Materials and reagents

Biological materials

1. Competent cells E. coli strain BL21 (DE3) (Tsingke, catalog number: DLC201)

2. Plasmid containing HS protein: pET-21d-HS (Figure 1)

Figure 1. Plasmid map for the HaloTag-SNAPTag (HS) constructs used in the experiment. (A) Plasmid map of the HS expression construct in the pET-21d vector. (B) Amino acid sequence of the HS fusion protein.

Reagents

1. Ampicillin (Macklin, catalog number: A6265)

2. Dimethyl sulfoxide (DMSO) (Macklin, catalog number: D6258)

3. Methanol (MeOH) (Macklin, catalog number: M813907)

4. 4-Ethynylanisole (4-EA) (Macklin, catalog number: M813630)

5. 1,4-Dioxane (Macklin, catalog number: D807836)

6. Magnesium chloride (MgCl₂) (Macklin, catalog number: M813763)

7. Sodium chloride (NaCl) (Macklin, catalog number: S805275)

8. Phosphate buffered saline (PBS), 1× (pH 7.4) (Servicebio, catalog number: G4202-500ML)

9. Acetonitrile (ACN) (Macklin, catalog number: A800362)

10. Glutathione (GSH) (Macklin, catalog number: G6268)

11. Copper(II) sulfate hydrate (CuSO₄·5H₂O) (Macklin, catalog number: 767480)

12. Sodium ascorbate (NaAsc) (Leyan, catalog number: LY-TRCS612200)

13. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Bomeibio, catalog number: PI5502-25g)

14. LB medium mix powder (Hopebio, catalog number: HB0128)

15. Nitric acid 65% (SCR, catalog number: 10014518)

Solutions

1. LB medium (see Recipes)

2. IPTG 1 M (see Recipes)

3. CuSO₄·5H₂O 1 M (see Recipes)

4. NaAsc 1 M (see Recipes)

5. GSH 2 M (see Recipes)

6. TTA-Cl₃ 15.7 mg/mL (see Recipes)

7. Tris-Cl₃ 10.4 mg/mL (see Recipes)

8. Rhodamine-labeled benzylguanine (Rho-BG) 15.7 mg/mL (see Recipes)

9. 5-Hydroxy-2-vinylphenol acrylate (HVPA) 20 mM (see Recipes)

10. Diallylpropylene dibenzoate (DAPB) 100 mM (see Recipes)

11. Ethynyldiphenylmethyl allyl ether (EnDA) 200 mM (see Recipes)

12. 3-azido-7-hydroxycoumarin (AzHCou) 25 mM (see Recipes)

13. 4-EA 50 mM (see Recipes)

Note: The structures of TTA-Cl₃, Tris-Cl₃, Rho-BG, HVPA, DAPB, EnDA, and AzHCou are shown in Figure 2.

Figure 2. Chemical structures of non-commercial reagents used in this article. TTA-Cl₃, Tris-Cl₃, and Rho-BG were synthesized following [17]; AzHCou was synthesized following [19]; HVPA was synthesized following [20]; and DAPB and EnDA were synthesized following [21].

Recipes

1. LB medium (500 mL)

Reagent	Final concentration	Quantity or volume
LB medium mix powder	25 g/L	12.5 g
ddH2O	n/a	500 mL
Total	n/a	500 mL

Note: Autoclave at 121 °C for 30 min and aliquot. Store at RT.

2. IPTG 1 M (2 mL)

Reagent	Final concentration	Quantity or volume
IPTG	1 M	476.6 mg
ddH2O	n/a	2 mL
Total	n/a	2 mL

Note: Sterilize by filtration and store at -20 °C for up to one year.

3. CuSO₄·5H₂O 1 M (1 mL)

Reagent	Final concentration	Quantity or volume
CuSO4·5H2O	1 M	249.7 mg
ddH2O	n/a	1 mL
Total	n/a	1 mL

4. NaAsc 1 M (1 mL)

Reagent	Final concentration	Quantity or volume
NaAsc	1 M	198.1 mg
ddH2O	n/a	1 mL
Total	n/a	1 mL

Note: Prepare the solution fresh each day and sterilize it by filtration before use.

