Figure 1. DNA-guided RNA cleavage activity

Slides:

Advertisements

Similar presentations

Figure 1. Schematic of the corA leader mRNA

Advertisements

Figure 2. BRCA2–RAD51 complexes organize into filament-like structures

Figure 1. RISC activity in BYL. ( A ) Quantity of AGO1 protein in BYL

Figure 1. AsCpf1 and LbCpf1-mediated gene editing in human cells

Figure 3. Definition of the outside distance, Dout, as the sum of the distances from each of the two protein binding sites to their corresponding.

Figure 1. A3B NTD mediates enzyme activity and oligomerization

Figure 2. Effects of 3′ and 5′ base mismatches on RNA templated DNA end-joining fidelity of PBCV-1 DNA ligase. For each PLP group, the contribution of.

Figure 1. Stabilization of the hairpin structure by non-nucleotide linkers (-L-) following enzymatic circularization. Structures of the stilbene diether.

From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase

Figure 1. PARP1 binds to abasic sites and DNA ends as a monomer

Figure 1. (A) The VEGF promoter PQS and scheme of G oxidation to OG, as well as (B) the proposed APE1-dependent pathway ... Figure 1. (A) The VEGF promoter.

Figure 1. Circular taxonomy tree based on the species that were sequenced in our study. Unless provided in the caption above, the following copyright applies.

Figure 1. Mechanisms of long dsRNA digestion by RNase III enzymes

Figure 1. Effect of recombinantly expressed RNase T on growth behavior of E. coli. Growth was assessed for E. coli ... Figure 1. Effect of recombinantly.

Figure 1: Axial T2 W images of penis showed a well-defined septated area of 2 cm in the posterior aspect of ... Figure 1: Axial T2 W images.

Figure 1. Aminoacylation of 3′-NH2-tRNA catalyzed by flexizymes

Figure 1. Effect of random T/A→dU/A substitutions on transcription by T7 RNAP using a 321 bp DNA transcription template ... Figure 1. Effect of random.

FIGURE 1 Histological diagnoses divided into 8-year time frames (n = 1208). Unless provided in the caption above, the following copyright applies to the.

Figure 1. Position and number of NLS improves genome editing by AsCas12a, LbCas12a and FnoCas12a. (A) General schematic ... Figure 1. Position and number.

Figure 1. Domain organization of IMP1

Figure 1. BRCA1-associated R-Loop accumulation at a non-coding region upstream of ESR1 locus. (A) Alignment of DRIP-seq ... Figure 1. BRCA1-associated.

Figure 1. Ratios of observed to expected numbers of exon boundaries aligning to boundaries of domain and disorder ... Figure 1. Ratios of observed to expected.

Figure 1. autoMLST workflow depicting placement and de novo mode

Figure 1. Chemical structures of DNA and tc-DNA

Figure 1. RNase III processes the 23S-5S rRNA precursor in vivo

Fig. 1 Mean change from baseline in ANC ± s. e

Point estimates with ... Point estimates with 95% CI. HR: hip replacement; KR: knee replacement. Unless provided in the caption above, the following copyright.

FIGURE 1 Participant flow diagram. Exercise Counseling Clinic (ECC).

Figure 6. The DNA lyase activity of hNTHL1 contributes to the processing of lesions in nucleosomes, even in the ... Figure 6. The DNA lyase activity of.

Figure 1. Analysis of human TRIM5α protein with Blast-Search and PhyML+SMS ‘One click’ workflow. (A) NGPhylogeny.fr ... Figure 1. Analysis of human TRIM5α.

Figure 2. Natural history of chlamydia transmission, with arrows showing the transitions between health states. Figure 2. Natural history of chlamydia.

Figure 1 Nelson-Aalen estimates of the cumulative incidence rates for patients on versus off IST. ON = optic neuritis; ... Figure 1 Nelson-Aalen estimates.

FIGURE 1 Study consort diagram

Figure 1. Illustration of DGR systems and their prediction using myDGR

Figure 1. The pipeline of Aggrescan3D 2.0 server.

Figure 1. Summary of experimental conditions and data normalization

Figure 1. EBOV VP35 has NTP-binding and NTPase activities

Figure 1. Prediction result for birch pollen allergen Bet v 1 (PDB: 1bv1), as obtained by comparison to the cherry ... Figure 1. Prediction result for.

