rna polymerase  (New England Biolabs)

Journal: bioRxiv

Article Title: Protein hyperacylation links mitochondrial dysfunction with nuclear organization

doi: 10.1101/2020.10.23.350892

Figure Lengend Snippet: Supplemental analysis of nucleosome stability on reconstituted DNA constructs, related to Figure 3 . (A,B) Analysis of nucleosome stability by monitoring T7 RNA polymerase transcription pausing and stopping during elongation at 4°C in vitro as described in methods. RNA transcripts were radiolabeled during preliminary open complex formation by transcription of a U-free cassette prior to cooling to 4°C and additional of all NTPs. Quantitation of two main stop sites near the center of the reconstituted nucleosome and normalization to the extent of template reconstitution (Nucleosome Obstacle Activity Per Reconstituted Template; NOAPRT) was as described in methods. Reduced NOAPRT values reflect less stable nucleosomes. In vitro nucleosome acylating agents and temperatures are indicated. (A) Example of results of single pilot study for multiple conditions. (B) Representative result and quantitation from independent studies (n = 3). (C) Agarose gel analysis of acylation-dependent effects on the stability of reconstituted nucleosomes, related to Figure 3K,L . Bands visualized with EtBr post-staining. Novel nucleosome species indicated as *.

Article Snippet: T7 RNA polymerase transcription assay through reconstituted nucleosome In vitro transcription reactions to assay nucleosome stability were performed by mapping radiolabeled RNA transcription products of T7 RNA polymerase elongation into a reconstituted nucleosome.

Techniques: Construct, In Vitro, Quantitation Assay, Activity Assay, Agarose Gel Electrophoresis, Staining

Journal: Nature Communications

Article Title: SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

doi: 10.1038/s41467-021-25337-5

Figure Lengend Snippet: A novel type III CRISPR-Cas tool for the sensitive detection of nucleic acids. a Denaturing PAGE resulting from activity assays performed with the CARF protein TTHB144 and a 5′ Cy5-labeled reporter RNA. Activation of TTHB144 due to cOAs produced by the reconstituted TtCmr-46 complex was monitored by offering fully complementary (T) target RNAs, or target RNA with mismatches in segments one (S1), four (S4) or by a single mismatch in the CAR (C1). A fully non-target RNA (NT) was used as a control. Results of the TTHB144 cleavage assay is representative of results obtained from three replicates (Supplementary Fig. S6 ). b Limit of detection (LOD) assay using reconstituted Cmr-46 on a synthetic SARS-CoV-2 E-gene and a fluorophore-quencher reporter RNA masking construct measured over time. A non-target RNA (NT) was used as a control. Data was obtained from three replicates. c Schematic overview of the 2-step reaction setup consisting out of (1) a (RT)-LAMP based pre-amplification step and (2) a T7-based in vitro transcription and type III CRISPR detection step. ‘F-Q’ represents the fluorophore-quencher reporter RNA. d Limit of detection assay using reconstituted Cmr-46 in the 2-step setup (depicted in panel c ), with a SARS-CoV-2 synthetic full RNA genome as target. Data was obtained from three replicates. e Detection of SARS-CoV-2 in human swab samples. Ct-values of qPCR analysis ( Orf1ab gene) of 81 samples are depicted on the X-axis with the true negative samples displayed as not determined (ND). See Supplementary Fig. S5 and Supplementary Table S2 for Ct-values of qPCR analysis of SARS-CoV-2 samples and respective Scope tool score. The data on the qPCR and SCOPE analysis was obtained from one replicate measurement. f One-pot LAMP-CRISPR limit of detection assay, using reconstituted Cmr-46, on a synthetic SARS-CoV-2 E-gene. Data was obtained from two replicates. All error bars in this figure represent the standard deviation of the mean. ‘Arb. units’ stands for arbitrary units. Source data are provided as a Source data file.

Article Snippet: Due to the maximum temperature tolerance of an appropriate, commercially available RNA polymerase (Hi-T7 RNAP, NEB), we performed a one-pot LAMP-CRISPR assay at a temperature of 55 °C.

