s pyogenes  (New England Biolabs)


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    Monarch RNA Cleanup Kit 10ug
    Description:
    The Monarch RNA Cleanup Kit 10 µg rapidly and reliably purifies and concentrates up to 10 μg of concentrated high quality RNA 25 nt from enzymatic reactions including labeling capping in vitro transcription IVT and DNase I treatment This kit utilizes a bind wash elute workflow with minimal incubation and spin times Our unique column design ensures zero buffer retention and no carryover of contaminants enabling elution of sample in volumes as low as 6 μl Eluted RNA is ready for use in a variety of downstream applications including RT PCR RNA Library Prep for NGS and transfection The protocol can also be modified to enable the purification of smaller RNA fragments 15 nts
    Catalog Number:
    t2030
    Price:
    52
    Size:
    10 preps
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    RNA Purification Kit Components
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    Structured Review

    New England Biolabs s pyogenes
    Monarch RNA Cleanup Kit 10ug
    The Monarch RNA Cleanup Kit 10 µg rapidly and reliably purifies and concentrates up to 10 μg of concentrated high quality RNA 25 nt from enzymatic reactions including labeling capping in vitro transcription IVT and DNase I treatment This kit utilizes a bind wash elute workflow with minimal incubation and spin times Our unique column design ensures zero buffer retention and no carryover of contaminants enabling elution of sample in volumes as low as 6 μl Eluted RNA is ready for use in a variety of downstream applications including RT PCR RNA Library Prep for NGS and transfection The protocol can also be modified to enable the purification of smaller RNA fragments 15 nts
    https://www.bioz.com/result/s pyogenes/product/New England Biolabs
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    s pyogenes - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Staphylococcus aureus Cas9 is a multiple-turnover enzyme"

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme

    Journal: RNA

    doi: 10.1261/rna.067355.118

    S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA
    Figure Legend Snippet: S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA

    Techniques Used:

    2) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    3) Product Images from "Variability within rare cell states enables multiple paths towards drug resistance"

    Article Title: Variability within rare cell states enables multiple paths towards drug resistance

    Journal: bioRxiv

    doi: 10.1101/2020.03.18.996660

    Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. A . Experimental approach for identifying the subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. These experiments began with approximately 400,000 WM989 A6-G3 cells transduced at an MOI ∼ 1.0 and allowed to divide for 6 days before splitting the culture into two groups. We treated one group with 4 μM DOT1L inhibitor (pinometostat) and the other with vehicle control (DMSO) for another 6 days. We then split each group again, fixing half as our “Carbon Copies” and treating the other half with 1 μM vemurafenib for ∼2.5 weeks. After vemurafenib treatment, we extracted genomic DNA from the remaining cells for barcode sequencing. B . We compared the abundance of each barcode identified in resistant cells pre-treated with DOT1L inhibitor versus resistant cells pre-treated with vehicle control as shown in A. This comparison revealed a subset of barcodes with a greater relative abundance in resistant cells pre-treated with DOT1L inhibitor than resistant cells pre-treated with vehicle control (blue points). We used these barcodes to design RNA FISH probes targeting cells requiring DOT1L inhibition to become vemurafenib resistant. A separate set of barcodes showed similar high abundance with or without DOT1L inhibition (orange points), which we used to design RNA FISH probes targeting primed cells not requiring DOT1L inhibition to become resistant. C . Using these probes, we labeled and sorted cells requiring DOT1L inhibition to become vemurafenib resistant (blue), primed cells not requiring DOT1L inhibition (orange), and non-primed cells (gray) from Carbon Copies for RNA sequencing. We separately sorted cells from Carbon Copies treated with DOT1L inhibitor and Carbon Copies treated with vehicle control (2 biological replicates each). D . To identify markers of cells that require DOT1L inhibition to become resistant, we used DESeq2 to compare their gene expression to non-primed cells (x-axis) and primed cells not requiring DOT1L inhibition (y-axis). In this analysis, we included cells sorted from all Carbon Copies (treated with DOT1L inhibitor or vehicle control) from 2 biological replicates and included DOT1L inhibitor treatment as a covariate in estimating log 2 fold changes. Red points correspond to genes differentially expressed in one or both comparisons (p-adjusted ≤0.1 and log 2 fold change ≥ 1). E . Expression of DEPTOR in transcripts per million (tpm) in the subpopulations isolated in B. Points indicate tpm values for experimental replicates. F . We used the same probe sets as in B. to identify cells in situ in Carbon Copies fixed prior to vemurafenib treatment, then measured single cell expression of DEPTOR, MGP, SOX10, MITF , and 6 priming markers by RNA FISH. Shown is the expression of DEPTOR in the indicated cell populations identified in the Carbon Copies treated with vehicle control. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. Above each boxplot is the proportion of cells with levels of DEPTOR RNA above the indicated threshold (∼95th percentile in non-primed cells). G . We applied the UMAP algorithm to visualize the single cell expression data from in situ Carbon Copies. These plots include 423 cells from the vehicle control treated Carbon Copy. In the upper left plot, points are colored according to the fate of each cell as determined by its barcode. For the remaining plots points are colored by the expression level of the indicated gene in that cell. These data correspond to 1 of 2 biological replicates (See Supp. Fig 13 for additional replicate).
    Figure Legend Snippet: Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. A . Experimental approach for identifying the subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. These experiments began with approximately 400,000 WM989 A6-G3 cells transduced at an MOI ∼ 1.0 and allowed to divide for 6 days before splitting the culture into two groups. We treated one group with 4 μM DOT1L inhibitor (pinometostat) and the other with vehicle control (DMSO) for another 6 days. We then split each group again, fixing half as our “Carbon Copies” and treating the other half with 1 μM vemurafenib for ∼2.5 weeks. After vemurafenib treatment, we extracted genomic DNA from the remaining cells for barcode sequencing. B . We compared the abundance of each barcode identified in resistant cells pre-treated with DOT1L inhibitor versus resistant cells pre-treated with vehicle control as shown in A. This comparison revealed a subset of barcodes with a greater relative abundance in resistant cells pre-treated with DOT1L inhibitor than resistant cells pre-treated with vehicle control (blue points). We used these barcodes to design RNA FISH probes targeting cells requiring DOT1L inhibition to become vemurafenib resistant. A separate set of barcodes showed similar high abundance with or without DOT1L inhibition (orange points), which we used to design RNA FISH probes targeting primed cells not requiring DOT1L inhibition to become resistant. C . Using these probes, we labeled and sorted cells requiring DOT1L inhibition to become vemurafenib resistant (blue), primed cells not requiring DOT1L inhibition (orange), and non-primed cells (gray) from Carbon Copies for RNA sequencing. We separately sorted cells from Carbon Copies treated with DOT1L inhibitor and Carbon Copies treated with vehicle control (2 biological replicates each). D . To identify markers of cells that require DOT1L inhibition to become resistant, we used DESeq2 to compare their gene expression to non-primed cells (x-axis) and primed cells not requiring DOT1L inhibition (y-axis). In this analysis, we included cells sorted from all Carbon Copies (treated with DOT1L inhibitor or vehicle control) from 2 biological replicates and included DOT1L inhibitor treatment as a covariate in estimating log 2 fold changes. Red points correspond to genes differentially expressed in one or both comparisons (p-adjusted ≤0.1 and log 2 fold change ≥ 1). E . Expression of DEPTOR in transcripts per million (tpm) in the subpopulations isolated in B. Points indicate tpm values for experimental replicates. F . We used the same probe sets as in B. to identify cells in situ in Carbon Copies fixed prior to vemurafenib treatment, then measured single cell expression of DEPTOR, MGP, SOX10, MITF , and 6 priming markers by RNA FISH. Shown is the expression of DEPTOR in the indicated cell populations identified in the Carbon Copies treated with vehicle control. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. Above each boxplot is the proportion of cells with levels of DEPTOR RNA above the indicated threshold (∼95th percentile in non-primed cells). G . We applied the UMAP algorithm to visualize the single cell expression data from in situ Carbon Copies. These plots include 423 cells from the vehicle control treated Carbon Copy. In the upper left plot, points are colored according to the fate of each cell as determined by its barcode. For the remaining plots points are colored by the expression level of the indicated gene in that cell. These data correspond to 1 of 2 biological replicates (See Supp. Fig 13 for additional replicate).

    Techniques Used: Inhibition, Sequencing, Fluorescence In Situ Hybridization, Labeling, RNA Sequencing Assay, Expressing, Isolation, In Situ

    Rewind identifies rare cell states giving rise to vemurafenib resistant colonies. A . Schematic of Rewind approach for isolating the initial primed WM989 A6-G3 melanoma cells that ultimately give rise to vemurafenib resistant colonies. For the experiment shown, we transduced ∼ 200,000 WM989 A6-G3 cells at an MOI ∼ 1.0 with our Rewind barcode library. After 11 days (∼4 population doublings) we divided the culture in two, fixing half in suspension as a Carbon Copy and treating the other half with 1 μM vemurafenib to select for resistant cells. After 3 weeks in vemurafenib, we extracted genomic DNA from the resistant cells that remain and identified their Rewind barcodes by targeted sequencing. We then designed RNA FISH probes targeting 60 of these barcodes and used these probes to specifically label cells primed to become resistant from our Carbon Copy. We then sorted these cells out from the population, extracted cellular RNA and performed RNA sequencing. B . To assess the sensitivity and specificity of the Rewind experiment in A, we performed targeted sequencing to identify barcodes from cDNA generated during RNA-seq library preparation. Bar graphs show the abundance (y-axis) and rank (x-axis) of each sequenced barcode (≥ 5 normalized reads). Red bars correspond to barcodes targeted by our probe set and gray bars correspond to “off-target” barcode sequences. Inset shows the proportion of barcodes targeted by our probeset detected in each group. These data correspond to 1 of 2 replicates. In the second replicate, 30 out of 50 probed barcodes were detected in the sorted primed population. C . We performed differential expression analysis using DESeq2 of primed vs. non-primed sorted cells. Shown is the mean expression level (TPM) for protein coding genes in primed cells (y-axis) and log 2 fold change in expression estimated using DESeq2 (x-axis) compared to non-primed cells. Colors indicate differentially expressed genes related to ECM Organization and Cell Migration (red), MAPK and PI3K/Akt signalling pathways (blue) and previously identified resistance markers (purple; Shaffer et al. 2017). Genes were assigned to categories based on a consensus of KEGG pathway and GO enrichment analyses (See Methods for details). D . We selected the most differentially expressed, cell surface ECM-related gene ( ITGA3 ) to validate as a predictive marker of vemurafenib resistance in WM989 A6-G3. After staining cells with a fluorescently labelled antibody targeting ITGA3, we sorted the brightest 0.5% (ITGA3-High) and remaining (ITGA3-Low) populations, then treated both with 1 μM vemurafenib. After approximately 18 days, we fixed the cells, stained nuclei with DAPI then imaged the entire wells to quantify the number of resistant colonies and cells. The data correspond to 1 of 3 biological replicates (See Supp. Fig. 4 for additional replicates).
    Figure Legend Snippet: Rewind identifies rare cell states giving rise to vemurafenib resistant colonies. A . Schematic of Rewind approach for isolating the initial primed WM989 A6-G3 melanoma cells that ultimately give rise to vemurafenib resistant colonies. For the experiment shown, we transduced ∼ 200,000 WM989 A6-G3 cells at an MOI ∼ 1.0 with our Rewind barcode library. After 11 days (∼4 population doublings) we divided the culture in two, fixing half in suspension as a Carbon Copy and treating the other half with 1 μM vemurafenib to select for resistant cells. After 3 weeks in vemurafenib, we extracted genomic DNA from the resistant cells that remain and identified their Rewind barcodes by targeted sequencing. We then designed RNA FISH probes targeting 60 of these barcodes and used these probes to specifically label cells primed to become resistant from our Carbon Copy. We then sorted these cells out from the population, extracted cellular RNA and performed RNA sequencing. B . To assess the sensitivity and specificity of the Rewind experiment in A, we performed targeted sequencing to identify barcodes from cDNA generated during RNA-seq library preparation. Bar graphs show the abundance (y-axis) and rank (x-axis) of each sequenced barcode (≥ 5 normalized reads). Red bars correspond to barcodes targeted by our probe set and gray bars correspond to “off-target” barcode sequences. Inset shows the proportion of barcodes targeted by our probeset detected in each group. These data correspond to 1 of 2 replicates. In the second replicate, 30 out of 50 probed barcodes were detected in the sorted primed population. C . We performed differential expression analysis using DESeq2 of primed vs. non-primed sorted cells. Shown is the mean expression level (TPM) for protein coding genes in primed cells (y-axis) and log 2 fold change in expression estimated using DESeq2 (x-axis) compared to non-primed cells. Colors indicate differentially expressed genes related to ECM Organization and Cell Migration (red), MAPK and PI3K/Akt signalling pathways (blue) and previously identified resistance markers (purple; Shaffer et al. 2017). Genes were assigned to categories based on a consensus of KEGG pathway and GO enrichment analyses (See Methods for details). D . We selected the most differentially expressed, cell surface ECM-related gene ( ITGA3 ) to validate as a predictive marker of vemurafenib resistance in WM989 A6-G3. After staining cells with a fluorescently labelled antibody targeting ITGA3, we sorted the brightest 0.5% (ITGA3-High) and remaining (ITGA3-Low) populations, then treated both with 1 μM vemurafenib. After approximately 18 days, we fixed the cells, stained nuclei with DAPI then imaged the entire wells to quantify the number of resistant colonies and cells. The data correspond to 1 of 3 biological replicates (See Supp. Fig. 4 for additional replicates).

