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    Proof of PNA-assisted AtMOC1-mediated cleavage of dsDNA. (a) Sketch providing an overview of PNA-guided AtMOC1-mediated double-strand break (DSB) formation. PNA invades the dsDNA in a sequence-specific manner and forms a loop. AtMOC1 recognizes and makes simultaneous nicks around the PNA invasion site, leading to the formation of DSBs. (b) Target regions 1 and 2 in pUC19 target-54 showing invasion of the top and bottom strands with γPNA1 and γPNA2 molecules, respectively. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of circular <t>(γPNA-invaded,</t> <t>noninvaded,</t> and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size controls included in Lanes 7 and 8. Lane 9 is the undigested plasmid control. <t>XmnI</t> was included in the circular plasmid cleavage reaction to release the fragment after AtMOC1-mediated DSB formation. A map of circular pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. (d) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. A map of XmnI-linearized pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. Lane M is the 1-kb plus DNA marker.
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    1) Product Images from "Harnessing Peptide Nucleic Acids and the Eukaryotic Resolvase MOC1 for Programmable, Precise Generation of Double-Strand DNA Breaks"

    Article Title: Harnessing Peptide Nucleic Acids and the Eukaryotic Resolvase MOC1 for Programmable, Precise Generation of Double-Strand DNA Breaks

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.3c05133

    Proof of PNA-assisted AtMOC1-mediated cleavage of dsDNA. (a) Sketch providing an overview of PNA-guided AtMOC1-mediated double-strand break (DSB) formation. PNA invades the dsDNA in a sequence-specific manner and forms a loop. AtMOC1 recognizes and makes simultaneous nicks around the PNA invasion site, leading to the formation of DSBs. (b) Target regions 1 and 2 in pUC19 target-54 showing invasion of the top and bottom strands with γPNA1 and γPNA2 molecules, respectively. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of circular (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size controls included in Lanes 7 and 8. Lane 9 is the undigested plasmid control. XmnI was included in the circular plasmid cleavage reaction to release the fragment after AtMOC1-mediated DSB formation. A map of circular pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. (d) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. A map of XmnI-linearized pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. Lane M is the 1-kb plus DNA marker.
    Figure Legend Snippet: Proof of PNA-assisted AtMOC1-mediated cleavage of dsDNA. (a) Sketch providing an overview of PNA-guided AtMOC1-mediated double-strand break (DSB) formation. PNA invades the dsDNA in a sequence-specific manner and forms a loop. AtMOC1 recognizes and makes simultaneous nicks around the PNA invasion site, leading to the formation of DSBs. (b) Target regions 1 and 2 in pUC19 target-54 showing invasion of the top and bottom strands with γPNA1 and γPNA2 molecules, respectively. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of circular (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size controls included in Lanes 7 and 8. Lane 9 is the undigested plasmid control. XmnI was included in the circular plasmid cleavage reaction to release the fragment after AtMOC1-mediated DSB formation. A map of circular pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. (d) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. A map of XmnI-linearized pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. Lane M is the 1-kb plus DNA marker.

    Techniques Used: Sequencing, Plasmid Preparation, Binding Assay, Marker

    AtMOC1-mediated cleavage of different XmnI-linearized targets with different nucleotide compositions around the γPNA1 and γPNA2 invasion sites. (a, b) Tables showing the nucleotide compositions at both ends of the γPNA1 and γPNA2 invasion sites in different pUC19 target plasmids. In the diagram, NN represents the nucleotides that are replaced with different nucleotides in different targets; the remaining pUC19 plasmid sequence is the same in all targets. (c, d) Gel images showing the AtMOC1-mediated cleavage of γPNA1- and γPNA2-invaded XmnI-linearized dsDNA, respectively (Lanes 1–8). Gel images also showing the cleavage of noninvaded XmnI-linearized targets (Lanes 9–16). Restriction enzyme size controls included in Lanes 17 in both gels. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1-mediated cleavage of different XmnI-linearized targets with different nucleotide compositions around the γPNA1 and γPNA2 invasion sites. (a, b) Tables showing the nucleotide compositions at both ends of the γPNA1 and γPNA2 invasion sites in different pUC19 target plasmids. In the diagram, NN represents the nucleotides that are replaced with different nucleotides in different targets; the remaining pUC19 plasmid sequence is the same in all targets. (c, d) Gel images showing the AtMOC1-mediated cleavage of γPNA1- and γPNA2-invaded XmnI-linearized dsDNA, respectively (Lanes 1–8). Gel images also showing the cleavage of noninvaded XmnI-linearized targets (Lanes 9–16). Restriction enzyme size controls included in Lanes 17 in both gels. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Plasmid Preparation, Sequencing, Marker

