellular proteins better than internal probes. However, their application is limited by nonspecific binding to intracellular components, resulting in weak target-specific signaling and higher background noise.
Recently, a fluorescent dye-labeled immunosensor known as Quenchbody (Q-body) has been successfully used to detect antigens in solutions or on the surface of cells. A Q body is essentially an antibody fragment capable of binding to a specific antigen.
In this context, researchers from Japan and Singapore led by Professor Hiroshi Ueda of Tokyo Institute of Technology (Tokyo Tech), Japan, recently reported the applicability of Q-bodies for imaging intracellular proteins in living cells. Their findings are now published in chemical sciences.
“Since the Q-body functions as a site-specific and antigen-dependent imaging tool, we hypothesized that it will display antigen-dependent switchable fluorescence when interacting with the target protein, allowing precise visualization of intracellular dynamics.We demonstrated this by synthesizing a Q-body for p53, a tumor suppressor biomarker protein that plays an important role in DNA repair, cell division and cell death,” says Professor Ueda.
The team synthesized a fluorescent dye-labeled “double” Q-body called “C11_Fab Q-body”, which showed better sensitivity and target specificity compared to conventional probes in p53-expressing human cancer cells. As p53 expression increases in cancer cells, they electroporated the Q body into several human cancer cell lines to validate their hypothesis.
Compared to a traditional probe that displayed continuous fluorescence signals even in the absence of p53, the Q-body probe displayed fluorescence signals in “fixed” cells (cells with denatured proteins to stop decay) expressing p53 . Moreover, the Q-body probe could visualize both wild type (control) and mutant type p53 in fixed cell samples.
Furthermore, the team observed fluorescence signals with 8-fold higher intensity in live human colon cancer cell lines with p53 expression compared to negatives. Interestingly, the Q body was long-term stable, displaying changes in fluorescence intensity with experimentally induced changes in p53 levels.
Flow cytometry revealed higher immunofluorescence with the Q body in cells expressing p53. Moreover, upon sorting, the fluorescence ratio and signal of these cells were significantly higher compared to the others (with or without Q-body).
What are the implications of these findings? Professor Ueda replies: “Existing techniques are unable to provide accurate imaging of less abundant intracellular targets with high specificity and sensitivity. In this context, our study demonstrates the potential of Q bodies in live cell imaging for a better visualization of dynamic intracellular changes., and provides an approach for antigen-specific intracellular sorting of live cells using a Q-body.”
In the future, we can expect the development of many more Q bodies to visualize several other intracellular biomarkers, paving the way for improved cellular therapeutic development and cancer research.
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Material provided by Tokyo Institute of Technology. Note: Content may be edited for style and length.