    Techniques Used: Sequencing, Fluorescence In Situ Hybridization, RNA Sequencing Assay, Generated, Expressing, Migration, Marker, Staining

    4) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: bioRxiv

    doi: 10.1101/815092

    High-temperature in vitro transcription does not affect antisense dsRNA by-product formation. A) Native gel electrophoresis analyses of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. B) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. C) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C vs . 50°C.
    Figure Legend Snippet: High-temperature in vitro transcription does not affect antisense dsRNA by-product formation. A) Native gel electrophoresis analyses of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. B) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. C) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C vs . 50°C.

    Techniques Used: In Vitro, Nucleic Acid Electrophoresis, Purification

    Antisense dsRNA by-product formation is template 3’ sequence dependent. A) dsRNA immunoblot using J2 antibody on IVT reactions with wild-type T7 at 37°C performed on modified 512B templates in which the 3’-terminal 25 bp sequence was moved to various positions within 512B template (512B-A to 512B-D). B) dsRNA immunoblot and native gel analysis of modified 512B templates (512B-A to 512B-D) using TsT7-1 at 37°C vs. 50°C. C) A chimeric template was generated in which 26 bp of the CLuc 3’-end sequence was added to the 3’-end of 512B template (denoted 512B::CLuc). Native gel electrophoresis analyses and dsRNA immunoblot analysis of IVT reactions of the 512B::CLuc chimeric template compared to the original unmodified 512B template. Reactions were performed with TsT7-1 at either 37°C or 50°C for one hour.
    Figure Legend Snippet: Antisense dsRNA by-product formation is template 3’ sequence dependent. A) dsRNA immunoblot using J2 antibody on IVT reactions with wild-type T7 at 37°C performed on modified 512B templates in which the 3’-terminal 25 bp sequence was moved to various positions within 512B template (512B-A to 512B-D). B) dsRNA immunoblot and native gel analysis of modified 512B templates (512B-A to 512B-D) using TsT7-1 at 37°C vs. 50°C. C) A chimeric template was generated in which 26 bp of the CLuc 3’-end sequence was added to the 3’-end of 512B template (denoted 512B::CLuc). Native gel electrophoresis analyses and dsRNA immunoblot analysis of IVT reactions of the 512B::CLuc chimeric template compared to the original unmodified 512B template. Reactions were performed with TsT7-1 at either 37°C or 50°C for one hour.

    Techniques Used: Sequencing, Modification, Generated, Nucleic Acid Electrophoresis

    Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.
    Figure Legend Snippet: Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, In Vitro

    5) Product Images from "An RNA polymerase ribozyme that synthesizes its own ancestor"

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1914282117

    In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.
    Figure Legend Snippet: In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.

    Techniques Used: In Vitro, Amplification, Functional Assay, Hybridization, Magnetic Beads, Polymerase Chain Reaction, Sequencing, Labeling

    6) Product Images from "Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis"

    Article Title: Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02487

    Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.
    Figure Legend Snippet: Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.

    Techniques Used: In Vitro, Agarose Gel Electrophoresis, Sequencing, Incubation, Imaging, Plasmid Preparation

    Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.
    Figure Legend Snippet: Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.

    Techniques Used: Sequencing, Incubation, Staining, RNA Sequencing Assay

    Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.
    Figure Legend Snippet: Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.

    Techniques Used: Sequencing, Incubation, Staining

    7) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    Antisense dsRNA by-product formation is template 3′ sequence dependent. ( A ) dsRNA immunoblot using J2 antibody on IVT reactions with wild-type T7 at 37°C performed on modified 512B templates in which the 3′-terminal 25 bp sequence was moved to various positions within 512B template (512B–A to 512B–D). ( B ) dsRNA immunoblot and native gel analysis of modified 512B templates (512B–A to 512B–D) using TsT7-1 at 37°C versus 50°C. ( C ) A chimeric template was generated in which 26 bp of the CLuc 3′-end sequence was added to the 3′-end of 512B template (denoted 512B::CLuc). Native gel electrophoresis analyses and dsRNA immunoblot analysis of IVT reactions of the 512B::CLuc chimeric template compared to the original unmodified 512B template. Reactions were performed with TsT7-1 at either 37°C or 50°C for 1 h.
    Figure Legend Snippet: Antisense dsRNA by-product formation is template 3′ sequence dependent. ( A ) dsRNA immunoblot using J2 antibody on IVT reactions with wild-type T7 at 37°C performed on modified 512B templates in which the 3′-terminal 25 bp sequence was moved to various positions within 512B template (512B–A to 512B–D). ( B ) dsRNA immunoblot and native gel analysis of modified 512B templates (512B–A to 512B–D) using TsT7-1 at 37°C versus 50°C. ( C ) A chimeric template was generated in which 26 bp of the CLuc 3′-end sequence was added to the 3′-end of 512B template (denoted 512B::CLuc). Native gel electrophoresis analyses and dsRNA immunoblot analysis of IVT reactions of the 512B::CLuc chimeric template compared to the original unmodified 512B template. Reactions were performed with TsT7-1 at either 37°C or 50°C for 1 h.

    Techniques Used: Sequencing, Modification, Generated, Nucleic Acid Electrophoresis

    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.
    Figure Legend Snippet: High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Purification

    8) Product Images from "Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex"

    Article Title: Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex

    Journal: bioRxiv

    doi: 10.1101/2020.02.02.931477

    Determination of Bicc1 Binding Motifs in RNA a Schematic representation of RNA Bind-n-Seq (RBNS), which determines RNA motifs enriched by target proteins with the use of a random RNA sequence library. 293FT cells were transfected with a plasmid for overexpression (O/E) of FLAG-tagged Bicc1. Cell lysates containing the Bicc1-FLAG protein were then mixed with a random RNA sequence library, and resulting RNA-protein complexes were isolated by immunoprecipitation with magnetic bead–conjugated antibodies to FLAG. Finally, the isolated RNA sequences were converted to a DNA library by RT-PCR for deep sequencing. b Analysis of the RBNS data set. The number of each k-mer (where k = 4, 5, or 6) RNA sequence was compared between cells transfected with the Bicc1-FLAG expression plasmid and those subjected to mock transfection (control). c A motif logo generated from aligned hexamers significantly enriched by Bicc1-FLAG. d Enriched 4-mer and 5-mer sequences sorted by relative frequency determined by comparison of the Bicc1-FLAG and control RBNS data. e Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3’-UTR of mouse mRNAs. A total of 31,16 5 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. f Histogram of motif frequency revealed by metagene analysis. The vertical black and blue lines indicate the averaged frequency of each target motif and the frequency of each target motif in the 200-nucleotide proximal region of the 3’-UTR of Dand5 mRNA. g Maps of GAC-containing motifs in the 3’-UTR of Dand5 mRNAs for the indicated species.
    Figure Legend Snippet: Determination of Bicc1 Binding Motifs in RNA a Schematic representation of RNA Bind-n-Seq (RBNS), which determines RNA motifs enriched by target proteins with the use of a random RNA sequence library. 293FT cells were transfected with a plasmid for overexpression (O/E) of FLAG-tagged Bicc1. Cell lysates containing the Bicc1-FLAG protein were then mixed with a random RNA sequence library, and resulting RNA-protein complexes were isolated by immunoprecipitation with magnetic bead–conjugated antibodies to FLAG. Finally, the isolated RNA sequences were converted to a DNA library by RT-PCR for deep sequencing. b Analysis of the RBNS data set. The number of each k-mer (where k = 4, 5, or 6) RNA sequence was compared between cells transfected with the Bicc1-FLAG expression plasmid and those subjected to mock transfection (control). c A motif logo generated from aligned hexamers significantly enriched by Bicc1-FLAG. d Enriched 4-mer and 5-mer sequences sorted by relative frequency determined by comparison of the Bicc1-FLAG and control RBNS data. e Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3’-UTR of mouse mRNAs. A total of 31,16 5 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. f Histogram of motif frequency revealed by metagene analysis. The vertical black and blue lines indicate the averaged frequency of each target motif and the frequency of each target motif in the 200-nucleotide proximal region of the 3’-UTR of Dand5 mRNA. g Maps of GAC-containing motifs in the 3’-UTR of Dand5 mRNAs for the indicated species.

    Techniques Used: Binding Assay, Sequencing, Transfection, Plasmid Preparation, Over Expression, Isolation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Generated

    9) Product Images from "Stochastic transcription in the p53‐mediated response to DNA damage is modulated by burst frequency"

    Article Title: Stochastic transcription in the p53‐mediated response to DNA damage is modulated by burst frequency

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20199068

    Single‐cell quantification of RNA expression by sm FISH highlights strong heterogeneity of p53 target gene expression p53 has been shown to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown. We selected p53 target genes that are involved in different cell fate programs ranging from apoptosis (BAX), DNA repair (DDB2) cell cycle arrest (CDKN1A), proliferation control (SESN1), and the regulation of the p53 network itself (PPM1D and MDM2). Induction of selected p53 target genes after DNA damage induction in A549 wild‐type and p53 knockdown cells. RNA levels were measured by qRT–PCR before and 3 h after treatment with 10 Gy IR. Fold changes relative to basal levels are shown for each cell line as mean and standard deviation from technical triplicates. Fluorescence microscopy images of smFISH probes labeled with CAL Fluor 610 (gray) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 μm distance; images were contrast‐ and brightness‐enhanced for better visualization. Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets show broad variability of RNA counts per cell for all genes in basal conditions. Dashed line: median; solid line: probability density estimate (see Data visualization section), CV: coefficient of variation, Fano: Fano factor, m: median, n : number of cells analyzed. Source data are available online for this figure.
    Figure Legend Snippet: Single‐cell quantification of RNA expression by sm FISH highlights strong heterogeneity of p53 target gene expression p53 has been shown to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown. We selected p53 target genes that are involved in different cell fate programs ranging from apoptosis (BAX), DNA repair (DDB2) cell cycle arrest (CDKN1A), proliferation control (SESN1), and the regulation of the p53 network itself (PPM1D and MDM2). Induction of selected p53 target genes after DNA damage induction in A549 wild‐type and p53 knockdown cells. RNA levels were measured by qRT–PCR before and 3 h after treatment with 10 Gy IR. Fold changes relative to basal levels are shown for each cell line as mean and standard deviation from technical triplicates. Fluorescence microscopy images of smFISH probes labeled with CAL Fluor 610 (gray) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 μm distance; images were contrast‐ and brightness‐enhanced for better visualization. Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets show broad variability of RNA counts per cell for all genes in basal conditions. Dashed line: median; solid line: probability density estimate (see Data visualization section), CV: coefficient of variation, Fano: Fano factor, m: median, n : number of cells analyzed. Source data are available online for this figure.

