Journal: Biochimica et biophysica acta

Article Title: A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system

doi: 10.1016/j.bbagrm.2017.12.008

Figure Lengend Snippet: DNA is shown in gray, except for PNA binding sequence, which is shown in turquoise; RNA is shown in black, except for sequence transcribed from PNA-binding insert, which is shown in dark blue; PNA is shown in magenta. PNA contains duplex- and triplex-forming moieties, connected by a flexible linker. Primary and secondary promoters are shown in brown-red and orange, respectively. Bent arrows indicate the start and the direction of transcription. RNA polymerase (RNAP) is shown as a gray oval.

Article Snippet: At the second step, 4 μl of the T7 transcription mixture, obtained from the first step were added to 11 μl of the mixture with the same composition as described above, except that it contained 1.7 units/μl of T3 RNAP (Promega corp, Madison, WI) instead of T7 RNAP, and in addition, it was supplemented with 10 μCi of radioactive (α- 32 P) CTP; and incubated for 30 min at 37°C.

Techniques: Binding Assay, Sequencing

Journal: Nature Communications

Article Title: Plug-and-play protein biosensors using aptamer-regulated in vitro transcription

doi: 10.1038/s41467-024-51907-4

Figure Lengend Snippet: a dART templates consist of a double-stranded promoter region (pink boxed region), a single-stranded aptamer domain on the template strand read by T7 RNAP (Apt), and a double-stranded output domain (O1). The single-stranded aptamer domain permits aptamer-ligand binding. b Secondary structure of the RNA transcript predicted by NUPACK . c The aptamer sequences of IFN-O1-dART, which binds IFN-γ and Dummy-O1-dART, whose aptamer domain does not have a tertiary structure or specific protein affinity. d dART transcripts react with an O1 DNA reporter via toehold-mediated strand displacement. e Reacted reporter kinetics from IFN-O1-dART and Dummy-O1-dART transcription with and without IFN-γ and potassium. f Simulated and experimental dose–response curves for 10 nM IFN-O1-dART with 0 to 1000 nM IFN-γ for K d,apparent = 8 nM (Supplementary Information Note ). The dashed line is a simulation for K d,apparent matching experiments. The shaded region shows simulation results for K d,apparent values spanning 1 to 20 nM (see “Methods”). g Experimentally measured (bold) and simulated (dashed) reacted reporter kinetics for 10 nM of IFN-O1-dART and 0 to 1000 nM of IFN-γ. h Simulated and experimental dose–response curves and i Experimental (bold) and simulated (dashed) reacted reporter kinetics ( h ) for 1 nM IFN-O1-dART and 0 to 100 nM of IFN-γ using the K d,apparent determined in ( f ). For ( f ) and ( h ), the reacted reporter concentration for each plot was measured at 120 min. Three technical replicates (blue) are plotted. Error bars indicate the average reacted reporter concentrations of three technical replicates per [IFN-γ] ± one s.d. ( N = 3). Supplementary Information Note describes the process for determining K d,apparent .

Article Snippet: T7 RNAP was purchased in bulk (300,000 U) from Cellscript (200 U μl −1 , C-T7300K) as well as from ThermoFisher Scientific (200 U μl −1 , EP0113).

Techniques: Ligand Binding Assay, Concentration Assay

Journal: Nature Communications

Article Title: Plug-and-play protein biosensors using aptamer-regulated in vitro transcription

doi: 10.1038/s41467-024-51907-4

Figure Lengend Snippet: a Schematic of coupled RNA transcription of dARTs by T7 RNAP and RNA degradation by RNase H. b The concentration of reacted reporter over time for an experiment with 10 nM IFN-O1-dART, 0 to 1000 nM of IFN-γ, 2 U µL −1 of T7 RNAP, and 2 × 10 −3 U µL −1 of RNase H. Shaded regions enclose the minimum and maximum values for independent trials ( N = 2; see “Methods”). c Steady-state analog outputs of IFN-O1-dART, Thr-O1-dART, IL6-O1-dART, and TNF-O1-dART with 5 to 1000 nM of their corresponding proteins after 240 min. Two individual technical replicates are plotted ( N = 2).

Article Snippet: T7 RNAP was purchased in bulk (300,000 U) from Cellscript (200 U μl −1 , C-T7300K) as well as from ThermoFisher Scientific (200 U μl −1 , EP0113).

