Genetically-encoded Fluorescent Sensors

Real-time in vivo signal measuring

Evrogen's genetically-encoded fluorescent sensors are complex chimeric proteins consisting of a fluorescent protein and a sensing polypeptide fused in a specific manner. The sensors demonstrate clearly detectable changes in fluorescence in response to corresponding cell events. The fluorescent biosensors allow precise targeting into various subcellular compartments or animal tissues and real-time in vivo measuring of signals in natural intracellular surroundings.

Genetically-encoded fluorescent indicators for monitoring changes in intracellular concentration of calcium ions (Ca²⁺) and hydrogen peroxide (H₂O₂), and sensors for early detection of caspase-3 apoptosis are offered.

Hydrogen Peroxide Sensor HyPer

• Ratiometric detection of intracellular H₂O₂ level changes
• High selectivity and sensitivity, no artifactual ROS generation
• Direct expression in cells, easy targeting to various subcellular compartments
• No exogenous chemical compounds required
• Recommended for monitoring H₂O₂ production inside living cells

HyPer is the first fully genetically encoded fluorescent sensor capable of detecting intracellular hydrogen peroxide (H₂O₂), one of the main ROS generated by cells [Belousov et al., 2006]. Developed on the basis of yellow fluorescent protein inserted into the regulatory domain of E. coli protein OxyR (OxyR-RD) [Choi et al., 2001], HyPer demonstrates submicromolar affinity to hydrogen peroxide and is insensitive to other oxidants tested, such as superoxide, oxidized glutathione, nitric oxide, and peroxinitrite. HyPer does not cause artifactual ROS generation and can be used for detection of fast changes of H₂O₂ concentration in different cell compartments under various physiological and pathological conditions.

Without H₂O₂ HyPer has two excitation peaks with maxima at 420 nm and 500 nm, and one emission peak with maximum at 516 nm. Upon exposure to H₂O₂, the excitation peak at 420 nm decreases proportionally to the increase in the peak at 500 nm, allowing ratiometric measurement of H₂O₂.

Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods. 2006; 3 (4):281-6. / pmid: 16554833

Choi H, Kim S, Mukhopadhyay P, Cho S, Woo J, Storz G, Ryu S. Structural basis of the redox switch in the OxyR transcription factor. Cell. 2001; 105 (1):103-13. / pmid: 11301006

Calcium Ion Sensor Case12

• High dynamic range detection of intracellular Ca²⁺ level changes
• High selectivity and sensitivity, relatively high pH stability
• Fast maturation, high brightness of fluorescent response
• Direct expression in cells, easy targeting to various subcellular compartments
• No exogenous chemical compounds required
• Recommended for monitoring changes in Ca²⁺ concentration inside living cells

Case12 is a high dynamic range genetically encoded fluorescent sensor for direct measurement of changes of intracellular Ca²⁺ under various physiological and pathological conditions [Souslova et al., 2007]. The sensor is sensitive to changes of calcium concentration in a physiological range from a hundred nanomoles to micromoles with a high signal-to-noise ratio. Binding of Ca²⁺ is fast and reversible, allowing monitoring of high-frequency Ca²⁺ oscillations. In response to Ca²⁺ concentration rise, Case12 shows up to 12-fold increase of fluorescence brightness. Fluorescence of Case12 is characterized by single excitation/emission maxima peaked at 491/516 nm.

Case12 is recommended for monitoring change of calcium concentration inside living cells during various physiological and pathological conditions.

Souslova EA, Belousov VV, Lock J, Stromblad S, Kasparov S, Bolshakov AP, Pinelis VG, Labas YA, Lukyanov S, Mayr LM, Chudakov DM. Single fluorescent protein-based Ca²⁺ sensors with increased dynamic range. BMC Biotechnol. 2007; 7 :37. / pmid: 17603870