    Techniques Used: RNA Expression, Fluorescence In Situ Hybridization, Expressing, Irradiation, Quantitative RT-PCR, Standard Deviation, Fluorescence, Microscopy, Labeling, Staining

    Smyd2 and Set8 activities affect p53 nuclear dynamics and promoter binding Western blot of acetylated p53 (K370/K382) in A549 Smyd2 and Set8 knockdown cells compared to wild‐type cell lines shows an increase in acetylation specifically at later time points in the DNA damage response. Dynamics of total p53 remained pulse like. GAPDH is shown as loading control. Amount of p53 bound to CDKN1A and MDM2 promoters in A549 Smyd2 (B) and Set8 (C) knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from two biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We observed an increase in promoter binding at later time points similar to the results after Nutlin‐3 treatment.
    Figure Legend Snippet: Smyd2 and Set8 activities affect p53 nuclear dynamics and promoter binding Western blot of acetylated p53 (K370/K382) in A549 Smyd2 and Set8 knockdown cells compared to wild‐type cell lines shows an increase in acetylation specifically at later time points in the DNA damage response. Dynamics of total p53 remained pulse like. GAPDH is shown as loading control. Amount of p53 bound to CDKN1A and MDM2 promoters in A549 Smyd2 (B) and Set8 (C) knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from two biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We observed an increase in promoter binding at later time points similar to the results after Nutlin‐3 treatment.

    Techniques Used: Binding Assay, Western Blot, Chromatin Immunoprecipitation, Quantitative RT-PCR

    Sm FISH ‐based analysis at the first and second p53 pulse after IR reveals gene‐specific stochastic expression patterns Schematic illustration of the life cycle of an mRNA and the rate constants that influence RNA abundance due to stochastic bursting according to previously published models of promoter activity. While burst frequency (bf) describes the switching of a promoter between a transcriptionally active and inactive state with the rate constants k on and k off, the burst size (bs) describes the number of RNAs transcribed in an active period. Additionally, degradation (δ) further influences RNA levels by reducing the cytoplasmic RNA pool. Illustration of promoter activity according to the random telegraph model. An increase in RNA levels per cell can be due to a higher burst frequency (more active promoter periods, a higher rate of transcription initiation), or an increase in burst size (a higher rate of RNA transcription in an active period). Additionally, also mixtures of both scenarios are possible. We used smFISH data to calculated promoter activity based on previously published models. An overview of the calculations characterizing stochastic gene expression is shown. X RNA : number of quantified RNAs/cell, n : number of genomic loci, f : fraction of active promoters (proxy for burst frequency bf), μ: transcription rate per cell [RNA/h] (proxy for burst size bs), δ RNA : RNA degradation rate per cell [1/h], M : polymerase occupancy [RNAs/h], v : RNAP2 speed (estimated as 3 kb/min), l : gene length, TSS: active TSS at the moment of measurement. Further details can be found in Materials and Methods section. Quantification of stochastic gene expression for the indicated p53 target genes before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (μ; proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity ( > 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates μ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2 C). Mean degradation rates of indicated RNAs in transcriptionally active cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as calculated from smFISH data. RNA stability is not changing in the measured time frame upon DNA damage. The plot displays the average RNA degradation rate per cell [1/h] over time after DNA damage, calculated from model (C) in actively transcribing cells for each gene. Based on promoter activity, we allocated target gene promoters along three archetypical expression patterns illustrated by a schematic triangle. Amount of p53 bound to indicated target gene promoters before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from 3 to 4 biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter (indicated by n.d.) Source data are available online for this figure.
    Figure Legend Snippet: Sm FISH ‐based analysis at the first and second p53 pulse after IR reveals gene‐specific stochastic expression patterns Schematic illustration of the life cycle of an mRNA and the rate constants that influence RNA abundance due to stochastic bursting according to previously published models of promoter activity. While burst frequency (bf) describes the switching of a promoter between a transcriptionally active and inactive state with the rate constants k on and k off, the burst size (bs) describes the number of RNAs transcribed in an active period. Additionally, degradation (δ) further influences RNA levels by reducing the cytoplasmic RNA pool. Illustration of promoter activity according to the random telegraph model. An increase in RNA levels per cell can be due to a higher burst frequency (more active promoter periods, a higher rate of transcription initiation), or an increase in burst size (a higher rate of RNA transcription in an active period). Additionally, also mixtures of both scenarios are possible. We used smFISH data to calculated promoter activity based on previously published models. An overview of the calculations characterizing stochastic gene expression is shown. X RNA : number of quantified RNAs/cell, n : number of genomic loci, f : fraction of active promoters (proxy for burst frequency bf), μ: transcription rate per cell [RNA/h] (proxy for burst size bs), δ RNA : RNA degradation rate per cell [1/h], M : polymerase occupancy [RNAs/h], v : RNAP2 speed (estimated as 3 kb/min), l : gene length, TSS: active TSS at the moment of measurement. Further details can be found in Materials and Methods section. Quantification of stochastic gene expression for the indicated p53 target genes before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (μ; proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity ( > 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates μ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2 C). Mean degradation rates of indicated RNAs in transcriptionally active cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as calculated from smFISH data. RNA stability is not changing in the measured time frame upon DNA damage. The plot displays the average RNA degradation rate per cell [1/h] over time after DNA damage, calculated from model (C) in actively transcribing cells for each gene. Based on promoter activity, we allocated target gene promoters along three archetypical expression patterns illustrated by a schematic triangle. Amount of p53 bound to indicated target gene promoters before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from 3 to 4 biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter (indicated by n.d.) Source data are available online for this figure.

    Techniques Used: Fluorescence In Situ Hybridization, Expressing, Activity Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Binding Assay

    The interplay of p53's C‐terminal lysine acetylation and methylation regulates transiently expressed target genes in response to IR A schematic illustration of p53's C‐terminal modifications and described functional implications, including key regulatory enzymes. Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML‐277, central lanes), and sustained p53 (10 Gy IR + Nutlin‐3, right lanes). See Fig 3 and Materials and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western blot and normalized to the abundance 3 h post‐IR. Means and propagated standard errors from three independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12 . The p53‐K370 methylase Smyd2 was down‐regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild‐type and knockdown cells by qRT–PCR. Mean levels and standard deviation from technical triplicates are indicated. Promoter activity of CDKN1A (E) and MDM2 (F) was quantified in Smyd2 knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong ( > 75% of TSS, solid colors) and weak (
    Figure Legend Snippet: The interplay of p53's C‐terminal lysine acetylation and methylation regulates transiently expressed target genes in response to IR A schematic illustration of p53's C‐terminal modifications and described functional implications, including key regulatory enzymes. Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML‐277, central lanes), and sustained p53 (10 Gy IR + Nutlin‐3, right lanes). See Fig 3 and Materials and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western blot and normalized to the abundance 3 h post‐IR. Means and propagated standard errors from three independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12 . The p53‐K370 methylase Smyd2 was down‐regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild‐type and knockdown cells by qRT–PCR. Mean levels and standard deviation from technical triplicates are indicated. Promoter activity of CDKN1A (E) and MDM2 (F) was quantified in Smyd2 knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong ( > 75% of TSS, solid colors) and weak (

    Techniques Used: Methylation, Functional Assay, Western Blot, Expressing, shRNA, Quantitative RT-PCR, Standard Deviation, Activity Assay

    10) Product Images from "Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics"

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics

    Journal: bioRxiv

    doi: 10.1101/2020.04.20.049437

    PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . Rings with an aromatic character are highlighted in red. B . Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P. aeruginosa (left). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (10 µg/mL) separately, then cells of both cultures were washed, mixed and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CP (see Methods and Fig. S5D for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) upon artificial induction of the mexGHI-opmD operon with arabinose (n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p
    Figure Legend Snippet: PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . Rings with an aromatic character are highlighted in red. B . Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P. aeruginosa (left). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (10 µg/mL) separately, then cells of both cultures were washed, mixed and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CP (see Methods and Fig. S5D for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) upon artificial induction of the mexGHI-opmD operon with arabinose (n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p

    Techniques Used: Expressing, Quantitative RT-PCR

    11) Product Images from "Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus"

    Article Title: Development of a recombinase polymerase amplification combined with lateral-flow dipstick assay for detection of bovine ephemeral fever virus

    Journal: Molecular and Cellular Probes

    doi: 10.1016/j.mcp.2017.12.003

    Analytical specificity of the BEFV LFD-RPA assay. (A) Specificity of the LFD-RPA assay. The specificity of the assay was assessed for other bovine viral pathogens with similar clinic and etiologies. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) The results of amplification products of the LFD-RPA on 2% agarose gel. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. (C) The quality detection of RNA/DNA of BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV. The RNA/DNA of BEFV, BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV prepared for specificity detection were undertook PCR reaction with viral specific primers ( Supplementary Table 1 ). The positive amplification results were shown in Lane 1, Lane 3, Lane 5, Lane 7, Lane 9, Lane 11, Lane 13, respectively. Lane 2, Lane 4, Lane 6, Lane 8, Lane 10, Lane 12, Lane 14 were negative controls with DNase-free water as template.
    Figure Legend Snippet: Analytical specificity of the BEFV LFD-RPA assay. (A) Specificity of the LFD-RPA assay. The specificity of the assay was assessed for other bovine viral pathogens with similar clinic and etiologies. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. Samples were tested in triplicate with one reaction displayed in figure for each triplicate. (B) The results of amplification products of the LFD-RPA on 2% agarose gel. Lane 1: positive control of BEFV; Lanes 2 to 7: BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV, respectively. (C) The quality detection of RNA/DNA of BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV. The RNA/DNA of BEFV, BVDV, IBRV, BPIV-3, BRSV, BcoV and VSV prepared for specificity detection were undertook PCR reaction with viral specific primers ( Supplementary Table 1 ). The positive amplification results were shown in Lane 1, Lane 3, Lane 5, Lane 7, Lane 9, Lane 11, Lane 13, respectively. Lane 2, Lane 4, Lane 6, Lane 8, Lane 10, Lane 12, Lane 14 were negative controls with DNase-free water as template.