5. GSH 2 M (2 mL)

Reagent	Final concentration	Quantity or volume
GSH	2 M	1.229 g
ddH2O	n/a	2 mL
Total	n/a	2 mL

Note: Sterilize by filtration and store at -20 °C for up to one year.

6. TTA-Cl₃ 15.7 mg/mL (1 mL)

Reagent	Final concentration	Quantity or volume
TTA-Cl3	20 mM	15.7 mg
DMSO	n/a	1 mL
Total	n/a	1 mL

Note: Sterilize by filtration.

7. Tris-Cl₃ 10.4 mg/mL (1 mL)

Reagent	Final concentration	Quantity or volume
Tris-Cl3	20 mM	10.4 mg
DMSO	n/a	1 mL
Total	n/a	1 mL

Note: Sterilize by filtration.

8. Rho-BG 15.7 mg/mL (1 mL)

Reagent	Final concentration	Quantity or volume
Rho-BG	15.7 mg/mL (20 mM)	15.7 mg
DMSO	n/a	1 mL
Total	n/a	1 mL

Note: Sterilize by filtration.

9. HVPA 20 mM (1 mL)

Reagent	Final concentration	Quantity or volume
HVPA	20 mM	3.80 mg
DMSO	n/a	1 mL
Total	n/a	1 mL

Note: Sterilize by filtration.

10. DAPB 100 mM (1 mL)

Reagent	Final concentration	Quantity or volume
HVPA	100 mM	36.44 mg
DMSO	n/a	1 mL
Total	n/a	1 mL

Note: Sterilize by filtration.

11. EnDA 200 mM (1 mL)

Reagent	Final concentration	Quantity or volume
HVPA	200 mM	49.67 mg
DMSO	n/a	1 mL
Total	n/a	1 mL

Note: Sterilize by filtration.

12. AzHCou 50 mM (0.5 mL)

Reagent	Final concentration	Quantity or volume
HVPA	50 mM	5.08 mg
DMSO	n/a	0.5 mL
Total	n/a	0.5 mL

Note: Sterilize by filtration.

13. 4-EA 50 mM (0.5 mL)

Reagent	Final concentration	Quantity or volume
HVPA	50 mM	3.30 mg
DMSO	n/a	0.5 mL
Total	n/a	0.5 mL

Note: Sterilize by filtration.

Laboratory supplies

1. Automatic pipettes (ThermoFisher Scientific)

2. Pipette tips (Servicebio)

3. 2 mL microcentrifuge tube (Servicebio, catalog number: EP-200-M）

4. 15 mL centrifuge tube (Servicebio, catalog number: EP-1500-BJ）

5. 12 mL bacteria culture tube (Bikeman Bio, catalog number: 110408020)

6. Syringe filter (0.22 µm) (Merck Millipore, catalog number: C134954)

Equipment

1. Floor incubator shaker with cooling (Zhichu, model: ZQLY-180)

2. Biochemical incubator (Lichen, model: SPX-250B)

3. Ice machine (Meiling, model: MZB-25ZS)

4. Clean bench (Lichen, model: SW-CJ-1FD)

5. Biological safety cabinet (Esco, model: AC2-4S1)

6. Microplate reader (Thermo Fisher Scientific, model: Multiskan FC)

7. pH meter (Oustor, model: PHS-3C)

8. Fully automatic autoclave sterilizer (STIK, model: MJ-78A)

9. Vortex mixer (Scientific Industries, model: Vortex-Genie 2)

10. Centrifuge (Eppendorf, model: 5424R/5810R)

11. Microvolume spectrophotometer (Thermo Fisher Scientific, model: NanoDrop-2000)

12. Ultrapure water system (Merck Millipore, model: Elix 3)

13. Ultrasonic cell crusher (Scientz, model: JY92-IIN)

14. Confocal microscope (Nikon, model: A1RMP)

15. Confocal microscope with FRAP module (Carl Zeiss Microscopy, model: LSM980)

15. Inductively coupled plasma-mass spectrometer (ICP-MS) (Agilent Technologies, model: 7900)

16. High-performance liquid chromatography (HPLC) (Waters, model: 600; and Agilent Technologies, model: 1260)

17. Transmission electron microscope (TEM) equipped with an adapter for energy-dispersive X-ray spectroscopy (EDS) (JEOL, model: JEM-2100 Plus)

Procedure