    AtMOC1-mediated cleavage and cleavage site identification. (a, b) Sketches representing the separate invasions of γPNA1 and γPNA2 into the pUC19 target-54, respectively. The circular pUC19 target-54 was invaded by γPNA1 or γPNA2 and independently cleaved with AtMOC1 + XmnI. Both released fragments were sequenced separately with forward and reverse primers. The sequencing traces in the figures reveal precise AtMOC1-mediated cleavage at the 5′ ends and 2-nt away from the γPNA1 or γPNA2 invasion sites in the top or bottom strands.
    Figure Legend Snippet: AtMOC1-mediated cleavage and cleavage site identification. (a, b) Sketches representing the separate invasions of γPNA1 and γPNA2 into the pUC19 target-54, respectively. The circular pUC19 target-54 was invaded by γPNA1 or γPNA2 and independently cleaved with AtMOC1 + XmnI. Both released fragments were sequenced separately with forward and reverse primers. The sequencing traces in the figures reveal precise AtMOC1-mediated cleavage at the 5′ ends and 2-nt away from the γPNA1 or γPNA2 invasion sites in the top or bottom strands.

    Techniques Used: Sequencing

    AtMOC1-mediated cleavage of γtcPNA-invaded XmnI-linearized dsDNA. (a) Map of XmnI-linearized pUC19 target-50 showing γtcPNA binding regions and restriction enzyme sites. (b) Diagram showing γtcPNA binding regions 1 and 2 in the pUC19 target-50 plasmid. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γtcPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1-mediated cleavage of γtcPNA-invaded XmnI-linearized dsDNA. (a) Map of XmnI-linearized pUC19 target-50 showing γtcPNA binding regions and restriction enzyme sites. (b) Diagram showing γtcPNA binding regions 1 and 2 in the pUC19 target-50 plasmid. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γtcPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Binding Assay, Plasmid Preparation, Marker

    AtMOC1-mediated cleavage of dsDNA targets invaded by different lengths of γPNA molecules. (a) Sketch showing the dsDNA target-containing complementary sequences for the different truncated γPNA1 and γPNA2 molecules. (b) Gel mobility shift assay of the invasion of different truncated γPNAs into the corresponding dsDNA targets. (c, d) Gel images showing AtMOC1-mediated cleavage of different circular and XmnI-linearized plasmids invaded independently by different lengths of truncated γPNA molecules (Lanes 1–8). Lane 9 in both the gels is the AtMOC1-mediated cleavage of noninvaded circular and linear targets. XmnI was added in all of the circular plasmid cleavage reactions to release the fragment after AtMOC1-mediated DSB formation. Lane 10 in both gels is the restriction enzyme size control. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1-mediated cleavage of dsDNA targets invaded by different lengths of γPNA molecules. (a) Sketch showing the dsDNA target-containing complementary sequences for the different truncated γPNA1 and γPNA2 molecules. (b) Gel mobility shift assay of the invasion of different truncated γPNAs into the corresponding dsDNA targets. (c, d) Gel images showing AtMOC1-mediated cleavage of different circular and XmnI-linearized plasmids invaded independently by different lengths of truncated γPNA molecules (Lanes 1–8). Lane 9 in both the gels is the AtMOC1-mediated cleavage of noninvaded circular and linear targets. XmnI was added in all of the circular plasmid cleavage reactions to release the fragment after AtMOC1-mediated DSB formation. Lane 10 in both gels is the restriction enzyme size control. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Mobility Shift, Plasmid Preparation, Marker