    Techniques Used: Recombinase Polymerase Amplification, Positive Control, Amplification, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    12) Product Images from "CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks"

    Article Title: CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

    Journal: eLife

    doi: 10.7554/eLife.55143

    Enzyme-concentration dependence of AsCas12a cis DNA cleavage kinetics. 250 pM of a cis duplex DNA target, radiolabeled on the 5' end of the NTS, was incubated with 240 nM crRNA and various concentrations of AsCas12a at 37°C for the following timepoints: 0 s, 5 s, 10 s, 20 s, 40 s, 10 m. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y = (y 0 -plateau)*exp(-k*x)+plateau), with y 0 constrained to 0. At the lower values of [AsCas12a], an early plateau indicates an underestimation of the true enzyme concentration, due to loss of enzyme across extensive serial dilution. Despite this experimental deficiency, these data show that enzyme-substrate association is not rate-limiting when [AsCas12a] is 100 nM—that is, when [AsCas12a] is 100 nM, association can be approximated as instantaneous, and cleavage rate constants reflect unimolecular processes that follow binding. Based on these experiments, initial cis cleavage events occur at ~0.12 s −1 at the reagent concentrations used for cleavage mapping experiments. Considering the steady-state kinetic measurements in Appendix 2—figure 1—figure supplement 2 , an upper limit on trans cleavage rate can be calculated as (k cat /K M )*[E], where [E] has an approximate upper limit of 10 nM (for radiolabeled-NTS experiments, in which cold TS is present at 10 nM, and assuming ssDNA-activated complexes have similar catalytic efficiency to dsDNA-activated complexes), yielding a rate of ~0.002 s −1 ( > 50 fold less than the cis cleavage rate). This trans cleavage rate estimate is an upper limit because radiolabeled DNA is mostly duplex and likely protected by the protein in non-duplex regions ( Swarts et al., 2017 ). Thus, trans cleavage probably only occurs to a detectable extent on PAM-distal DNA fragments that are released from the enzyme following cis cleavage.
    Figure Legend Snippet: Enzyme-concentration dependence of AsCas12a cis DNA cleavage kinetics. 250 pM of a cis duplex DNA target, radiolabeled on the 5' end of the NTS, was incubated with 240 nM crRNA and various concentrations of AsCas12a at 37°C for the following timepoints: 0 s, 5 s, 10 s, 20 s, 40 s, 10 m. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y = (y 0 -plateau)*exp(-k*x)+plateau), with y 0 constrained to 0. At the lower values of [AsCas12a], an early plateau indicates an underestimation of the true enzyme concentration, due to loss of enzyme across extensive serial dilution. Despite this experimental deficiency, these data show that enzyme-substrate association is not rate-limiting when [AsCas12a] is 100 nM—that is, when [AsCas12a] is 100 nM, association can be approximated as instantaneous, and cleavage rate constants reflect unimolecular processes that follow binding. Based on these experiments, initial cis cleavage events occur at ~0.12 s −1 at the reagent concentrations used for cleavage mapping experiments. Considering the steady-state kinetic measurements in Appendix 2—figure 1—figure supplement 2 , an upper limit on trans cleavage rate can be calculated as (k cat /K M )*[E], where [E] has an approximate upper limit of 10 nM (for radiolabeled-NTS experiments, in which cold TS is present at 10 nM, and assuming ssDNA-activated complexes have similar catalytic efficiency to dsDNA-activated complexes), yielding a rate of ~0.002 s −1 ( > 50 fold less than the cis cleavage rate). This trans cleavage rate estimate is an upper limit because radiolabeled DNA is mostly duplex and likely protected by the protein in non-duplex regions ( Swarts et al., 2017 ). Thus, trans cleavage probably only occurs to a detectable extent on PAM-distal DNA fragments that are released from the enzyme following cis cleavage.

    Techniques Used: Concentration Assay, Incubation, Serial Dilution, Binding Assay

    Comparing the substrate specificities of Cas12a trans -active holoenzyme and S1 nuclease. Top panel : Enzymes were first titrated in an activity assay with a radiolabeled ssDNA substrate to determine what concentration to use in substrate specificity assays. A ssDNA oligo (the same one shown in the other panels) was 5'-radiolabeled and incubated with varying concentrations of either AsCas12a/crRNA/pre-cleaved DNA activator (all components held equimolar at the indicated concentration) or S1 nuclease for 30 minutes at 30°C, followed by quenching. Products were resolved by denaturing PAGE and quantified from the phosphorimage. “U” on the x-axis of S1 nuclease refers to the units defined by the enzyme manufacturer. To achieve 90% cleavage of the ssDNA substrate in the given time course, 115 nM AsCas12a holoenzyme or 0.513 U/µL S1 nuclease was required. Middle panels : Phosphorimage of AsCas12a or S1 nuclease cleavage products, resolved by denaturing PAGE. Trans -active AsCas12a holoenzyme (115 nM of each component: protein, crRNA, pre-cleaved activator) or S1 nuclease (0.513 U/µL) was incubated with 1 nM of the indicated substrate for 2 hours at 30°C prior to quenching. Substrate (a) was a single-stranded DNA oligonucleotide with no homology to the crRNA. To generate substrates (b) through (l), substrate (a) was hybridized to a variety of unlabeled complementary DNA oligonucleotides. Substrate (c) contained a nick. Substrates (d), (e), and (f) contained gaps of 1, 4, and 8 nt, respectively. Substrates (g), (h), and (i) contained bubbles of 1, 4, and 8 nt, respectively. Substrates (j), (k), and (l) contained bulges of 1, 4, and 8 nt, respectively. Bottom panel : Quantifications of cleavage from phosphorimages. “Fraction cleaved” is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane).
    Figure Legend Snippet: Comparing the substrate specificities of Cas12a trans -active holoenzyme and S1 nuclease. Top panel : Enzymes were first titrated in an activity assay with a radiolabeled ssDNA substrate to determine what concentration to use in substrate specificity assays. A ssDNA oligo (the same one shown in the other panels) was 5'-radiolabeled and incubated with varying concentrations of either AsCas12a/crRNA/pre-cleaved DNA activator (all components held equimolar at the indicated concentration) or S1 nuclease for 30 minutes at 30°C, followed by quenching. Products were resolved by denaturing PAGE and quantified from the phosphorimage. “U” on the x-axis of S1 nuclease refers to the units defined by the enzyme manufacturer. To achieve 90% cleavage of the ssDNA substrate in the given time course, 115 nM AsCas12a holoenzyme or 0.513 U/µL S1 nuclease was required. Middle panels : Phosphorimage of AsCas12a or S1 nuclease cleavage products, resolved by denaturing PAGE. Trans -active AsCas12a holoenzyme (115 nM of each component: protein, crRNA, pre-cleaved activator) or S1 nuclease (0.513 U/µL) was incubated with 1 nM of the indicated substrate for 2 hours at 30°C prior to quenching. Substrate (a) was a single-stranded DNA oligonucleotide with no homology to the crRNA. To generate substrates (b) through (l), substrate (a) was hybridized to a variety of unlabeled complementary DNA oligonucleotides. Substrate (c) contained a nick. Substrates (d), (e), and (f) contained gaps of 1, 4, and 8 nt, respectively. Substrates (g), (h), and (i) contained bubbles of 1, 4, and 8 nt, respectively. Substrates (j), (k), and (l) contained bulges of 1, 4, and 8 nt, respectively. Bottom panel : Quantifications of cleavage from phosphorimages. “Fraction cleaved” is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane).

    Techniques Used: Activity Assay, Concentration Assay, Incubation, Polyacrylamide Gel Electrophoresis

    Effect of PAM-distal mismatches on non-target-strand and target-strand cleavage kinetics and position with bubbled DNA targets. 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM of DNA target at 37°C for 0 s, 15 s, 2 min, 10 min, and 1 hr, prior to quenching and resolution by denaturing PAGE. Each time series corresponds to a different DNA target, bearing varying numbers of PAM-distal mismatches with respect to the crRNA. Indicated above each time series is the number of base pairs of complementarity between the TS and the crRNA spacer, starting with the base immediately adjacent to the PAM. For the time series lacking an asterisk, the DNA target was fully duplex (as in Figure 3—figure supplement 3 ). For the time series that bear asterisks, the DNA target contained a bubble across the region of crRNA:TS complementarity (as illustrated in Figure 3B ), which stabilized the R-loop. In the top panel, the NTS was 5'-radiolabeled. In the bottom panel, the TS was 5'-radiolabeled.
    Figure Legend Snippet: Effect of PAM-distal mismatches on non-target-strand and target-strand cleavage kinetics and position with bubbled DNA targets. 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM of DNA target at 37°C for 0 s, 15 s, 2 min, 10 min, and 1 hr, prior to quenching and resolution by denaturing PAGE. Each time series corresponds to a different DNA target, bearing varying numbers of PAM-distal mismatches with respect to the crRNA. Indicated above each time series is the number of base pairs of complementarity between the TS and the crRNA spacer, starting with the base immediately adjacent to the PAM. For the time series lacking an asterisk, the DNA target was fully duplex (as in Figure 3—figure supplement 3 ). For the time series that bear asterisks, the DNA target contained a bubble across the region of crRNA:TS complementarity (as illustrated in Figure 3B ), which stabilized the R-loop. In the top panel, the NTS was 5'-radiolabeled. In the bottom panel, the TS was 5'-radiolabeled.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    Cleavage at phosphorothioates can be selectively slowed by substitution of CaCl 2 for MgCl 2 . 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM radiolabeled duplex DNA target at 37°C, followed by quenching (at timepoints 0, 5 s, 15 s, 1 min, 5 min, 10 min, 30 min, 1 hr) and resolution by denaturing PAGE. Substrate diagrams are colored as in Appendix 2—figure 2—figure supplement 1 . The top panel shows the experiment done in cleavage buffer with 5 mM MgCl 2 . The bottom panel shows the experiment done in cleavage buffer with 5 mM CaCl 2 —at the end of each time course on this gel, the 1-hr timepoint of the MgCl 2 experiment is included for visual comparison. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y = (y 0 -plateau)*exp(-k*x)+plateau), with y 0 constrained to 0. The plateau value was constrained to 1 for those time courses that did not exceed fraction cleaved = 0.5 by the 1-hr timepoint. Rate constants (with 95% confidence intervals) are shown in the table below the gels. It is unclear why cleavage of a phosphorothioated TS occurs more rapidly than cleavage of a phosphorothioated NTS, although it is conceivable that this is an intrinsic feature of the enzyme cleavage pathway when the chemical transformation is rate-limiting. Considering only effects on the NTS, the calcium substitution decreases the phosphodiester cleavage rate by a factor of 57 and decreases the phosphorothioate cleavage rate by a factor of 730, resulting in a 13-fold increase in selectivity for phosphodiesters over phosphorothioates and yielding kinetics slow enough to resolve by manual pipetting.
    Figure Legend Snippet: Cleavage at phosphorothioates can be selectively slowed by substitution of CaCl 2 for MgCl 2 . 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM radiolabeled duplex DNA target at 37°C, followed by quenching (at timepoints 0, 5 s, 15 s, 1 min, 5 min, 10 min, 30 min, 1 hr) and resolution by denaturing PAGE. Substrate diagrams are colored as in Appendix 2—figure 2—figure supplement 1 . The top panel shows the experiment done in cleavage buffer with 5 mM MgCl 2 . The bottom panel shows the experiment done in cleavage buffer with 5 mM CaCl 2 —at the end of each time course on this gel, the 1-hr timepoint of the MgCl 2 experiment is included for visual comparison. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y = (y 0 -plateau)*exp(-k*x)+plateau), with y 0 constrained to 0. The plateau value was constrained to 1 for those time courses that did not exceed fraction cleaved = 0.5 by the 1-hr timepoint. Rate constants (with 95% confidence intervals) are shown in the table below the gels. It is unclear why cleavage of a phosphorothioated TS occurs more rapidly than cleavage of a phosphorothioated NTS, although it is conceivable that this is an intrinsic feature of the enzyme cleavage pathway when the chemical transformation is rate-limiting. Considering only effects on the NTS, the calcium substitution decreases the phosphodiester cleavage rate by a factor of 57 and decreases the phosphorothioate cleavage rate by a factor of 730, resulting in a 13-fold increase in selectivity for phosphodiesters over phosphorothioates and yielding kinetics slow enough to resolve by manual pipetting.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Transformation Assay