Techniques: Concentration Assay

Journal: Nature Communications

Article Title: Plug-and-play protein biosensors using aptamer-regulated in vitro transcription

doi: 10.1038/s41467-024-51907-4

Figure Lengend Snippet: a Circuit diagram for an amplified IFN-C1’-dART/Ref-C1-dART comparator. The Ref-C1-RNA output, which is high when IFN-γ exceeds a threshold concentration, activates transcription of a genelet that transcribes O4, which is detected by a reporter. b The reactions through which Ref-C1-RNA coactivates G1O4 to produce the O4 output. c Reacted reporter kinetics of IFN-AC-50-O4, which consists of 25 nM Ref-C1-dART, 50 nM IFN-C1’-dART, 100 nM G1O4:B1, 200 nM A1, and 2000 nM O4 DNA reporter, for 0 nM or 50 nM IFN-γ. d Reacted reporter kinetics of the IFN-AC-1-O4, which consists of 0.5 nM of Ref-C1-dART, 1 nM IFN-C1’-dART, 20 nM of G1O4:B1, and 100 nM of A1, and 250 nM of O4 DNA reporter for IFN-γ inputs from 0 to 4 nM. 4 U µL −1 T7 RNAP and 4 × 10 −3 U µL −1 RNase H were used in IFN-AC-1-O4. e Comparison of the dose–response curves of IFN-AC-1-O4, IFN-C-30-O1, IFN-C-50-O1, and IFN-C-100-O1. Three individual technical replicates are plotted. Lines connect the average reacted reporter concentrations of three technical replicates per [IFN-γ]. Shaded regions indicate the average value of three technical replicates ± one s.d. ( N = 3).

Article Snippet: T7 RNAP was purchased in bulk (300,000 U) from Cellscript (200 U μl −1 , C-T7300K) as well as from ThermoFisher Scientific (200 U μl −1 , EP0113).

Techniques: Amplification, Concentration Assay, Comparison

Journal: Nature Communications

Article Title: Dynamic self-assembly of compartmentalized DNA nanotubes

doi: 10.1038/s41467-021-23850-1

Figure Lengend Snippet: a Inactive tiles are activated by an RNA trigger transcribed using a synthetic gene and subsequently assemble into nanotubes. b A computational model illustrates that by increasing the concentration of synthetic gene template, we can speed up the assembly kinetics and increase the equilibrium fraction of assembled tiles (modeling details in Supplementary Note ). Increasing amounts of gene template are represented with darker shades of blue. c Inactive tiles at 500 nM concentration were encapsulated with transcription mix, 2.5% w/v PEG, and 2.5% v/v RNAP and incubated at 37 °C. d – g Representative fluorescence microscopy images of droplet samples in which we titrated the amount of synthetic gene transcribing RNA trigger (insets) with plots of mean skewness (purple) and kurtosis (orange) for sampled droplets. The rate of increase of skewness and kurtosis correlates with the concentration of DNA template producing the trigger RNA strand. Data were presented as mean values ± standard deviation. Images were processed using the droplet detection code as described in Supplementary Note . The number of droplets sampled and a histogram data of radii for sampled droplets are reported in Supplementary Note and . Source data for this figure is provided as a source data file. Scale bars: 20 μm.

Article Snippet: T7 RNAP was purchased from Lucigen ® and RNase H was purchased from Promega TM .

Techniques: Concentration Assay, Incubation, Fluorescence, Microscopy, Standard Deviation

Journal: Nature Communications

Article Title: Dynamic self-assembly of compartmentalized DNA nanotubes

doi: 10.1038/s41467-021-23850-1

Figure Lengend Snippet: a Schematic of the reactions occurring in a sample that includes inactive tiles, DNA template transcribing the RNA trigger (promoting growth of nanotubes), and RNase H (promoting degradation of nanotubes). b Computational prediction showing that loss of activity of RNAP against unchanged RNase H activity yields a temporal pulse in the fraction of assembled tiles, whose peak and duration depend on the amount of RNase H. Increasing amounts of RNase H are represented with darker shades of pink. Modeling details are in Supplementary Note . c – f Representative fluorescence microscopy images of the RNase H titration experiments (insets) with plots of mean skewness (purple) and kurtosis (orange) for sampled droplets. Inactive tiles at 500 nM concentration were encapsulated with 100 nM gene template, 2.5% w/v PEG, 2.5% v/v RNAP, and 0.025 U/µL–0.1 U/µL RNase H. These experimental results show that, when RNA trigger transcription and degradation components are simultaneously present, a pulse of nanotube polymerization is observed. Eventually RNase H causes disassembly of the nanotubes at a speed that depends on the RNase H concentration, presumably due to a decrease of RNAP activity. Skewness and kurtosis are plotted in shades of purple and orange, respectively. Data were presented as mean values ± standard deviation. Data extracted using droplet detection code as described in Supplementary Note S4.13. The number of droplets sampled and histograms of radii for sampled droplets are in Supplementary Notes and . (Also see: Supplementary Movie ). Source data for this figure is provided as a source data file. Scale bars: 20 μm.

Article Snippet: T7 RNAP was purchased from Lucigen ® and RNase H was purchased from Promega TM .

Techniques: Activity Assay, Fluorescence, Microscopy, Titration, Concentration Assay, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

doi: 10.1016/j.omtn.2020.06.023

Figure Lengend Snippet: PAGE of Products of Transcripts from Templates Containing l -T at Different Positions of the Transcribed Region in the Template Strand Transcription reactions were carried out using T7 RNAP on templates T/N, T+4/N, T+7/N, T+8/N, T+10/N, and T+12/N. The amounts of T7 RNAP of four lanes with the same template were 0, 25, 50, and 100 U/mL, respectively. The template concentration of each lane was 1 μM. The positions of the migration of the DNA templates and the RNA products were marked on the left on the gel. The DNA template used in each reaction is indicated at the top of each lane.

