RESULTS

This project aims to develop a novel, high-performance genetically encoded fluorescent probe for nitric oxide (NO) using protein engineering strategies. The probe is designed based on the regulatory domain of the bacterial transcription factor NorR, which will be rationally engineered and evolutionarily optimized through directed evolution before being fused with a fluorescent protein.

This approach enables highly sensitive and specific fluorescent responses to changes in NO concentration. Furthermore, using molecular biology techniques, the probe will be precisely targeted to specific subcellular locations—such as the cytoplasm and mitochondria—to achieve in situ, high spatiotemporal resolution imaging of NO dynamics in living cells.

The successful development of this probe will provide a crucial tool for elucidating the central role of NO in physiological and pathological processes. It also holds promise for applications in early diagnosis of suboptimal health conditions, evaluation of therapeutic efficacy, and the development of targeted drugs, thereby advancing the field of precision medicine.

To develop novel nitric oxide (NO) biosensors, we inserted the YFP, GFP, and mCherry sequences into the pRSETa-NorR vector using BamHI and KpnI restriction cloning (see Fig. 1 for construction workflow). This engineered vector provides an N-terminal 6×His tag to facilitate subsequent protein purification and contains a T7 promoter for high-level expression in host cells.

Fig.1 Vector construction of NorR-YFP_pRSETa, NorR-mCherry_pRSETa and NorR-GFP_pRSETa

A: PCR of YFP sequences; B: PCR of mCherry sequence; C: PCR of GFP sequences; D: The pRSETa vector after double enzyme digestion.

Following transformation of the ligation product into DH5α competent cells, overnight cultivation yielded abundant transformant colonies (Fig.2). Preliminary screening of the clones was performed by colony PCR using vector-specific primers (T7 promoter and terminator primers). As shown in Fig.3, all randomly selected clones produced a single DNA band of the expected size, indicating preliminary success in the insertion of the target fragment into the vector.

Fig.2 The screening plate for the transformation of recombinant plasmid by DH5α

Fig.3 PCR Verification of the recombinant plasmid

To obtain final confirmation, the purified plasmid constructs were subjected to Sanger sequencing. The sequencing results indicated that no mutations were introduced into the recombinant vectors, confirming the successful construction of the plasmids.

Based on the successfully constructed plasmids, the NorR-fluorescent protein fusion was expressed in E. coli BL21(DE3) cells. The bacterial culture was induced with 0.42 mM IPTG and incubated at 18°C with shaking at 220 rpm for 24 hours. After centrifugation, the cell pellets were resuspended in HEPES buffer and stored at –80°C. For purification, the resuspended cells were disrupted by sonication followed by centrifugation. The supernatant was incubated with nickel magnetic beads at 4°C for 3 hours to allow binding of the His-tagged fusion protein. Stepwise elution was performed using imidazole concentrations ranging from 50 to 500 mM. Fractions eluted with 100 mM and 300 mM imidazole were collected for further analysis. The purified proteins are shown in Fig. 4.

Fig.4 The purification results of recombinant proteins NorR-YFP, NorR-GFP and NorR-mCherry

4.1Functional Comparison of Different Fluorescent Probes

After obtaining the three fluorescent probes—YFP, GFP, and mCherry—we systematically evaluated their nitric oxide (NO) binding capabilities to identify the optimal performer. YFP and GFP were analyzed using a dual-excitation ratiometric method, measuring fluorescence intensity at 515 nm under excitations of 420 nm and 485 nm, followed by ratio calculation. mCherry was assessed by direct fluorescence intensity measurement under 590 nm excitation and 645 nm emission. Each probe was incubated with varying concentrations of the NO donor Furoxan. The ratio of fluorescence intensity after adding the NO donor to that under baseline (zero NO) conditions was used as the evaluation metric (also applied in subsequent experiments). As shown in Fig.5, with increasing Furoxan concentration, both YFP and GFP exhibited significant ratiometric changes in a dose-dependent manner, eventually reaching saturation. In contrast, mCherry showed no notable change in fluorescence ratio across the concentration range, indicating its inability to effectively respond to NO. Further comparison revealed that YFP demonstrated a markedly greater response amplitude than GFP, suggesting higher sensitivity. Therefore, YFP was selected as the NO-sensing probe for subsequent studies.

Fig.5 Fluorescence response curves of the YFP-, GFP- and mCherry-NO probes

4.2Functional Validation of the NorR-YFP Fluorescent Probe

After identifying YFP as the optimal probe, we conducted systematic functional validation of the NorR-YFP fusion protein to comprehensively evaluate its performance under practical application conditions. Key parameters including pH stability, thermal stability, concentration-dependent response, binding specificity, detection reliability in plasma, and subcellular localization capability were assessed to ensure the probe's accuracy and reliability in complex biological environments.

First, the thermal and pH stability of NorR-YFP were examined. Each reaction system contained 100 μL of 1 μmol/L NorR-YFP protein. The experimental group was treated with 100 μM Furoxan (NO donor), while the control group received an equal volume of buffer. As illustrated in Fig.6, a significant increase in the fluorescence ratio was observed after Furoxan-induced NO release. The probe exhibited consistent responsiveness within the pH range of 7.2–8.0 and the temperature range of 20–40°C, demonstrating robust environmental adaptability and detection stability.

Fig.6 pH value and temperature stability of NorR-YFP

To assess the binding specificity of NorR-YFP, we introduced 100 μM of Furoxan, CO, H₂O₂, NaNO₂, and NaHS, respectively, and measured corresponding changes in the fluorescence ratio. As shown in Fig. 7, a rapid and significant increase in the fluorescence ratio occurred only in the Furoxan group, with no notable changes observed in other groups, confirming highly specific responsiveness of the probe to nitric oxide (NO).

Fig.7 Specificity assessment of NorR-YFP toward various reactive species

To establish the quantitative relationship between fluorescence intensity of NorR-YFP and NO concentration, the protein was treated with different concentrations of the NO donor Furoxan. A solution of 100 μL of 1 μmol/L protein was used, and changes in the fluorescence ratio were dynamically monitored over 40 minutes. A positive correlation was observed between Furoxan concentration and the fluorescence signal of the NorR-YFP sensor (Fig. 8), indicating a clear dose-response relationship to NO.

Fig.8 Real-time Monitoring of NorR-YFP Fluorescence Kinetics upon Furoxan Stimulation