    Kinetics of target-strand cleavage in DNA targets with various sequences in the R-loop flank. Experiment performed as described in legend to Figure 3C . 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM of DNA target at 25°C for 0 s, 15 s, 30 s, 1 min, 2 min, 4 min, or 10 min, prior to quenching and resolution by denaturing PAGE. All DNA targets were 5'-radiolabeled on the TS. The NTS was pre-gapped from positions 14–18 but complementary to the TS at positions 1–13 and 19–20. In each lane, the DNA target was varied to contain different sequences in the R-loop flank, which either formed a perfect duplex (substrates A, C, and E) or contained a 3-bp NTS:TS mismatch (substrates B, D, and F). ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y=(y 0 -plateau)*exp(-k*x)+plateau), with y 0 constrained to 0. A representative replicate (n = 3) is shown. The value of k obs ± SD for each time course is as follows: A [0.092 ± 0.012 s −1 ], B [0.145 ± 0.007 s −1 ], C [0.0059 ± 0.0006 s −1 ], D [0.137 ± 0.002 s −1 ], E [0.024 ± 0.002 s −1 ], F [0.061 ± 0.013 s −1 ]. The rate constants for B and D should be interpreted with caution due to poor sampling of informative timepoints.
    Figure Legend Snippet: Kinetics of target-strand cleavage in DNA targets with various sequences in the R-loop flank. Experiment performed as described in legend to Figure 3C . 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM of DNA target at 25°C for 0 s, 15 s, 30 s, 1 min, 2 min, 4 min, or 10 min, prior to quenching and resolution by denaturing PAGE. All DNA targets were 5'-radiolabeled on the TS. The NTS was pre-gapped from positions 14–18 but complementary to the TS at positions 1–13 and 19–20. In each lane, the DNA target was varied to contain different sequences in the R-loop flank, which either formed a perfect duplex (substrates A, C, and E) or contained a 3-bp NTS:TS mismatch (substrates B, D, and F). ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y=(y 0 -plateau)*exp(-k*x)+plateau), with y 0 constrained to 0. A representative replicate (n = 3) is shown. The value of k obs ± SD for each time course is as follows: A [0.092 ± 0.012 s −1 ], B [0.145 ± 0.007 s −1 ], C [0.0059 ± 0.0006 s −1 ], D [0.137 ± 0.002 s −1 ], E [0.024 ± 0.002 s −1 ], F [0.061 ± 0.013 s −1 ]. The rate constants for B and D should be interpreted with caution due to poor sampling of informative timepoints.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Sampling

    Concentration dependence of various modes of DNase activity. In biochemical reactions containing DNA, crRNA, and AsCas12a, a cut in a DNA molecule could be attributed to one of three distinct modes of RuvC DNase activity: cis cleavage of an enzyme’s own bound R-loop; trans cleavage (by an R-loop-activated complex) of free DNA or DNA in a different complex; or trans cleavage by an excess of DNA-free Cas12a/crRNA complex (which is shown in this figure to be catalytically competent, albeit inefficient). Various concentrations of crRNA, AsCas12a, and DNA activator were incubated with 2 nM of a radiolabeled single-stranded DNA oligonucleotide for 1 hr at 37°C prior to quenching and resolution by denaturing PAGE. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). We show here that both DNA-activated and DNA-free modes of trans cleavage are dependent upon enzyme concentration in the nanomolar range—that is, unlike cis cleavage, which occurs with saturated binding kinetics at the concentrations used (see Appendix 2—figure 1—figure supplement 3 ), trans cleavage remains concentration-dependent in this range. Therefore, DNase cleavage products that appear with kinetics that are independent of enzyme concentration can be uniquely attributed to PAM-dependent cis cleavage (see Appendix 2—figure 1—figure supplement 5 ).
    Figure Legend Snippet: Concentration dependence of various modes of DNase activity. In biochemical reactions containing DNA, crRNA, and AsCas12a, a cut in a DNA molecule could be attributed to one of three distinct modes of RuvC DNase activity: cis cleavage of an enzyme’s own bound R-loop; trans cleavage (by an R-loop-activated complex) of free DNA or DNA in a different complex; or trans cleavage by an excess of DNA-free Cas12a/crRNA complex (which is shown in this figure to be catalytically competent, albeit inefficient). Various concentrations of crRNA, AsCas12a, and DNA activator were incubated with 2 nM of a radiolabeled single-stranded DNA oligonucleotide for 1 hr at 37°C prior to quenching and resolution by denaturing PAGE. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). We show here that both DNA-activated and DNA-free modes of trans cleavage are dependent upon enzyme concentration in the nanomolar range—that is, unlike cis cleavage, which occurs with saturated binding kinetics at the concentrations used (see Appendix 2—figure 1—figure supplement 3 ), trans cleavage remains concentration-dependent in this range. Therefore, DNase cleavage products that appear with kinetics that are independent of enzyme concentration can be uniquely attributed to PAM-dependent cis cleavage (see Appendix 2—figure 1—figure supplement 5 ).

    Techniques Used: Concentration Assay, Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay

    Non-target-strand cleavage precedes target-strand cleavage for AsCas12a and Cas12i1. 100 nM AsCas12a or Cas12i1 was incubated with 120 nM cognate crRNA and 2 nM radiolabeled duplex DNA target for 1 hr at 37°C, followed by quenching, denaturing PAGE, and phosphorimaging. For AsCas12a, the reaction was conducted in 5 mM CaCl 2 . For Cas12i1, the reaction was conducted in 5 mM MgCl 2 . In the duplex diagrams, red shading indicates the presence of a phosphorothioate (PS) tract across the standard cleavage sites. Blue indicates phosphodiester (PO) linkages within the TS. Gray indicates phosphodiester linkages within the NTS. For both AsCas12a and Cas12i1, the nature of the linkages in the TS has no apparent effect on NTS cleavage. However, the presence of phosphorothioates in the NTS inhibits cleavage of both the NTS and the TS. Trace TS cleavage is observed for Cas12i1 in the PS-NTS condition (lane 6)—it is unclear whether this is due to TS cleavage prior to NTS cleavage or to incomplete duplex formation, as the trace cleavage event is shifted to the site cleaved during ssDNA-targeting (lane 7).
    Figure Legend Snippet: Non-target-strand cleavage precedes target-strand cleavage for AsCas12a and Cas12i1. 100 nM AsCas12a or Cas12i1 was incubated with 120 nM cognate crRNA and 2 nM radiolabeled duplex DNA target for 1 hr at 37°C, followed by quenching, denaturing PAGE, and phosphorimaging. For AsCas12a, the reaction was conducted in 5 mM CaCl 2 . For Cas12i1, the reaction was conducted in 5 mM MgCl 2 . In the duplex diagrams, red shading indicates the presence of a phosphorothioate (PS) tract across the standard cleavage sites. Blue indicates phosphodiester (PO) linkages within the TS. Gray indicates phosphodiester linkages within the NTS. For both AsCas12a and Cas12i1, the nature of the linkages in the TS has no apparent effect on NTS cleavage. However, the presence of phosphorothioates in the NTS inhibits cleavage of both the NTS and the TS. Trace TS cleavage is observed for Cas12i1 in the PS-NTS condition (lane 6)—it is unclear whether this is due to TS cleavage prior to NTS cleavage or to incomplete duplex formation, as the trace cleavage event is shifted to the site cleaved during ssDNA-targeting (lane 7).

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    Concentration dependence of phosphodiester-mapped cleavage events. Each graph follows the kinetics of appearance/disappearance of a cleavage product at the indicated phosphodiester. ‘Fraction cleaved’ is defined as (band volume for a given cleavage product)/(total volume in lane). For example, ‘NTS 5', 11/12’ displays the fraction of 5'-radiolabeled NTS that was detected to have been cleaved at the 11/12 dinucleotide for each timepoint. Blue circles are equivalent to the data shown in Appendix 2—figure 1 , with error bars denoting standard deviation across three replicates. Red squares and green triangles indicate variants of this experiment in which the total concentration of either DNA-bound holoenzyme (red squares) or AsCas12a/crRNA (green triangles) was decreased. The fact that almost all species exhibit equivalent kinetics of appearance/disappearance in all three conditions (with the potential exception of the phosphodiesters on the portion of the PAM-distal NTS fragment that remains single-stranded after release by the enzyme, which exhibit slight concentration-dependence on longer timescales) indicates that we are observing mostly cis cleavage events, as explained in Appendix 2—figure 1—figure supplement 4 .
    Figure Legend Snippet: Concentration dependence of phosphodiester-mapped cleavage events. Each graph follows the kinetics of appearance/disappearance of a cleavage product at the indicated phosphodiester. ‘Fraction cleaved’ is defined as (band volume for a given cleavage product)/(total volume in lane). For example, ‘NTS 5', 11/12’ displays the fraction of 5'-radiolabeled NTS that was detected to have been cleaved at the 11/12 dinucleotide for each timepoint. Blue circles are equivalent to the data shown in Appendix 2—figure 1 , with error bars denoting standard deviation across three replicates. Red squares and green triangles indicate variants of this experiment in which the total concentration of either DNA-bound holoenzyme (red squares) or AsCas12a/crRNA (green triangles) was decreased. The fact that almost all species exhibit equivalent kinetics of appearance/disappearance in all three conditions (with the potential exception of the phosphodiesters on the portion of the PAM-distal NTS fragment that remains single-stranded after release by the enzyme, which exhibit slight concentration-dependence on longer timescales) indicates that we are observing mostly cis cleavage events, as explained in Appendix 2—figure 1—figure supplement 4 .

    Techniques Used: Concentration Assay, Standard Deviation

    Determinants of altered target-strand cleavage kinetics and position. 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM duplex DNA target radiolabeled on the 5' end of the target strand at 37°C for 0 s, 15 s, 1 min, 4 min, 15 min, or 1 hr, prior to quenching and resolution by denaturing PAGE. The 20-nt target sequence immediately adjacent to the PAM is shown below the crRNA spacer sequence used in each experiment. Red letters indicate TS:crRNA mismatches. Green letters indicate compensatory changes in the crRNA to restore a 20-nt match. The final timepoint of each reaction is reproduced in the gel on the right, for side-by-side comparison of the cleavage site distributions. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y = (y 0 -plateau)*exp(-k*x)+plateau), with y 0 constrained to 0 and the plateau value constrained to ≤1. A representative replicate is shown. The value of k obs for each time course is as follows: A (0.12 s −1 ), B (0.0093 s −1 ), C (0.0094 s −1 ), D (0.16 s −1 ), E (0.015 s −1 ). The precise value of k obs for A and D should be interpreted with caution due to poor sampling of informative timepoints.
    Figure Legend Snippet: Determinants of altered target-strand cleavage kinetics and position. 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM duplex DNA target radiolabeled on the 5' end of the target strand at 37°C for 0 s, 15 s, 1 min, 4 min, 15 min, or 1 hr, prior to quenching and resolution by denaturing PAGE. The 20-nt target sequence immediately adjacent to the PAM is shown below the crRNA spacer sequence used in each experiment. Red letters indicate TS:crRNA mismatches. Green letters indicate compensatory changes in the crRNA to restore a 20-nt match. The final timepoint of each reaction is reproduced in the gel on the right, for side-by-side comparison of the cleavage site distributions. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y = (y 0 -plateau)*exp(-k*x)+plateau), with y 0 constrained to 0 and the plateau value constrained to ≤1. A representative replicate is shown. The value of k obs for each time course is as follows: A (0.12 s −1 ), B (0.0093 s −1 ), C (0.0094 s −1 ), D (0.16 s −1 ), E (0.015 s −1 ). The precise value of k obs for A and D should be interpreted with caution due to poor sampling of informative timepoints.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Sequencing, Sampling

