Journal: Nucleic Acids Research

Article Title: Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries

doi: 10.1093/nar/gky1228

Figure Lengend Snippet: Dual luciferase reporter monitoring A-to-I editing. The reporter utilizes the natural GluA2 editing substrate in which the R/G editing site has been modified into a stop codon (U A G) that upon editing is recoded into a tryptophan codon (U G G). A firefly luciferase reporter gene upstream of the edited site monitors translation and a nanoluciferase reporter gene downstream is used to measure read-through after editing. Editing is measured as the ratio between luminescence from nanoluciferase and firefly luciferase.

Article Snippet: For Nanoluciferase bioluminescence determination, 3 μl nanoluciferase substrate from Nano-Glo Luciferase Assay System (Promega) was added manually in a separate well containing 30 μl yeast culture from the same sample.

Techniques: Luciferase, Modification

Journal: Nucleic Acids Research

Article Title: Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries

doi: 10.1093/nar/gky1228

Figure Lengend Snippet: Highly detectable signal of editing in the reporter when transfected into HEK293 cells with cotransfected ADAR1 or ADAR2. Editing was determined as the ratio between luminescence from nanoluciferase and firefly luciferase (Nluc/FFL) expressed from the reporter plasmid. ( A ) Nluc to FFL signal ratio increased in a linear fashion when the reporter was cotransfected with increasing amounts of ADAR1 (left, R 2 = 0.993, n = 3) and ADAR2 (right, R 2 = 0.986, n = 3) expression vector. Error bars represent standard deviation. ( B ) Editing was verified by Sanger sequencing, and editing levels (G/(A+G) × 100) correlated logarithmically with the amount of cotransfected ADAR1 (left, R 2 = 0.997, n = 3) and ADAR2 (right, R 2 = 0.959, n = 3). Error bars represent standard deviation. ( C ) Reporter signal as a function of editing levels indicating that the Nluc/FFL ratio was exponentially dependent on editing percentage when the reporter was edited either by ADAR1 (left, R 2 = 0.991, n = 3) or by ADAR2 (right, R 2 = 0.983, n = 3). Error bars represent standard deviation.

Article Snippet: For Nanoluciferase bioluminescence determination, 3 μl nanoluciferase substrate from Nano-Glo Luciferase Assay System (Promega) was added manually in a separate well containing 30 μl yeast culture from the same sample.

Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Standard Deviation, Sequencing

Journal: Nucleic Acids Research

Article Title: Sensitive ADAR editing reporter in cancer cells enables high-throughput screening of small molecule libraries

doi: 10.1093/nar/gky1228

Figure Lengend Snippet: The transfected reporter sensing endogenous editing in different human cancer cell lines. Editing was determined as the ratio between luminescence from nanoluciferase and firefly luciferase (Nluc/FFL) in: HEK293, transformed human embryonic kidney cells; HeLa, cervical cancer cells; MCF7, breast cancer cells and MDA-MB-231, breast cancer cells. Positive control: the edited site (U A G) was mutated to Trp (U G G), mimicking 100% editing. Negative control: deletion of the editing complementary sequence prevented editing and was used as a negative control. Below, editing verified by Sanger sequencing of total RNA after RT-PCR and shown as a dual A and G peak in the chromatogram. Arrows indicate site of editing. Error bars indicate standard deviation ( n = 3).

Article Snippet: For Nanoluciferase bioluminescence determination, 3 μl nanoluciferase substrate from Nano-Glo Luciferase Assay System (Promega) was added manually in a separate well containing 30 μl yeast culture from the same sample.

Techniques: Transfection, Luciferase, Transformation Assay, Positive Control, Negative Control, Sequencing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation