NS3a(BBa_25HYFBUH)

The hepatitis C virus (HCV) NS3/4A protease (NS3a) is a critical enzyme for viral replication and a well-established drug target. Its activity can be competitively inhibited by compounds such as the small-molecule drug Grazoprevir¹ and the short-peptide ANR, which bind to its active site.

Beyond its biological role, NS3a has emerged as a foundational component in synthetic biology due to these specific, drug-regulatable interactions. Its core utility lies in a dual-control mechanism: it naturally forms a high-affinity (nanomolar) complex with ANR, which Grazoprevir can efficiently dissociate². Concurrently, the same drug acts as a molecular bridge, enabling NS3a to recruit a computationally designed protein, GNCR³. This unique capacity for both chemically induced dissociation (CIDiss) from ANR and chemically induced dimerization (CIDimer) with GNCR makes NS3a an exceptionally versatile platform for engineering sophisticated genetic switches.

Figure 1. Schematic illustration of the mechanism of Grazoprevir induced NS3a-ANR dissociation or NS3a-GNCR interaction

Cell Culture and Transfection

HEK293T cells (ATCC CRL-3216) were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO₂. Cells were seeded at 5 × 10⁴ per well in 24-well plates and transfected 12 h later using a PEI-based protocol (PEI:DNA = 5:1, w/w). The transfection mix was replaced with fresh medium 6 h post-transfection. Unless indicated otherwise, transfection was performed at 16 h after seeding 5 × 10⁴ mammalian cells into each well of a 24-well plate. The cell culture medium was replaced with fresh medium (not containing transfection reagents) at 6 h after transfection. HEK-293T cells were transfected using a PEI-based protocol at a PEI:DNA ratio of 5:1 (w/w) and in a transfection volume of 50 μL native serum-free DMEM per well.

Reporter Quantification

Secreted NanoLuc levels were measured from cell culture supernatants using the Nano-Glo Luciferase Assay System (Promega, N1120). All measurements were performed in biological quadruplicates (n = 4). Statistical analyses used two-tailed unpaired t-tests (P < 0.05 considered significant)

For the CIDiss switch based on Grazoprevir-induced NS3a:ANR dissociation, HEK cells were co-transfected with three plasmids: PCMV-IgK-Fluc-TMD-NS3a-BGHPA (BBa_2506II6Z), P_CMV-ANR-TetR-NLS-VP64-BGHPA (BBa_2596NLG0), and TCE-IgK-Nluc-BGHPA (BBa_25EPQC3P) at a 1:2:6 DNA ratio (corresponding to 50 ng, 100 ng, and 300 ng per well, respectively). At 24 hours post-transfection, cells were treated with 10 µM Grazoprevir (experimental group) or an equivalent volume of DMSO (vehicle control). Supernatants were harvested 24 hours after drug treatment for NanoLuc luciferase quantification. The results showed a significant increase in NanoLuc activity in Grazoprevir-treated cells compared to the control, confirming the successful establishment of a functional NS3a-based CIDiss system capable of small molecule-regulated gene expression.

Figure 2. NS3a's function in CIDiss switch configurations. NanoLuc levels in culture supernatants 24 hours after Grazoprevir (10 µM) or DMSO treatment. Data shown as mean ± SD (n = 4); ***P < 0.001.

For the CIDimer switch based on Grazoprevir-induced NS3a:GNCR interaction, HEK cells were co-transfected with three plasmids: PCMV-TetR-NLS-NS3a-BGHPA (BBa_25NDCL0K), PCMV-GNCR-GSlinker-NLS-VP64-BGHPA (BBa_25PW9O16), and TCE-IgK-Nluc-BGHPA (BBa_25EPQC3P) at a 2:2:1 DNA ratio (corresponding to 200 ng, 200 ng, and 100 ng per well, respectively). At 24 hours post-transfection, cells were treated with 10 µM Grazoprevir (experimental group) or an equivalent volume of DMSO (vehicle control). Supernatants were harvested 24 hours after drug treatment for NanoLuc luciferase quantification. The results showed a significant increase in NanoLuc activity in Grazoprevir-treated cells compared to the control, confirming the successful establishment of a functional NS3a-based CIDimer system capable of small molecule-regulated gene expression.

Figure 3. NS3a's function in CIDimer switch configurations. NanoLuc levels in culture supernatants 24 hours after Grazoprevir (10 µM) or DMSO treatment. Data shown as mean ± SD (n = 4); ***P < 0.001

In our project, NS3a (BBa_25HYFBUH) was directly incorporated into the core response module and collaborated with the anchoring module to jointly construct a modular secretion control system(chemSPARK ,Figure 4A). By leveraging the Grazoprevir-induced NS3a:ANR dissociation mechanism, we successfully developed a molecular switch capable of rapidly responding to drug signals and precisely controlling the secretion of target proteins. This fully demonstrates the powerful functionality and application potential of this foundational component within complex genetic circuits.

HEK293T cells were co-transfected with plasmids carrying P_CMV–IgK–(Nluc–FS)3–TMD–(NS3a)2–BGHPA (BBa_25WEPAKX) and plasmids carrying P_CMV-ANR–mCherry–LifeAct–BGHPA (BBa_25NYLBFF) at a 1:3 DNA ratio (100 ng : 300 ng per well).After 48 h, cells were treated with 10 µM Grazoprevir (experimental group) or DMSO (control). Supernatants were collected 2 h post-treatment for NanoLuc quantification. Cells treated with Grazoprevir showed a significant increase in NanoLuc secretion, confirming that NS3a–ANR dissociation releases pre-anchored vesicles and activates rapid protein secretion (Figure 4B).

Figure 4. NanoLuc levels in culture supernatants 2 h after Grazoprevir (10 µM) or DMSO treatment. Data shown as mean ± SD (n = 4); ***P < 0.001.

Limitations:
In both transcriptional and secretory regulation contexts, the NS3a-based system functions as a drug-inducible module whose activity depends on the continued presence of its small-molecule inducer. Prolonged activation may be affected by drug metabolism, proteolytic degradation of the NS3a complex, or potential cytotoxic and off-target effects of the compound. These factors must be carefully balanced when designing long-term or high-sensitivity applications.

Comparisons:
Compared with optogenetic systems such as mMaple3 and CarH, which achieve noninvasive and precise light-based control, the NS3a-based system trades optical precision for pharmacological convenience. By relying on a diffusible small molecule rather than light, it overcomes limitations in tissue penetration and experimental setup, enabling stable and systemic activation suitable for in vivo or clinical scenarios that demand long-duration control.

Future Directions:
A key advantage of the NS3a platform is its use of clinically approved drugs such as Grazoprevir, which confers excellent biocompatibility and translational potential. This feature positions the NS3a-based switch as a valuable tool for drug-responsive disease modeling and high-throughput screening in mammalian cells. Beyond research use, it offers a foundation for drug-controllable therapeutic systems, allowing precise regulation of gene expression or secretion in engineered cells. Whether applied to long-term transcriptional modulation or rapid post-translational secretion, NS3a-based regulation provides a flexible and clinically relevant strategy for next-generation smart cell therapies.