    dCas12a ribonucleoprotein binds tightly to pre-gapped/pre-unwound targets despite PAM-distal mismatches. The affinity of dAsCas12a/crRNA for various cognate DNA targets was assessed by a filter-binding assay. ‘Pre-gapped’ indicates the presence of a 5-nt gap in the non-target strand (see Appendix 2). ‘Pre-unwound’ indicates the presence of a stretch of NTS:TS mismatches in the DNA substrate. In Figure 3A , protospacer 3 is annotated as ‘DNA substrate 1;’ protospacer 4 is annotated as ‘DNA substrate 2;’ and crRNA 3 is the depicted crRNA. For each combination of crRNA/DNA target, crRNA was titrated in a solution with fixed [dAsCas12a] (400 nM), [DNA probe] (100 pM), and [non-specific DNA competitor] (500 nM). The identities of the titrant/fixed component were inverted in this experiment (as compared to all other binding experiments) because crRNA can form a stable complex with pre-unwound DNA targets in the absence of protein. Keeping [dAsCas12a] at 400 nM favored the formation of (dAsCas12a/crRNA):DNA complexes over crRNA:DNA complexes (which would be indistinguishable from free DNA in the filter binding assay). In the presence of high [apo protein], 500 nM non-specific DNA competitor (a duplex with a short ssDNA overhang) was also included to disfavor non-specific interactions between radiolabeled DNA and apo protein. The value of ‘fraction bound’ was 0 at [crRNA]=0 for all substrates (not shown due to the logarithmic x-axis). For all pre-unwound DNA targets, the fraction bound was essentially concentration-independent across all nonzero concentrations tested, suggesting that the lowest concentration tested had already saturated the specific binding interaction being probed. The high stability is in line with thermodynamic expectations for an interaction involving hybridization of two complementary 18-nt or 20-nt oligonucleotides (T m > 40°C) ( Kibbe, 2007 ). The fact that the saturated bound fraction is less than 1 could be due to (1) a common feature of filter-binding assays in which the process of physical separation disrupts bound species or (2) a stable population of protein-free crRNA:DNA complexes. In any case, the important conclusion to be drawn from these data is that each protospacer exhibits the same fraction bound regardless of the presence of mismatches at positions 19 and 20 in the crRNA. Thus, the crRNA-dependent effects seen in Figure 3A and Figure 3—figure supplement 2 must emerge from fundamental differences in conformational dynamics and not from differences in binding occupancy of Cas12a/crRNA on the DNA probe.
    Figure Legend Snippet: dCas12a ribonucleoprotein binds tightly to pre-gapped/pre-unwound targets despite PAM-distal mismatches. The affinity of dAsCas12a/crRNA for various cognate DNA targets was assessed by a filter-binding assay. ‘Pre-gapped’ indicates the presence of a 5-nt gap in the non-target strand (see Appendix 2). ‘Pre-unwound’ indicates the presence of a stretch of NTS:TS mismatches in the DNA substrate. In Figure 3A , protospacer 3 is annotated as ‘DNA substrate 1;’ protospacer 4 is annotated as ‘DNA substrate 2;’ and crRNA 3 is the depicted crRNA. For each combination of crRNA/DNA target, crRNA was titrated in a solution with fixed [dAsCas12a] (400 nM), [DNA probe] (100 pM), and [non-specific DNA competitor] (500 nM). The identities of the titrant/fixed component were inverted in this experiment (as compared to all other binding experiments) because crRNA can form a stable complex with pre-unwound DNA targets in the absence of protein. Keeping [dAsCas12a] at 400 nM favored the formation of (dAsCas12a/crRNA):DNA complexes over crRNA:DNA complexes (which would be indistinguishable from free DNA in the filter binding assay). In the presence of high [apo protein], 500 nM non-specific DNA competitor (a duplex with a short ssDNA overhang) was also included to disfavor non-specific interactions between radiolabeled DNA and apo protein. The value of ‘fraction bound’ was 0 at [crRNA]=0 for all substrates (not shown due to the logarithmic x-axis). For all pre-unwound DNA targets, the fraction bound was essentially concentration-independent across all nonzero concentrations tested, suggesting that the lowest concentration tested had already saturated the specific binding interaction being probed. The high stability is in line with thermodynamic expectations for an interaction involving hybridization of two complementary 18-nt or 20-nt oligonucleotides (T m > 40°C) ( Kibbe, 2007 ). The fact that the saturated bound fraction is less than 1 could be due to (1) a common feature of filter-binding assays in which the process of physical separation disrupts bound species or (2) a stable population of protein-free crRNA:DNA complexes. In any case, the important conclusion to be drawn from these data is that each protospacer exhibits the same fraction bound regardless of the presence of mismatches at positions 19 and 20 in the crRNA. Thus, the crRNA-dependent effects seen in Figure 3A and Figure 3—figure supplement 2 must emerge from fundamental differences in conformational dynamics and not from differences in binding occupancy of Cas12a/crRNA on the DNA probe.

    Techniques Used: Filter-binding Assay, Binding Assay, Concentration Assay, Hybridization

    Phosphorimage and quantification of non-target-strand gap-dependence experiments, in MgCl 2 , with radiolabeled trans substrate. Extent of trans cleavage by wild type AsCas12a in the presence of various DNA activator variants, as resolved by denaturing PAGE. Cas12a ternary complex (final concentrations: 100 nM AsCas12a, 120 nM crRNA, pre-hybridized 120 nM TS/240 nM NTS) was assembled with each of the indicated NTS variants and combined with 2 nM (final concentration) radiolabeled non-specific trans ssDNA target in cleavage buffer (5 mM MgCl 2 ). These reactions were then incubated for 30 min at 37°C prior to quenching and resolution by denaturing PAGE. Control lanes on the left contain some combination of intact NTS/TS with phosphodiesters (PO) or phosphorothioates (PS) across the standard cleavage sites; ‘pc’ stands for pre-cleaved (only PAM-proximal cleavage products: NTS truncated after nt 13, TS truncated after nt 22). Reactions without NTS contained 120 nM of a non-specific DNA oligonucleotide to account for substrate competition. All lanes indicated by a letter (C–Y) contained an NTS variant (see Appendix 2—figure 2—figure supplement 4 ) along with a TS containing phosphorothioates across the standard cleavage sites. When a given category has more than one substrate (e.g., 3-nt gap includes substrates M, N, O, and P), the first listed substrate (M) is shown as a blue bar, the second (N) as a red bar, the third (O) as a green bar, and the fourth (P) as a pink bar. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane).
    Figure Legend Snippet: Phosphorimage and quantification of non-target-strand gap-dependence experiments, in MgCl 2 , with radiolabeled trans substrate. Extent of trans cleavage by wild type AsCas12a in the presence of various DNA activator variants, as resolved by denaturing PAGE. Cas12a ternary complex (final concentrations: 100 nM AsCas12a, 120 nM crRNA, pre-hybridized 120 nM TS/240 nM NTS) was assembled with each of the indicated NTS variants and combined with 2 nM (final concentration) radiolabeled non-specific trans ssDNA target in cleavage buffer (5 mM MgCl 2 ). These reactions were then incubated for 30 min at 37°C prior to quenching and resolution by denaturing PAGE. Control lanes on the left contain some combination of intact NTS/TS with phosphodiesters (PO) or phosphorothioates (PS) across the standard cleavage sites; ‘pc’ stands for pre-cleaved (only PAM-proximal cleavage products: NTS truncated after nt 13, TS truncated after nt 22). Reactions without NTS contained 120 nM of a non-specific DNA oligonucleotide to account for substrate competition. All lanes indicated by a letter (C–Y) contained an NTS variant (see Appendix 2—figure 2—figure supplement 4 ) along with a TS containing phosphorothioates across the standard cleavage sites. When a given category has more than one substrate (e.g., 3-nt gap includes substrates M, N, O, and P), the first listed substrate (M) is shown as a blue bar, the second (N) as a red bar, the third (O) as a green bar, and the fourth (P) as a pink bar. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Concentration Assay, Incubation, Variant Assay

    13) Product Images from "Development of RNA-based assay for rapid detection of SARS-CoV-2 in clinical samples"

    Article Title: Development of RNA-based assay for rapid detection of SARS-CoV-2 in clinical samples

    Journal: bioRxiv

    doi: 10.1101/2020.06.30.172833

    Sensitivity of developed assay to detect SARS-CoV-2 RNA. Assay sensitivity was determined using different concentrations (concentration are in ng) of IVT synthesized SARS-CoV-2 RNA (a-b) , or nCOVID synthetic DNA (c-d) . Absorption spectra (a and c) corresponding to aggregated colloids exhibit a clear red-shift in peak with broadening, indicating successful hybridization. Optical pictures (b d) of the assay performed to demonstrate the color change, an extra pair of vials (on the left) to show color change with a higher amount (10ng) of target nucleic acid for the reference.
    Figure Legend Snippet: Sensitivity of developed assay to detect SARS-CoV-2 RNA. Assay sensitivity was determined using different concentrations (concentration are in ng) of IVT synthesized SARS-CoV-2 RNA (a-b) , or nCOVID synthetic DNA (c-d) . Absorption spectra (a and c) corresponding to aggregated colloids exhibit a clear red-shift in peak with broadening, indicating successful hybridization. Optical pictures (b d) of the assay performed to demonstrate the color change, an extra pair of vials (on the left) to show color change with a higher amount (10ng) of target nucleic acid for the reference.

    Techniques Used: Concentration Assay, Synthesized, Hybridization

    14) Product Images from "Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis"

    Article Title: Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02487

    Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.
    Figure Legend Snippet: Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.

    Techniques Used: In Vitro, Agarose Gel Electrophoresis, Sequencing, Incubation, Imaging, Plasmid Preparation

    Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.
    Figure Legend Snippet: Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.

    Techniques Used: Sequencing, Incubation, Staining, RNA Sequencing Assay

    Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.
    Figure Legend Snippet: Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.

    Techniques Used: Sequencing, Incubation, Staining

    15) Product Images from "High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus"

    Article Title: High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus

    Journal: Genetics

    doi: 10.1534/genetics.120.303269

    Oncopeltus CRISPR/Cas9 mutagenesis workflow. (A) (i) Genomic structure of Of-w ; exon boundaries are based on transcriptome data and the alignment of a cDNA sequenced in this study to the genome; however, exon 4 was not found in the genome. The dsRNA target region (519 bp) used in this study is shown as an orange bar (exons only). (ii) The target location gRNAs (A–C) used in this study are in red. (iii) The primers used to PCR-amplify exon 2 for our two genotyping assays are shown as black arrows. The amplicon is 513 bp and includes some intronic regions surrounding the exon. Stars mark the predicted loci of Cas9 cleavage and thus likely mutation; the predicted Surveyor cleavage product sizes (in base pairs) for each gRNA are shown above the strands. (B) Gels showing representative results from the Surveyor digest and the heteroduplex mobility assays used to genotype G1s. (i) In the Surveyor assay, PCR products were digested with Surveyor nuclease, resulting in cleavage in samples derived from heterozygous individuals. All gRNA A individuals screened here (lanes 1–7) show the expected cleavage products (∼100 and 413 bp), as do all gRNA B individuals (lanes 8–9; ∼277 and 236 bp, asterisks), identifying all individuals shown here as heterozygotes. (ii) In the heteroduplex mobility assay, 5 μl of PCR product was electrophoresed on a 4% agarose gel for > 5 hr at 80 V, allowing visualization of heteroduplexes, which migrate more slowly than homoduplexes, in samples from heterozygotes (lanes 11–14); thus, samples from homozygous individuals (WT) should produce only the 513-bp band (lane 10). (C) Sequenced mutant alleles induced by (i) gRNA-A/Cas9 and (ii) gRNA-B/Cas9. Red and blue lettering indicate the PAM site and insertions, respectively. (i) The mutant allele from individual A6-17 has a 16-bp insertion (blue) that adds a premature stop codon (red box); the allele from individual A12-10 has a 15-bp insertion (blue) and 1-bp deletion that induces a frameshift. (ii) The allele from individual B4-4 has a 7-bp deletion, resulting in a frameshift; the allele from individual B5-16 is complex, showing substitutions (blue) replacing sequence (underlined) 5′ and 3′ of the PAM site (red), including mutation of the splicing donor site (black box). cDNA, complementary DNA; CRISPR, clustered regularly interspaced short palindromic repeats; dsRNA, double-stranded RNA; gRNA, guide RNA; PAM, protospacer-adjacent motif; WT, wild-type.
    Figure Legend Snippet: Oncopeltus CRISPR/Cas9 mutagenesis workflow. (A) (i) Genomic structure of Of-w ; exon boundaries are based on transcriptome data and the alignment of a cDNA sequenced in this study to the genome; however, exon 4 was not found in the genome. The dsRNA target region (519 bp) used in this study is shown as an orange bar (exons only). (ii) The target location gRNAs (A–C) used in this study are in red. (iii) The primers used to PCR-amplify exon 2 for our two genotyping assays are shown as black arrows. The amplicon is 513 bp and includes some intronic regions surrounding the exon. Stars mark the predicted loci of Cas9 cleavage and thus likely mutation; the predicted Surveyor cleavage product sizes (in base pairs) for each gRNA are shown above the strands. (B) Gels showing representative results from the Surveyor digest and the heteroduplex mobility assays used to genotype G1s. (i) In the Surveyor assay, PCR products were digested with Surveyor nuclease, resulting in cleavage in samples derived from heterozygous individuals. All gRNA A individuals screened here (lanes 1–7) show the expected cleavage products (∼100 and 413 bp), as do all gRNA B individuals (lanes 8–9; ∼277 and 236 bp, asterisks), identifying all individuals shown here as heterozygotes. (ii) In the heteroduplex mobility assay, 5 μl of PCR product was electrophoresed on a 4% agarose gel for > 5 hr at 80 V, allowing visualization of heteroduplexes, which migrate more slowly than homoduplexes, in samples from heterozygotes (lanes 11–14); thus, samples from homozygous individuals (WT) should produce only the 513-bp band (lane 10). (C) Sequenced mutant alleles induced by (i) gRNA-A/Cas9 and (ii) gRNA-B/Cas9. Red and blue lettering indicate the PAM site and insertions, respectively. (i) The mutant allele from individual A6-17 has a 16-bp insertion (blue) that adds a premature stop codon (red box); the allele from individual A12-10 has a 15-bp insertion (blue) and 1-bp deletion that induces a frameshift. (ii) The allele from individual B4-4 has a 7-bp deletion, resulting in a frameshift; the allele from individual B5-16 is complex, showing substitutions (blue) replacing sequence (underlined) 5′ and 3′ of the PAM site (red), including mutation of the splicing donor site (black box). cDNA, complementary DNA; CRISPR, clustered regularly interspaced short palindromic repeats; dsRNA, double-stranded RNA; gRNA, guide RNA; PAM, protospacer-adjacent motif; WT, wild-type.