Techniques: CRISPR, Polyacrylamide Gel Electrophoresis, Activity Assay, Labeling, Activation Assay, Produced, Cleavage Assay, Construct, Amplification, In Vitro, Detection Assay, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: medRxiv

Article Title: Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific Viral Diagnostic

doi: 10.1101/2020.10.14.20212670

Figure Lengend Snippet: CRISPR-Csm-based detection of SARS-CoV-2 in clinical nasopharyngeal swab samples. ( A ) Schematic comparing RT-qPCR to RT-LAMP-CRISPR-Csm based detection. In RT-qPCR reaction , reverse transcribed template is amplified in PCR reaction that cycles between different temperatures. As Taq DNA polymerase (DNA pol.) synthesizes nascent DNA strand it degrades the probe annealed to amplified template and releases the fluorophore (green star) from the quencher (black hexagon). In CRISPR-Csm detection , the viral RNA is reverse transcribed, and resulting DNA is amplified in an RT-LAMP reaction to produce transcription templates for T7 RNA polymerase, in one pot. An aliquot of the RT-LAMP-T7 reaction (40 min) is then mixed with CRISPR-Csm reaction (20 min). ( B ) Nasopharyngeal swabs from 24 individuals were tested with RT-qPCR and RT-LAMP-T7 CRISPR-Csm. Top panel shows threshold cycle (Ct) values for RT-qPCR. Swabs with Ct values below 40 for both N1 and N2 CDC diagnostic primers are considered positive for SARS-CoV-2 RNA (nd – signal is not detected over during 45 cycles). Vertical dashed line highlights the Ct value corresponding to a concentration of 10 6 SARS-CoV-2 RNA copies/mL ( Supplemental Fig. 4 ). Bottom panel shows CRISPR-Csm based detection in the same swab samples targeting nucleocapsid (N) gene, as shown in (A). Data is shown as fold change in fluorescence compared to a no-template control reaction. Technical repeats are shown in triplicate. * - swab sample test result was inconclusive both for CDC assay and CRISPR-Csm detection.

Article Snippet: RT-LAMP-T7 CRISPR-Csm Isothermal amplification of nucleic acids in swab samples was performed by combining RT-LAMP (NEB) with in vitro transcription in a single reaction using Hi-T7 RNA polymerase (NEB).

Techniques: CRISPR, Quantitative RT-PCR, Amplification, Polymerase Chain Reaction, Diagnostic Assay, Concentration Assay, Fluorescence, CDC Assay

Journal: medRxiv

Article Title: Inexpensive and colorimetric RNA detection by E. coli cell-free protein synthesis platform at room temperature

doi: 10.1101/2021.11.29.21267025

Figure Lengend Snippet: Optimization and screening of isothermal RNA amplification platforms. (A) Non-specific products were detected with NASBA reaction, in parallel with previous reports 30 (B) Homemade NASBA reaction additive screening. (C) Influence of random RNA duplex on T7 RNA polymerase efficiency. Please note that more pronounced RNA bands were observed with the additions of the random duplex, in comparison to (A) and (B). (D) Influence of different reaction conditions and temperature cycling. (D) Room temperature amplifications by NASBA and RT-RPA. (Note that prior to loading onto gel, RT-RPA reactions –but not NASBA– were pre-incubated at 95 °C for 3 min and immediately placed on ice, in order to dissociate the SSB from the amplicons.) We also note that under reported reaction conditions, our initial attempts for RT-RCA did not yield amplification.

Article Snippet: Final reaction mixture contained 1x reaction buffer, deoxyribonucleotide mix (1 mM each), ribonucleotide mix (2 mM each), forward and reverse primers ([final] = 1 µM each), 0.01 U of Yeast inorganic pyrophosphatase (YIPP, New England Biolabs), 0.4 U RiboLock RNase inhibitor (ThermoFisher), 0.08 U RNaseH (New England Biolabs), RevertAid Reverse Transcriptase 128 U (ThermoFisher), 32 U Hi®-T7 RNA polymerase (New England Biolabs), 500 nM random duplex primer pair, 1 µL of template RNA (varying amounts), 10% (v/v) DMSO, filled up to 10 µL of final volume with RNase/DNase-free water.

Techniques: Amplification, Recombinase Polymerase Amplification, Incubation