Article Snippet: T7 RNAP was purchased from Beijing Biolink Biotechnology (NEB, Beijing, China).

Techniques: Concentration Assay, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

doi: 10.1016/j.omtn.2020.06.023

Figure Lengend Snippet: PAGE of Transcripts from Templates Containing l -T at Different Positions of the Non-transcribed Region in the Template Strand Transcription reactions were carried out using T7 RNAP on templates T/N, T−3/N, and T−10/N. The amounts of T7 RNAP of four lanes with the same template were 0, 25, 50, and 100 U/mL, respectively. The template concentration of each lane was 1 μM. The positions of the migration of the DNA templates and the RNA products were marked on the left on the gel. The DNA template used in each reaction is indicated at the top of each lane.

Article Snippet: T7 RNAP was purchased from Beijing Biolink Biotechnology (NEB, Beijing, China).

Techniques: Concentration Assay, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

doi: 10.1016/j.omtn.2020.06.023

Figure Lengend Snippet: PAGE of Transcripts from Templates Containing l -T at Different Positions of the Non-template Strand Transcription reactions were carried out using T7 RNAP on templates T/N, T/N−2, T/N−8, and T/N+11. The amounts of T7 RNAP of four lanes with the same template were 0, 25, 50, and 100 U/mL, respectively. The template concentration of each lane was 1 μM. The positions of the migration of the DNA templates and the RNA products were marked on the left on the gel. The DNA template used in each reaction is indicated at the top of each lane.

Article Snippet: T7 RNAP was purchased from Beijing Biolink Biotechnology (NEB, Beijing, China).

Techniques: Concentration Assay, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

doi: 10.1016/j.omtn.2020.06.023

Figure Lengend Snippet: PAGE of Transcripts from Templates Containing Two l -Ts at Different Positions of the Transcribed Region in the Template Strand Transcription reactions were carried out using T7 RNAP on templates T/N, T+7+8/N, and T+8+10/N. The positions of the migration of the DNA templates and the RNA products were marked on the left on the gel. The DNA template used in each reaction is indicated at the top of each lane.

Article Snippet: T7 RNAP was purchased from Beijing Biolink Biotechnology (NEB, Beijing, China).

Techniques: Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

doi: 10.1016/j.omtn.2020.06.023

Figure Lengend Snippet: Running Start Assays of Transcription Reactions Conducted with Three Templates (T/N, T+4/N, and T+7/N) by T7 RNAP (A) Analysis of transcripts of the three templates at various times on denaturing gels. (B) Kinetic curves of transcripts from different templates. A plot of the amount of 17-mer versus time for reactions carried out on the three templates and fit to A(1 − e −kobs t ) is shown, where A is the amount of 17-mer transcripts at long time points, and k obs is the time constant for productive transcription initiation (as measured by the appearance of the 17-mer transcripts). ▪, T/N; ●, T+4/N; ▲, T+7/N.

Article Snippet: T7 RNAP was purchased from Beijing Biolink Biotechnology (NEB, Beijing, China).

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

doi: 10.1016/j.omtn.2020.06.023

Figure Lengend Snippet: Michaelis-Menten Plots of the Transcription Reaction (Transcript Formation) by T7 RNAP as a Function of the Concentration of NTP with Templates ▪, T/N; ●, T+4/N; ▲, T+7/N. The concentration of T7 RNAP was kept at 5 U/μL.

Article Snippet: T7 RNAP was purchased from Beijing Biolink Biotechnology (NEB, Beijing, China).

Techniques: Concentration Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

doi: 10.1016/j.omtn.2020.06.023

Figure Lengend Snippet: Fidelity of Incorporation of NTP Opposite l -T in a Natural Template (T 2 /N 2 and T 2 +7/N 2 ) in a Transcription Reaction Catalyzed by T7 RNAP Sequences of templates (A) and PAGE of transcripts (B) in the fidelity analysis. The NTP added is indicated on the bottom of each lane. Transcription experiments were performed with a standard reaction system except that the NTP mixtures were substituted to a single nucleotide.

Article Snippet: T7 RNAP was purchased from Beijing Biolink Biotechnology (NEB, Beijing, China).

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

doi: 10.1016/j.omtn.2020.06.023

Figure Lengend Snippet: Insights into NTP Addition from 3D Structure of T7 RNAP (A–F) Conformation of the active center of T7 RNAP before (A) and after (B) NTP insertion or in complex with template T+4/N (C, d -T; D, l -T; PDB: 1QLN ) or template T+7/N (E, d -T; F, l -T; PDB: 3E2E ).

Article Snippet: T7 RNAP was purchased from Beijing Biolink Biotechnology (NEB, Beijing, China).

Techniques: