Hee-Jin Jeong

Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence

Recently, we described a novel reagentless fluorescent biosensor strategy named Quenchbody, which functions via the antigen-dependent removal of the quenching effect on a fluorophore that is attached to a single-chain antibody variable region. To explore the practical utility of Quenchbodies, we prepared antibody Fab fragments that were fluorolabeled at either one or two of the N-terminal regions, using a cell-free translation-mediated position-specific protein labeling system. Unexpectedly, the Fab fragment labeled at the heavy chain N-terminal region demonstrated a deeper quenching and antigen-dependent release compared to that observed using scFv. Moreover, when the Fab was fluorolabeled at the two N-termini with either the same dye or with two different dyes, an improved response due to enhanced quenching via dye-dye interactions was observed. On the basis of this approach, several targets, including peptides, proteins, and haptens, as well as narcotics, were quantified with a higher response up to 50-fold. In addition, differentiation of osteosarcoma to osteoblasts was successfully imaged using a similarly fluorolabeled recombinant Fab protein prepared from E. coli. Due to its versatility, this “Ultra-Quenchbody” is expected to exhibit a range of applications from in vitro diagnostics to the live imaging of various targets in situ.

Detection of vimentin serine phosphorylation by multicolor Quenchbodies

Protein phosphorylation is a key event in intracellular signal transduction, and fluorescent biosensor for the specific phosphorylation event in a target protein is considered highly useful as a tool of cellular biology and drug screening. Vimentin, the most abundant intermediate filament protein, is phosphorylated at its specific serine (Ser) residues in a cell cycle dependent manner. Its structural and functional characteristics are modified by the phosphorylation, which affects biological properties of the cell. Here we present the detection of the vimentin Ser71 phosphorylation (PS71) and the vimentin Ser82 phosphorylation (PS82) using a novel fluorescent biosensor Quenchbody, which works on the principle of antigen-dependent removal of a quenching effect by intrinsic tryptophan residues on a carboxytetramethylrhodamine (TAMRA) dye incorporated at the N-terminal region of single chain antibody variable region. First, we found that rhodamine 6G (R6G)-labeled Quenchbody shows superior response than TAMRA-labeled one. Next, we made several Quenchbodies to detect PS71 and PS82. After optimization of reaction conditions, the fluorescence intensity of V(H)-V(L) type PS71 Quenchbody labeled with R6G at two positions was increased to 4.0-fold in an antigen dependent manner. Furthermore, the fluorescence intensity of doubly R6G-labeled V(L)-V(H) type PS82 Quenchbody was increased to 6.7-fold immediately after adding antigen peptide, also suggesting deeper quenching due to H-dimer formation between the dyes. Due to its simplicity, the Quenchbody-based phosphorylation biosensors will be widely applicable to in vitro diagnostics, drug screening and imaging in a rapid, simple and high-sensitive manner.

Strategy for Making a Superior Quenchbody to Proteins: Effect of the Fluorophore Position

Antibody-based sensors have made outstanding contributions to the fields of molecular biology and biotechnology. Our group recently developed a novel powerful fluorescent immunosensor strategy named Quenchbody (Q-body), which has been applied to the detection of a range of antigens in a rapid, simple, and sensitive manner. However, there were some Q-bodies whose fluorescence response was limited, especially for detecting protein antigens. With the aim of improving this issue, here we made twelve types of Q-bodies incorporated with different number and position of TAMRA fluorophore in the single chain Fv of HyHEL-10, an anti-hen egg lysozyme antibody, as a model. By measuring the fluorescence intensity and its antigen dependency, it was revealed that VL-VH type Q-bodies labeled at a non-CDR loop region of the VL shows the highest fluorescence response. This position locates close to the quenching Trp35 in VL, while it is far from Trp residues in the bound antigen. This result clearly suggests the importance of dye position to maximize the fluorescence quenching and antigen-dependent de-quenching. The discovery may open a way to make many other Q-bodies with superior response.

One-pot construction of Quenchbodies using antibody-binding proteins

Fluorolabeled antibody-binding proteins were constructed based on Staphylococcus protein A and Streptococcus protein G domains, and used as an adaptor to convert the Fab fragment of interest to a Q-body, a fluorescent biosensor that exhibits antigen-dependent fluorescence enhancement. Without having to perform the tedious procedure of genetically introducing a fluorescent dye molecule into a cloned Fab fragment, we successfully converted both a cloned anti-osteocalcin Fab fragment and a commercially available anti-vimentin (Fab)2 fragment to a Q-body using this method. This method is not only a simpler way for constructing Q-bodies but also a convenient alternative to finding a suitable antibody that has a greater potential to become an excellent biosensor.

A Signal-On Fluorosensor Based on Quench-Release Principle for Sensitive Detection of Antibiotic Rapamycin

An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose “Q’-body”, which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q’-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q’-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.

Development of a Quenchbody for the Detection and Imaging of the Cancer-Related Tight-Junction-Associated Membrane Protein Claudin

Claudins (CLs) are membrane proteins found in tight junctions and play a major role in establishing the intercellular barrier. However, some CLs are abnormally overexpressed on tumor cells and are valid clinical biomarkers for cancer diagnosis. Here, we constructed antibody Fab fragment-based Quenchbodies (Q-bodies) as effective and reliable fluorescent sensors for detecting and visualizing CLs on live tumor cells. The variable region genes for anti-CL1 and anti-CL4 antibodies were used to express recombinant Fab fragments, and clones recognizing CL4 with high affinity were selected for making Q-bodies. When two fluorescent dyes were conjugated to the N-terminal tags attached to the Fab, the fluorescent signal was significantly increased after adding nanomolar-levels of purified CL4. Moreover, addition of the Q-body to CL4-expressing cells including CL4-positive cancer cells led to a clear fluorescence signal with low background, even without washing steps. Our findings suggested that such Q-bodies would serve as a potent tool for specifically illuminating membrane targets expressed on cancer cells, both in vitro and in vivo.