Journal: Microbiology Spectrum

Article Title: Development of FRET-based cap-snatching endonuclease assay

doi: 10.1128/spectrum.03289-24

Figure Lengend Snippet: Examination of RNA endonuclease activity by RT-qPCR: (A) A schematic showing a linearized plasmid containing a T7 promoter or a PCR product with flanking T7 promoter is transcribed in vitro using a T7 transcription kit. The resulting RNA (test RNA) is purified and incubated with the endonuclease domain (endo.) in the absence or presence of the inhibitor at 37°C for 1 h. RNA lysis buffer is added to terminate the reaction, followed by the addition of 0.5 µg of total cellular RNA before the RNA purification is carried out. The test RNA is quantified by RT-qPCR using β-actin as internal control. (B–D). Test RNA (350 ng) was incubated with either phosphate-buffered saline (PBS; mock) or bacterially expressed and purified N protein (50 nM), RNase A (50 U), endonuclease domain, shown as Endo. (50 nM), the endonuclease D97A point mutant, shown as Endo.D97A (50 nM), in RNA digestion buffer at 37°C for 1 h in the absence (B) or presence of 200 µM DPBA (C) or 100 U of RNasin (D). The purified proteins used in this assay were dissolved in 1× PBS. The purified test RNA was quantified by RT-qPCR using β-actin as internal control. The quantified test RNA levels were normalized relative to mock and plotted along the Y -axis. (E–G). The experiment was repeated exactly as in (B)–(D), except that the PCR-amplified DNA was used instead of RNA. After the incubation of reaction mixtures at 37°C for 1 h, the DNA was purified by a plasmid min-prep kit without the addition of RNA lysis buffer or 0.5 µg of total cellular RNA, as shown in(A). The eluted DNA (5 µL) from each sample was quantified by real-time PCR, and the DNA levels were normalized to mock control (G) and plotted along the Y -axis.

Article Snippet: Purified RNA (350 ng) was then incubated with either a bacterially expressed and purified endonuclease domain, the endonuclease D97A point mutant, or the N protein at a concentration of 50 nM, or with 50 U of RNase A, in 50-µL of RNA digestion buffer (10 mM Tris-HCl, pH 8.0, and 1 mM MnCl₂) at 37°C for 1 h. Reactions were terminated by adding 300 µL of RNA lysis buffer from the RNA purification kit (Zymo), followed by incubation at room temperature for 10 min. Additionally, 0.5 µg of total RNA extracted from HEK293T cells was added to each reaction tube, immediately followed by RNA purification using the RNeasy kit ( , schematic).

Techniques: Activity Assay, Quantitative RT-PCR, Plasmid Preparation, In Vitro, Purification, Incubation, Lysis, Control, Saline, Mutagenesis, Amplification, Real-time Polymerase Chain Reaction

Journal: Microbiology Spectrum

Article Title: Development of FRET-based cap-snatching endonuclease assay

doi: 10.1128/spectrum.03289-24

Figure Lengend Snippet: FRET-based endonuclease assay. (A) A 20-nucleotide-long RNA (5′FAM-CUCCUCAUUUUUCGCUAGUU-IB3′), labeled with 6-FAM fluorophore at the 5′ terminus and Iowa Black (IB) quencher at the 3′ terminus, is referred to from here onward as FRET RNA. The FRET RNA is cleaved by the endonuclease domain, generating the dequenched fluorescence signal. (B) FRET RNA sample (120 nM) in digestion buffer (10 mM tris HCl, pH 8.0, and 1 mM MnCl2) was excited at 465 nm, and the emission spectrum was recorded from 495 to 650 nm (black line). The FRET RNA was then incubated with the purified cap-snatching endonuclease domain (20 nM), and the spectrum in the same wavelength range was again recorded (red line). The cartoon on the right shows that the treatment of FRET RNA with endonuclease domain (shown as sizer) causes the RNA cleavage, resulting in the generation of dequenched fluorescence signal (green). (C) The FRET RNA sample (120 nM) in digestion buffer was treated with either N protein (20 nM) or mutant endonuclease domain having D97A point mutation (20 nM) or with increasing concentrations (12, 20, or 23 nM) of the wild-type endonuclease domain, and the fluorescence intensity at 520 nm was recorded over time.

Article Snippet: Purified RNA (350 ng) was then incubated with either a bacterially expressed and purified endonuclease domain, the endonuclease D97A point mutant, or the N protein at a concentration of 50 nM, or with 50 U of RNase A, in 50-µL of RNA digestion buffer (10 mM Tris-HCl, pH 8.0, and 1 mM MnCl₂) at 37°C for 1 h. Reactions were terminated by adding 300 µL of RNA lysis buffer from the RNA purification kit (Zymo), followed by incubation at room temperature for 10 min. Additionally, 0.5 µg of total RNA extracted from HEK293T cells was added to each reaction tube, immediately followed by RNA purification using the RNeasy kit ( , schematic).

Techniques: Labeling, Fluorescence, Incubation, Purification, Mutagenesis

Journal: Microbiology Spectrum

Article Title: Development of FRET-based cap-snatching endonuclease assay

doi: 10.1128/spectrum.03289-24

Figure Lengend Snippet: Characterization of endonuclease activity. (A–F) The FRET RNA (A–C) or FRET DNA (D-F) at a concentration of 120 nM in digestion buffer were incubated with either 1× PBS (mock) or 20 nM N protein (negative control) or 50 U of RNase A (positive control) or 20 nM endonuclease domain, shown as an Endo. or 20 nM endonuclease D97A mutant, shown as Endo.D97A, at 25°C in the absence (A, D) or presence of either 200 µM DPBA (B. E) or 100 U of RNasin (C, F). The recorded fluorescence signal ( F 520 ) was plotted along the Y -axis. (G) The FRET RNA was diluted from 600 to 15.6 nM and incubated with 20 nM endonuclease domain at 25°C for 30 min. The fluorescence signal of FRET RNA samples treated with endonuclease domain was divided by the corresponding fluorescence signal of FRET RNA samples without endonuclease treatment. The ratio was plotted versus FRET RNA concentration.

Article Snippet: Purified RNA (350 ng) was then incubated with either a bacterially expressed and purified endonuclease domain, the endonuclease D97A point mutant, or the N protein at a concentration of 50 nM, or with 50 U of RNase A, in 50-µL of RNA digestion buffer (10 mM Tris-HCl, pH 8.0, and 1 mM MnCl₂) at 37°C for 1 h. Reactions were terminated by adding 300 µL of RNA lysis buffer from the RNA purification kit (Zymo), followed by incubation at room temperature for 10 min. Additionally, 0.5 µg of total RNA extracted from HEK293T cells was added to each reaction tube, immediately followed by RNA purification using the RNeasy kit ( , schematic).

Techniques: Activity Assay, Concentration Assay, Incubation, Negative Control, Positive Control, Mutagenesis, Fluorescence