    Techniques Used: CRISPR, Mutagenesis, Polymerase Chain Reaction, Amplification, Derivative Assay, Agarose Gel Electrophoresis, Sequencing

    16) Product Images from "Staphylococcus aureus Cas9 is a multiple-turnover enzyme"

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme

    Journal: RNA

    doi: 10.1261/rna.067355.118

    S. aureus Cas9 is a multiple turnover enzyme. A total of 25 nM SpyCas9 (black) or SauCas9 (red) was preincubated with 100 nM sgRNA for 15 min prior to the addition of 250 nM DNA. The amount of HNH-cleaved non-PAM strand of the DNA, divided by the enzyme concentration, was plotted versus time. ( A ) DNA1, no decoy PAMs. SauCas9: amplitude = 3.6 ± 0.3; k exp = 0.010 ± 0.001 min −1 ; k lin = 1.6 × 10 −3 ± 6 × 10 −4 min −1 ( R 2 = 0.98). SpyCas9: amplitude = 1.0 ± 0.1; k lin = 2.4 × 10 −4 ± 9 × 10 −5 min −1 ( R 2 = 0.95). ( B ) DNA2, 2 decoy PAMs. SauCas9: amplitude = 3.5 ± 0.60; k exp = 0.012 ± 0.001 min −1 ; k lin = 1.5 × 10 −3 ± 6 × 10 −4 min −1 ( R 2 = 0.96). SpyCas9: amplitude = 1.2 ± 0.2; k lin = 3.5 × 10 −4 ± 8 × 10 −5 min −1 ( R 2 = 0.83). ( C ) DNA3, 5 decoy PAMs. SauCas9: amplitude = 3.4 ± 0.6; k exp = 0.013 ± 0.001 min −1 ; k lin = 8.6 × 10 −4 ± 3 × 10 −4 min −1 ( R 2 = 0.96). SpyCas9: amplitude = 1.1 ± 0.2; k lin = 1.5 × 10 −4 ± 7 × 10 −5 min −1 ( R 2 = 0.83). ( D ) Summary of the data in A–C for convenience. ND, the rate of the burst could not be resolved by manual quenching. All of the values are the result of at least three independent experiments and are reported as mean ± average deviation.
    Figure Legend Snippet: S. aureus Cas9 is a multiple turnover enzyme. A total of 25 nM SpyCas9 (black) or SauCas9 (red) was preincubated with 100 nM sgRNA for 15 min prior to the addition of 250 nM DNA. The amount of HNH-cleaved non-PAM strand of the DNA, divided by the enzyme concentration, was plotted versus time. ( A ) DNA1, no decoy PAMs. SauCas9: amplitude = 3.6 ± 0.3; k exp = 0.010 ± 0.001 min −1 ; k lin = 1.6 × 10 −3 ± 6 × 10 −4 min −1 ( R 2 = 0.98). SpyCas9: amplitude = 1.0 ± 0.1; k lin = 2.4 × 10 −4 ± 9 × 10 −5 min −1 ( R 2 = 0.95). ( B ) DNA2, 2 decoy PAMs. SauCas9: amplitude = 3.5 ± 0.60; k exp = 0.012 ± 0.001 min −1 ; k lin = 1.5 × 10 −3 ± 6 × 10 −4 min −1 ( R 2 = 0.96). SpyCas9: amplitude = 1.2 ± 0.2; k lin = 3.5 × 10 −4 ± 8 × 10 −5 min −1 ( R 2 = 0.83). ( C ) DNA3, 5 decoy PAMs. SauCas9: amplitude = 3.4 ± 0.6; k exp = 0.013 ± 0.001 min −1 ; k lin = 8.6 × 10 −4 ± 3 × 10 −4 min −1 ( R 2 = 0.96). SpyCas9: amplitude = 1.1 ± 0.2; k lin = 1.5 × 10 −4 ± 7 × 10 −5 min −1 ( R 2 = 0.83). ( D ) Summary of the data in A–C for convenience. ND, the rate of the burst could not be resolved by manual quenching. All of the values are the result of at least three independent experiments and are reported as mean ± average deviation.

    Techniques Used: Concentration Assay

    S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA
    Figure Legend Snippet: S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA

    Techniques Used:

    S. aureus Cas9 does not exhibit detectable post-cleavage trimming activity on cleaved DNA. ( A , B ) Representative capillary electrophoresis of 110mer DNA labeled with 5′-FAM (blue trace) on the PAM-containing strand and 5′-ROX (red trace) on the non-PAM strand hydrolyzed by ( A ) SpyCas9 or ( B ) SauCas9 at reaction time points of 15 sec, 10 min, 30 min, and 120 min. Data are plotted as relative fluorescence versus DNA oligomer length. The trend was consistently observed over the course of our experimentation ( n > 10) with SpyCas9 and SauCas9.
    Figure Legend Snippet: S. aureus Cas9 does not exhibit detectable post-cleavage trimming activity on cleaved DNA. ( A , B ) Representative capillary electrophoresis of 110mer DNA labeled with 5′-FAM (blue trace) on the PAM-containing strand and 5′-ROX (red trace) on the non-PAM strand hydrolyzed by ( A ) SpyCas9 or ( B ) SauCas9 at reaction time points of 15 sec, 10 min, 30 min, and 120 min. Data are plotted as relative fluorescence versus DNA oligomer length. The trend was consistently observed over the course of our experimentation ( n > 10) with SpyCas9 and SauCas9.

    Techniques Used: Activity Assay, Electrophoresis, Labeling, Size-exclusion Chromatography, Fluorescence

    17) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: bioRxiv

    doi: 10.1101/815092

    Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.
    Figure Legend Snippet: Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, In Vitro

    18) Product Images from "An RNA polymerase ribozyme that synthesizes its own ancestor"

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1914282117

    In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.
    Figure Legend Snippet: In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.

    Techniques Used: In Vitro, Amplification, Functional Assay, Hybridization, Magnetic Beads, Polymerase Chain Reaction, Sequencing, Labeling

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    New England Biolabs monarch rna cleanup kit
    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc <t>RNA</t> synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on <t>512B::CLuc</t> chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.
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    New England Biolabs monarch dna cleanup columns
    Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. A . Experimental approach for identifying the subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. These experiments began with approximately 400,000 WM989 A6-G3 cells transduced at an MOI ∼ 1.0 and allowed to divide for 6 days before splitting the culture into two groups. We treated one group with 4 μM DOT1L inhibitor (pinometostat) and the other with vehicle control (DMSO) for another 6 days. We then split each group again, fixing half as our “Carbon Copies” and treating the other half with 1 μM vemurafenib for ∼2.5 weeks. After vemurafenib treatment, we extracted genomic <t>DNA</t> from the remaining cells for <t>barcode</t> sequencing. B . We compared the abundance of each barcode identified in resistant cells pre-treated with DOT1L inhibitor versus resistant cells pre-treated with vehicle control as shown in A. This comparison revealed a subset of barcodes with a greater relative abundance in resistant cells pre-treated with DOT1L inhibitor than resistant cells pre-treated with vehicle control (blue points). We used these barcodes to design RNA FISH probes targeting cells requiring DOT1L inhibition to become vemurafenib resistant. A separate set of barcodes showed similar high abundance with or without DOT1L inhibition (orange points), which we used to design RNA FISH probes targeting primed cells not requiring DOT1L inhibition to become resistant. C . Using these probes, we labeled and sorted cells requiring DOT1L inhibition to become vemurafenib resistant (blue), primed cells not requiring DOT1L inhibition (orange), and non-primed cells (gray) from Carbon Copies for RNA sequencing. We separately sorted cells from Carbon Copies treated with DOT1L inhibitor and Carbon Copies treated with vehicle control (2 biological replicates each). D . To identify markers of cells that require DOT1L inhibition to become resistant, we used DESeq2 to compare their gene expression to non-primed cells (x-axis) and primed cells not requiring DOT1L inhibition (y-axis). In this analysis, we included cells sorted from all Carbon Copies (treated with DOT1L inhibitor or vehicle control) from 2 biological replicates and included DOT1L inhibitor treatment as a covariate in estimating log 2 fold changes. Red points correspond to genes differentially expressed in one or both comparisons (p-adjusted ≤0.1 and log 2 fold change ≥ 1). E . Expression of DEPTOR in transcripts per million (tpm) in the subpopulations isolated in B. Points indicate tpm values for experimental replicates. F . We used the same probe sets as in B. to identify cells in situ in Carbon Copies fixed prior to vemurafenib treatment, then measured single cell expression of DEPTOR, MGP, SOX10, MITF , and 6 priming markers by RNA FISH. Shown is the expression of DEPTOR in the indicated cell populations identified in the Carbon Copies treated with vehicle control. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. Above each boxplot is the proportion of cells with levels of DEPTOR RNA above the indicated threshold (∼95th percentile in non-primed cells). G . We applied the UMAP algorithm to visualize the single cell expression data from in situ Carbon Copies. These plots include 423 cells from the vehicle control treated Carbon Copy. In the upper left plot, points are colored according to the fate of each cell as determined by its barcode. For the remaining plots points are colored by the expression level of the indicated gene in that cell. These data correspond to 1 of 2 biological replicates (See Supp. Fig 13 for additional replicate).
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    Image Search Results


    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Journal: RNA

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1261/rna.073858.119

    Figure Lengend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Article Snippet: For the 512B transcripts, reactions were cleaned up with the Monarch RNA Cleanup Kit (New England Biolabs).