Figure 1. Using Voronoi tessellation to define contacts

Figure 1. Designed cotranscriptional RNA structures

Figure 1. Analysis of the AP lyase activity of BsuLigD

Figure 4. RLS spectra of (A) TMPipEOPP and (B) OMHEPzEOPP in the presence of different concentrations of KRAS. The RLS ... Figure 4. RLS spectra of (A)

Figure 1. PaintOmics 3 workflow diagram

Figure 1. Concept of poly(A) tail labeling for translation and localization analyses of reporter mRNAs. Azido-modified ... Figure 1. Concept of poly(A)

Figure 1. Schematic diagram of solar energy and coal-fired power generation system. Unless provided in the caption above, the following copyright applies.

Figure 1. MERMAID web server interface (Start page, Parameter page): MERMAID provides two ways to submit a protein ... Figure 1. MERMAID web server interface.

Figure 1. Yvis platform overview

Figure 1. The framework of NetGO with seven steps

Figure 1. Workflow of the HawkDock server that is divided into three major steps: (i) input of unbound or bound protein ... Figure 1. Workflow of the HawkDock.

Figure 2. Result page of a Primer3Plus Cloning run showing the left and right primers in blue and yellow. The included ... Figure 2. Result page of a Primer3Plus.

Fig. 1. —Synteny analysis of melon chromosome 1 (brown) and cucumber chromosome 7 (green) based on melon-cucumber ... Fig. 1. —Synteny analysis of melon.

Figure 4. MTase JHP1050 inactivation causes phenotypic effects that vary between strains: growth, viability and ... Figure 4. MTase JHP1050 inactivation.

Figure 1. Scheme of a phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) for selection ... Figure 1. Scheme of a phosphorothioated-terminal.

Fig. 2. —Phylogenetic relationships and motif compositions of some representative MORC genes in plants and animals. ... Fig. 2. —Phylogenetic relationships.

Figure 1. 3C analysis of HEM3, BLM10, and SEN1 genes in rpb4Δ and isogenic wild type cells. (A) Schematic ... Figure 1. 3C analysis of HEM3, BLM10, and.

Figure 1 Genetic results. No case had more than one diagnostic result

Figure 1. CSB does not affect the recruitment of OGG1 to oxidative DNA damage. (A) Representative stills of time-lapse ... Figure 1. CSB does not affect.

Figure 1. (A) Baseline contrast-enhanced CT scan of melanoma patient presenting with metastases in the liver and lymph ... Figure 1. (A) Baseline contrast-enhanced.

Figure 1. The overlap between Ensembl/GENCODE, RefSeq and UniProtKB genes. The number of genes classified as coding in ... Figure 1. The overlap between.

Figure 1. Illustration of aggregation functions on the local network of 1-decene. 1-decene is marked as target. ... Figure 1. Illustration of aggregation.

Figure 6. Protein-protein interactions

Figure 1. Optimization of the variant calling algorithm, ADIScan1, by tangential conversion of read depth ratios ... Figure 1. Optimization of the variant.

Figure 1. GWAS Catalog associations for coronary artery disease plotted across all chromosomes. Associations added ... Figure 1. GWAS Catalog associations.

Figure 1 The workflow of CAR development from a hybridoma

Figure 1. Crystal structures of Rim1

Source:Zimnisky (2014). Source:Zimnisky (2014).

Figure 1 Mechanisms of mitral regurgitation.

Figure 1. Removal of the 2B subdomain activates Rep monomer unwinding

Figure 5. The endonucleolytic product from PfuPCNA/MR activity is displaced from dsDNA. Results from real-time ... Figure 5. The endonucleolytic product.

Figure 4. In vitro mo5U forming activity of TrmR

Presentation transcript:

