Luminescence counter

These identified aptamers were validated by the luciferase reporter assay as a secondary screen. Aptamers were synthesized by Sangon Biotech and cloned into the BsmBI sites of the vector phU6‐gLuc sgRNA‐scaffold‐1.2 BsmBI‐ scaffold‐polyT‐EF1a‐NLS‐linker‐EcoRI‐WT RBD‐EcoRI‐linker‐VPH. HEK293T/dCas9 cells were seeded at 2.5 × 10⁴ cells per well into 96‐well assay plates. And transient transfection was performed the next day using PEI with plasmids expressing 9 × gLuc target firefly/gaussia luciferase, hU6 ‐gLuc sgRNA‐scaffold‐1.2 aptamer‐scaffold‐polyT‐EF1a‐NLS‐linker‐EcoRI‐WT RBD‐EcoRI‐linker‐VPH, and a renilla luciferase expression plasmid as transfection control. The phU6‐gLuc sgRNA‐scaffold‐1.2 BsmBI‐ scaffold‐polyT‐EF1a‐NLS‐linker‐EcoRI‐WT RBD‐EcoRI‐linker‐VPH plasmid was used as a negative control. A total of 200 ng of DNA was transfected, and each plasmid was transfected in equal amounts. The medium was replaced with fresh medium 6 h after transfection. The firefly/gaussia and renilla luciferase activities were recorded sequentially by Luminescence Counter (Perkin Elmer) 48 h later.

HEK293 cell membrane integrity was evaluated using an LDH Cytotoxicity Detection Kit according to the manufacturer’s instructions. Briefly, cells were exposed to various concentrations of graphene oxide for 24 h, following which dead-cell protease activity was assessed using a previously described method [28 (link)]. The cytotoxicity assay was used to evaluate the cytotoxic effects of graphene oxide on HEK293 cells. Cytotoxicity was determined based on the reaction of intracellular proteases with a luminogenic peptide substrate (alanyl-alanyl-phenylalanyl-aminoluciferin). Luminescence was measured using a Luminescence Counter (Perkin Elmer, Waltham, MA, USA).

As described previously [42 (link)], the aminoacylation assays were performed at room temperature with 50 nM enzyme in 50 μL reaction mixture contains 50 mM Hepes (pH 7.5), 20 mM KCl, 5 mM MgCl₂, 4 mM ATP, 2 mM DTT, 5 μg/mL pyrophosphatase, 20 μM cold L-serine, 1.34 μM [3H]-serine (1 mCi/mL), and 200 μM yeast total tRNA (Roche Diagnostics, Indianapolis, Indiana, USA). The reaction was initiated by adding 50 nM enzyme into the reaction mixture. Aliquots of 5 μL were applied into 100 μL quench solution (300 mM NaOAc (pH 3.0), 1 mg/mL DNA, and 100 mM EDTA) in the Multi Screen 96-well filter plate (0.45 μm pore size hydrophobic, low-protein-binding membrane; Merck Millipore) at different time points of the reaction. After that, 100 μL of 20% (w/v) trichloroacetic acid (TCA) was added to precipitate the nucleic acids in each well. Plate was then washed 4 times with 200 μL/well of 5% TCA containing 100 mM cold L-serine, followed by 1-time wash with 95% ethanol. Air-dried plate was then treated with 70 μL/well of 100 mM NaOH to elute tRNAs. The eluted tRNAs were centrifuged into a 96-well flexible PET microplate (PerkinElmer, Santa Clara, California, USA) with 150 μL/well of Supermix scintillation mixture. After mixing, the radioactivity in each well of the plate was measured in a PerkinElmer 1450 Liquid Scintillation Counter and Luminescence Counter.

Dead-cell protease activity was assessed as previously described [44 (link)]. The cytotoxicity assay was used to evaluate the cytotoxic effects of AgNPs in HCT116 cells. Cytotoxicity was determined using the reaction of intracellular protease with a luminogenic peptide substrate (alanyl-alanylphenylalanyl-aminoluciferin). Luminescence was measured using a Luminescence Counter (Perkin Elmer, Waltham, MA, USA).