    Techniques: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. A . Experimental approach for identifying the subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. These experiments began with approximately 400,000 WM989 A6-G3 cells transduced at an MOI ∼ 1.0 and allowed to divide for 6 days before splitting the culture into two groups. We treated one group with 4 μM DOT1L inhibitor (pinometostat) and the other with vehicle control (DMSO) for another 6 days. We then split each group again, fixing half as our “Carbon Copies” and treating the other half with 1 μM vemurafenib for ∼2.5 weeks. After vemurafenib treatment, we extracted genomic DNA from the remaining cells for barcode sequencing. B . We compared the abundance of each barcode identified in resistant cells pre-treated with DOT1L inhibitor versus resistant cells pre-treated with vehicle control as shown in A. This comparison revealed a subset of barcodes with a greater relative abundance in resistant cells pre-treated with DOT1L inhibitor than resistant cells pre-treated with vehicle control (blue points). We used these barcodes to design RNA FISH probes targeting cells requiring DOT1L inhibition to become vemurafenib resistant. A separate set of barcodes showed similar high abundance with or without DOT1L inhibition (orange points), which we used to design RNA FISH probes targeting primed cells not requiring DOT1L inhibition to become resistant. C . Using these probes, we labeled and sorted cells requiring DOT1L inhibition to become vemurafenib resistant (blue), primed cells not requiring DOT1L inhibition (orange), and non-primed cells (gray) from Carbon Copies for RNA sequencing. We separately sorted cells from Carbon Copies treated with DOT1L inhibitor and Carbon Copies treated with vehicle control (2 biological replicates each). D . To identify markers of cells that require DOT1L inhibition to become resistant, we used DESeq2 to compare their gene expression to non-primed cells (x-axis) and primed cells not requiring DOT1L inhibition (y-axis). In this analysis, we included cells sorted from all Carbon Copies (treated with DOT1L inhibitor or vehicle control) from 2 biological replicates and included DOT1L inhibitor treatment as a covariate in estimating log 2 fold changes. Red points correspond to genes differentially expressed in one or both comparisons (p-adjusted ≤0.1 and log 2 fold change ≥ 1). E . Expression of DEPTOR in transcripts per million (tpm) in the subpopulations isolated in B. Points indicate tpm values for experimental replicates. F . We used the same probe sets as in B. to identify cells in situ in Carbon Copies fixed prior to vemurafenib treatment, then measured single cell expression of DEPTOR, MGP, SOX10, MITF , and 6 priming markers by RNA FISH. Shown is the expression of DEPTOR in the indicated cell populations identified in the Carbon Copies treated with vehicle control. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. Above each boxplot is the proportion of cells with levels of DEPTOR RNA above the indicated threshold (∼95th percentile in non-primed cells). G . We applied the UMAP algorithm to visualize the single cell expression data from in situ Carbon Copies. These plots include 423 cells from the vehicle control treated Carbon Copy. In the upper left plot, points are colored according to the fate of each cell as determined by its barcode. For the remaining plots points are colored by the expression level of the indicated gene in that cell. These data correspond to 1 of 2 biological replicates (See Supp. Fig 13 for additional replicate).

    Journal: bioRxiv

    Article Title: Variability within rare cell states enables multiple paths towards drug resistance

    doi: 10.1101/2020.03.18.996660

    Figure Lengend Snippet: Rewind identifies a distinct subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. A . Experimental approach for identifying the subpopulation of cells that require DOT1L inhibition to become vemurafenib resistant. These experiments began with approximately 400,000 WM989 A6-G3 cells transduced at an MOI ∼ 1.0 and allowed to divide for 6 days before splitting the culture into two groups. We treated one group with 4 μM DOT1L inhibitor (pinometostat) and the other with vehicle control (DMSO) for another 6 days. We then split each group again, fixing half as our “Carbon Copies” and treating the other half with 1 μM vemurafenib for ∼2.5 weeks. After vemurafenib treatment, we extracted genomic DNA from the remaining cells for barcode sequencing. B . We compared the abundance of each barcode identified in resistant cells pre-treated with DOT1L inhibitor versus resistant cells pre-treated with vehicle control as shown in A. This comparison revealed a subset of barcodes with a greater relative abundance in resistant cells pre-treated with DOT1L inhibitor than resistant cells pre-treated with vehicle control (blue points). We used these barcodes to design RNA FISH probes targeting cells requiring DOT1L inhibition to become vemurafenib resistant. A separate set of barcodes showed similar high abundance with or without DOT1L inhibition (orange points), which we used to design RNA FISH probes targeting primed cells not requiring DOT1L inhibition to become resistant. C . Using these probes, we labeled and sorted cells requiring DOT1L inhibition to become vemurafenib resistant (blue), primed cells not requiring DOT1L inhibition (orange), and non-primed cells (gray) from Carbon Copies for RNA sequencing. We separately sorted cells from Carbon Copies treated with DOT1L inhibitor and Carbon Copies treated with vehicle control (2 biological replicates each). D . To identify markers of cells that require DOT1L inhibition to become resistant, we used DESeq2 to compare their gene expression to non-primed cells (x-axis) and primed cells not requiring DOT1L inhibition (y-axis). In this analysis, we included cells sorted from all Carbon Copies (treated with DOT1L inhibitor or vehicle control) from 2 biological replicates and included DOT1L inhibitor treatment as a covariate in estimating log 2 fold changes. Red points correspond to genes differentially expressed in one or both comparisons (p-adjusted ≤0.1 and log 2 fold change ≥ 1). E . Expression of DEPTOR in transcripts per million (tpm) in the subpopulations isolated in B. Points indicate tpm values for experimental replicates. F . We used the same probe sets as in B. to identify cells in situ in Carbon Copies fixed prior to vemurafenib treatment, then measured single cell expression of DEPTOR, MGP, SOX10, MITF , and 6 priming markers by RNA FISH. Shown is the expression of DEPTOR in the indicated cell populations identified in the Carbon Copies treated with vehicle control. Each point corresponds to an individual cell. Error bars indicate 25th and 75th percentiles of distributions. Above each boxplot is the proportion of cells with levels of DEPTOR RNA above the indicated threshold (∼95th percentile in non-primed cells). G . We applied the UMAP algorithm to visualize the single cell expression data from in situ Carbon Copies. These plots include 423 cells from the vehicle control treated Carbon Copy. In the upper left plot, points are colored according to the fate of each cell as determined by its barcode. For the remaining plots points are colored by the expression level of the indicated gene in that cell. These data correspond to 1 of 2 biological replicates (See Supp. Fig 13 for additional replicate).

    Article Snippet: We purified the ligated barcode ClampFISH probes using Monarch DNA cleanup columns (NEB) according to the manufacturer’s protocol.

    Techniques: Inhibition, Sequencing, Fluorescence In Situ Hybridization, Labeling, RNA Sequencing Assay, Expressing, Isolation, In Situ

    Rewind identifies rare cell states giving rise to vemurafenib resistant colonies. A . Schematic of Rewind approach for isolating the initial primed WM989 A6-G3 melanoma cells that ultimately give rise to vemurafenib resistant colonies. For the experiment shown, we transduced ∼ 200,000 WM989 A6-G3 cells at an MOI ∼ 1.0 with our Rewind barcode library. After 11 days (∼4 population doublings) we divided the culture in two, fixing half in suspension as a Carbon Copy and treating the other half with 1 μM vemurafenib to select for resistant cells. After 3 weeks in vemurafenib, we extracted genomic DNA from the resistant cells that remain and identified their Rewind barcodes by targeted sequencing. We then designed RNA FISH probes targeting 60 of these barcodes and used these probes to specifically label cells primed to become resistant from our Carbon Copy. We then sorted these cells out from the population, extracted cellular RNA and performed RNA sequencing. B . To assess the sensitivity and specificity of the Rewind experiment in A, we performed targeted sequencing to identify barcodes from cDNA generated during RNA-seq library preparation. Bar graphs show the abundance (y-axis) and rank (x-axis) of each sequenced barcode (≥ 5 normalized reads). Red bars correspond to barcodes targeted by our probe set and gray bars correspond to “off-target” barcode sequences. Inset shows the proportion of barcodes targeted by our probeset detected in each group. These data correspond to 1 of 2 replicates. In the second replicate, 30 out of 50 probed barcodes were detected in the sorted primed population. C . We performed differential expression analysis using DESeq2 of primed vs. non-primed sorted cells. Shown is the mean expression level (TPM) for protein coding genes in primed cells (y-axis) and log 2 fold change in expression estimated using DESeq2 (x-axis) compared to non-primed cells. Colors indicate differentially expressed genes related to ECM Organization and Cell Migration (red), MAPK and PI3K/Akt signalling pathways (blue) and previously identified resistance markers (purple; Shaffer et al. 2017). Genes were assigned to categories based on a consensus of KEGG pathway and GO enrichment analyses (See Methods for details). D . We selected the most differentially expressed, cell surface ECM-related gene ( ITGA3 ) to validate as a predictive marker of vemurafenib resistance in WM989 A6-G3. After staining cells with a fluorescently labelled antibody targeting ITGA3, we sorted the brightest 0.5% (ITGA3-High) and remaining (ITGA3-Low) populations, then treated both with 1 μM vemurafenib. After approximately 18 days, we fixed the cells, stained nuclei with DAPI then imaged the entire wells to quantify the number of resistant colonies and cells. The data correspond to 1 of 3 biological replicates (See Supp. Fig. 4 for additional replicates).

    Journal: bioRxiv

    Article Title: Variability within rare cell states enables multiple paths towards drug resistance

    doi: 10.1101/2020.03.18.996660

    Figure Lengend Snippet: Rewind identifies rare cell states giving rise to vemurafenib resistant colonies. A . Schematic of Rewind approach for isolating the initial primed WM989 A6-G3 melanoma cells that ultimately give rise to vemurafenib resistant colonies. For the experiment shown, we transduced ∼ 200,000 WM989 A6-G3 cells at an MOI ∼ 1.0 with our Rewind barcode library. After 11 days (∼4 population doublings) we divided the culture in two, fixing half in suspension as a Carbon Copy and treating the other half with 1 μM vemurafenib to select for resistant cells. After 3 weeks in vemurafenib, we extracted genomic DNA from the resistant cells that remain and identified their Rewind barcodes by targeted sequencing. We then designed RNA FISH probes targeting 60 of these barcodes and used these probes to specifically label cells primed to become resistant from our Carbon Copy. We then sorted these cells out from the population, extracted cellular RNA and performed RNA sequencing. B . To assess the sensitivity and specificity of the Rewind experiment in A, we performed targeted sequencing to identify barcodes from cDNA generated during RNA-seq library preparation. Bar graphs show the abundance (y-axis) and rank (x-axis) of each sequenced barcode (≥ 5 normalized reads). Red bars correspond to barcodes targeted by our probe set and gray bars correspond to “off-target” barcode sequences. Inset shows the proportion of barcodes targeted by our probeset detected in each group. These data correspond to 1 of 2 replicates. In the second replicate, 30 out of 50 probed barcodes were detected in the sorted primed population. C . We performed differential expression analysis using DESeq2 of primed vs. non-primed sorted cells. Shown is the mean expression level (TPM) for protein coding genes in primed cells (y-axis) and log 2 fold change in expression estimated using DESeq2 (x-axis) compared to non-primed cells. Colors indicate differentially expressed genes related to ECM Organization and Cell Migration (red), MAPK and PI3K/Akt signalling pathways (blue) and previously identified resistance markers (purple; Shaffer et al. 2017). Genes were assigned to categories based on a consensus of KEGG pathway and GO enrichment analyses (See Methods for details). D . We selected the most differentially expressed, cell surface ECM-related gene ( ITGA3 ) to validate as a predictive marker of vemurafenib resistance in WM989 A6-G3. After staining cells with a fluorescently labelled antibody targeting ITGA3, we sorted the brightest 0.5% (ITGA3-High) and remaining (ITGA3-Low) populations, then treated both with 1 μM vemurafenib. After approximately 18 days, we fixed the cells, stained nuclei with DAPI then imaged the entire wells to quantify the number of resistant colonies and cells. The data correspond to 1 of 3 biological replicates (See Supp. Fig. 4 for additional replicates).

    Article Snippet: We purified the ligated barcode ClampFISH probes using Monarch DNA cleanup columns (NEB) according to the manufacturer’s protocol.

    Techniques: Sequencing, Fluorescence In Situ Hybridization, RNA Sequencing Assay, Generated, Expressing, Migration, Marker, Staining