Figure 1. DNA-guided RNA cleavage activity Figure 1. DNA-guided RNA cleavage activity. (A) Schematic of cleavage assay using ss gRNA or gDNA. RNA-free AGO207 is ... Figure 1. DNA-guided RNA cleavage activity. (A) Schematic of cleavage assay using ss gRNA or gDNA. RNA-free AGO207 is shown with AGO207 bound to endogenous E. coli RNA that co-purifies with the protein (black and gray strands). Either gRNA or gDNA is loaded into the RNA-free population of AGO207. Following complex formation, a 5′ labeled 60-nt unstructured RNA or DNA target whose sequence perfectly matches the guide is added to the reaction. Yellow circle indicates <sup>32</sup>P radiolabel. (B) Schematic of guide and target pairs used in the cleavage assay described in (A) showing combinations of gRNA or gDNA bound to a complementary cap-labeled RNA target or a 5′ end-labeled DNA target. Yellow circle indicates radiolabel. Black arrowhead indicates cleavage site. (C) Cleavage assay using guide and target pairs shown in (B). Black bars indicate average of three independent replicates and dots indicate cleavage percentage of each individual replicate relative to the canonical gRNA targeting the RNA substrate. Inset shows low-level cleavage of DNA targets by either RISC or DISC. (D) Analysis of cleavage site by RISC and DISC. AGO207 was programmed with either a 23-nt gRNA or gDNA followed by addition of a perfectly matched cap-labeled target RNA. Substrates and products were resolved on 16% denaturing PAGE alongside base-hydrolyzed polyuridine RNA. Inset shows expanded view of cleavage products, demonstrating that RISC and DISC both cleave the RNA substrate at the same position. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 46, Issue 16, 12 June 2018, Page e98, https://doi.org/10.1093/nar/gky496 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Optimization of gDNAs Figure 2. Optimization of gDNAs. (A) Evaluating the effect of the 5′ nt on cleavage activity. AGO207 was programmed ... Figure 2. Optimization of gDNAs. (A) Evaluating the effect of the 5′ nt on cleavage activity. AGO207 was programmed with gDNAs whose 5′ nt was T, A, G or C followed by addition of a cap-labeled RNA target that perfectly matches all guides from positions 2–23. Black bars indicate average cleavage of three independent replicates and dots indicate cleavage percentage of each individual replicate. (B) Optimizing gDNA length. (Left) miR-20a-derived gDNA sequences with 5′ T were varied in length from 15 – 25 nt. Yellow circle indicates cap-label. Black arrowheads indicate cleavage site. (Right) Quantification of cleavage products using gDNAs of different length. Black bars indicate average of three independent replicates and dots indicate cleavage percentage of each individual replicate. (C) Evaluating sequence-specificity of DISC. (Top) Sequences of gRNA or gDNA paired with a cap-labeled RNA target that contains a dinucleotide mismatch at a non-permissive site known to abrogate cleavage by yeast AGO. Yellow circle indicates cap-label on target strand. Black arrowheads indicate the cleavage site at t10 and t11 (positions 10 and 11 counting from the 5′ end of the guide). (Bottom) AGO207 was pre-incubated with either a gRNA or gDNA followed by addition of either a perfectly matched target or a mismatched target. Substrates and products were resolved by 16% denaturing PAGE. Black arrowhead indicates migration of cleavage product. (D) Single- and dinucleotide mismatch assay. (Left) Schematic of gDNAs (bottom) and target RNA (top) with mismatches in the guide shown in red and bold. Dotted line flanked by black arrowhead indicates cleavage site. Yellow circle indicates cap-label on target strand. (Right) Black bars on plot represent average cleavage by DISC of three independent replicates and gray dots represent individual replicates. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 46, Issue 16, 12 June 2018, Page e98, https://doi.org/10.1093/nar/gky496 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Cleavage of highly structured HIV-1 ΔDIS 5′UTR RNA by DISC Figure 3. Cleavage of highly structured HIV-1 ΔDIS 5′UTR RNA by DISC. (A) Schematic of DISC-mediated cleavage assay. ... Figure 3. Cleavage of highly structured HIV-1 ΔDIS 5′UTR RNA by DISC. (A) Schematic of DISC-mediated cleavage assay. The target RNA sequence was divided into 23-nt segments (shown in different colors) spanning the entire sequence, with each segment targeted by a different gDNA. AGO207 molecules that are bound to endogenous E. coli RNA are not shown for clarity. Following DISC-assembly, a 5′ end-labeled HIV-1 ΔDIS 5′ UTR transcript was added to the mixture to initiate cleavage by DISC. (B) Substrates and products generated by the assay described in (A) were resolved by denaturing PAGE (8%) revealing cleavability of the viral RNA by DISC. (C) Secondary structure of HIV-1 ΔDIS 5′UTR predicted by SHAPE. Colored circles at nucleotide positions indicate SHAPE reactivity. Guide DNAs targeting the different TRs of the HIV transcript are indicated by light blue bars and arrows (TR1 – TR14). Arrowheads indicate cleavage sites by DISC. The color of each arrowhead reflects the distribution of cleavage percentages by DISC shown in (B) and (D) (white, 0–12.5%; yellow, 12.5–25%; orange, 25–37.5%; red, 37.5–50%). (D) Quantification of DISC-mediated cleavage of each TR. Purple circles indicate nucleotides that are unpaired and green circles indicate nucleotides involved in G•U base pairs. Dotted line flanked by black arrowheads indicates cleavage site. Black bars on plot represent average cleavage by DISC of three independent replicates and gray dots represent individual replicates. Asterisk for set using gDNA13 indicates average of two replicates. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 46, Issue 16, 12 June 2018, Page e98, https://doi.org/10.1093/nar/gky496 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Comparing cleavage activity of DISC and RNase H Figure 4. Comparing cleavage activity of DISC and RNase H. (A) Schematic of matched and mismatched guide and target ... Figure 4. Comparing cleavage activity of DISC and RNase H. (A) Schematic of matched and mismatched guide and target pairs used to target four TRs across the HIV-1 ΔDIS 5′UTR RNA. For each pair, the HIV-1 ΔDIS 5′UTR sequence is shown on top and the perfectly matched gDNA strand is shown on the bottom. Circle indicates target position complementary to the first position of the guide that does not pair due to structural restrains by the protein. Black arrowheads indicate cleavage site. Mismatches between the guide and target strands are indicated by a black box around the bases of the guide that are mutated to the bases shown below the box. (B) Quantified cleavage products from the assay using matched and mismatched guide and target pairs described in (A) are plotted with solid bars representing the average of three replicates and circles representing individual replicates. Cleavage that was not detectable by the assay is indicated by ‘nd’. (C and D) Comparing DISC (circles) and RNase H (triangles) cleavage of the unstructured 60-nt target (C) or of a structured 352-nt RNA target (D). Bars indicate average cleavage of three replicates. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 46, Issue 16, 12 June 2018, Page e98, https://doi.org/10.1093/nar/gky496 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. Detection of multiple cleavage events in a single reaction Figure 5. Detection of multiple cleavage events in a single reaction. (A) Secondary structure of HIV-1 ΔDIS 5′UTR with ... Figure 5. Detection of multiple cleavage events in a single reaction. (A) Secondary structure of HIV-1 ΔDIS 5′UTR with TRs colored for clarity. Colored arrowheads indicate cleavage sites on each TR. (B) Schematic of cleavage assay using multiple gDNAs targeting different TRs of HIV-1 ΔDIS 5′UTR. For clarity, AGO207 molecules bound to endogenous E. coli RNA are not shown. AGO207 was loaded with five gDNAs (gDNA4–gDNA8). Following assembly of the DISCs, a <sup>32</sup>P body-labeled HIV-1 ΔDIS 5′UTR RNA was added to the mixture to initiate cleavage. Cartoon secondary structure showing cleavage products with the 5′ products colored black and 3′ products colored gray. Colored gDNAs show complementary regions between guide and target strands. (C) Multiple DISC-mediated cleavage. Substrates and products formed at the indicated time points were separated by denaturing PAGE (8%). Reactions using only one guide are shown in lanes 3–7 as reference for product migration. The one-pot reaction time course is shown in lanes 8–14. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 46, Issue 16, 12 June 2018, Page e98, https://doi.org/10.1093/nar/gky496 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6. High-throughput detection of DISC-mediated cleavage events Figure 6. High-throughput detection of DISC-mediated cleavage events. (A) Schematic of high-throughput assay to detect ... Figure 6. High-throughput detection of DISC-mediated cleavage events. (A) Schematic of high-throughput assay to detect accessibility of structured RNAs. Guide DNAs spanning the target sequence were mixed together to assemble a mixed population of assorted DISCs. For clarity, AGO207 molecules bound to endogenous E. coli RNA are not shown. An unlabeled HIV-1 ΔDIS 5′UTR was added to the mixture to initiate cleavage. Cleaved RNA products were used as templates to prime reverse transcription using a fluorophore-labeled DNA primer. The cDNA products were detected by capillary electrophoresis yielding an electropherogram of peaks whose positions reflect DISC cleavage sites. (B) Electropherogram of DISC-cleavage sites programmed using 11 gDNAs with 23-nt increments. The average of three individual replicates was plotted as a single trace. Cleavage events were detectable at different sites with varying sensitivity. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, Volume 46, Issue 16, 12 June 2018, Page e98, https://doi.org/10.1093/nar/gky496 The content of this slide may be subject to copyright: please see the slide notes for details.