    AtMOC1 activity assay using the γPNA1-invaded target at different temperatures and time points. (a, b) Gel images showing the activity of AtMOC1 on γPNA1-invaded circular and XmnI-linearized pUC19 target-54 at temperatures ranging from 4 to 90 °C (Lanes 1–11). Lane 12 in (a) and (b) gels showing AtMOC1-mediated cleavage of noninvaded circular and XmnI-linearized targets at 37 °C. XmnI was added to all of the circular plasmid cleavage reactions to release a fragment after AtMOC1-mediated cleavage. Restriction enzyme size control included in lane 13 in (a) and (b) gels. (c, d) Gel images showing AtMOC1 activity on γPNA1-invaded circular and XmnI-linearized pUC19 target-54 at different time points, independently (Lanes 1–7). Lane 8 in (c) and (d) gels showing AtMOC1-mediated cleavage of noninvaded circular and XmnI-linearized targets after 30 min. XmnI was added to all of the circular plasmid cleavage reactions to release a fragment after AtMOC1-mediated cleavage. Restriction enzyme size control included in lane 9 in (c) and (d) gels. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1 activity assay using the γPNA1-invaded target at different temperatures and time points. (a, b) Gel images showing the activity of AtMOC1 on γPNA1-invaded circular and XmnI-linearized pUC19 target-54 at temperatures ranging from 4 to 90 °C (Lanes 1–11). Lane 12 in (a) and (b) gels showing AtMOC1-mediated cleavage of noninvaded circular and XmnI-linearized targets at 37 °C. XmnI was added to all of the circular plasmid cleavage reactions to release a fragment after AtMOC1-mediated cleavage. Restriction enzyme size control included in lane 13 in (a) and (b) gels. (c, d) Gel images showing AtMOC1 activity on γPNA1-invaded circular and XmnI-linearized pUC19 target-54 at different time points, independently (Lanes 1–7). Lane 8 in (c) and (d) gels showing AtMOC1-mediated cleavage of noninvaded circular and XmnI-linearized targets after 30 min. XmnI was added to all of the circular plasmid cleavage reactions to release a fragment after AtMOC1-mediated cleavage. Restriction enzyme size control included in lane 9 in (c) and (d) gels. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Activity Assay, Plasmid Preparation, Marker

    AtMOC1-mediated multiplex cleavage of γPNA-invaded circular and linear plasmids. (a) Circular and XmnI-linearized pUC19 multiplex target-151 containing γPNA1 and γPNA4 binding regions. Restriction sites and the fragments released after multiplex cleavage are indicated in the plasmid maps. Target regions 1 and 2 are the sequences that can be invaded by γPNA1 and γPNA4 molecules, respectively. AtMOC1 cleavage sites are indicated by arrows. (b, c) Gel images showing the AtMOC1-mediated multiplex cleavage of circular and XmnI-linearized plasmids, respectively. Lanes 1–3 in both gels are the only invasion of targets with γPNA1, γPNA4, and γPNA1+γPNA4, respectively. Lanes 4–6 in both gels are the AtMOC1-mediated cleavage of γPNA1, γPNA4, and γPNA1+γPNA4 invaded targets, respectively. Lane 7 in both gels shows the AtMOC1-mediated cleavage of noninvaded targets. Lanes 8–11 in both the gels are restriction digestions as the size controls for AtMOC1-mediated multiplex cleavage. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1-mediated multiplex cleavage of γPNA-invaded circular and linear plasmids. (a) Circular and XmnI-linearized pUC19 multiplex target-151 containing γPNA1 and γPNA4 binding regions. Restriction sites and the fragments released after multiplex cleavage are indicated in the plasmid maps. Target regions 1 and 2 are the sequences that can be invaded by γPNA1 and γPNA4 molecules, respectively. AtMOC1 cleavage sites are indicated by arrows. (b, c) Gel images showing the AtMOC1-mediated multiplex cleavage of circular and XmnI-linearized plasmids, respectively. Lanes 1–3 in both gels are the only invasion of targets with γPNA1, γPNA4, and γPNA1+γPNA4, respectively. Lanes 4–6 in both gels are the AtMOC1-mediated cleavage of γPNA1, γPNA4, and γPNA1+γPNA4 invaded targets, respectively. Lane 7 in both gels shows the AtMOC1-mediated cleavage of noninvaded targets. Lanes 8–11 in both the gels are restriction digestions as the size controls for AtMOC1-mediated multiplex cleavage. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Multiplex Assay, Binding Assay, Plasmid Preparation, Marker

    xmni  (New England Biolabs)


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    New England Biolabs xmni
    Proof of PNA-assisted AtMOC1-mediated cleavage of dsDNA. (a) Sketch providing an overview of PNA-guided AtMOC1-mediated double-strand break (DSB) formation. PNA invades the dsDNA in a sequence-specific manner and forms a loop. AtMOC1 recognizes and makes simultaneous nicks around the PNA invasion site, leading to the formation of DSBs. (b) Target regions 1 and 2 in pUC19 target-54 showing invasion of the top and bottom strands with γPNA1 and γPNA2 molecules, respectively. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of <t>circular</t> <t>(γPNA-invaded,</t> noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size controls included in Lanes 7 and 8. Lane 9 is the undigested plasmid control. <t>XmnI</t> was included in the circular plasmid cleavage reaction to release the fragment after AtMOC1-mediated DSB formation. A map of circular pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. (d) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. A map of XmnI-linearized pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. Lane M is the 1-kb plus DNA marker.
    Xmni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    86/100 stars

    Images

    1) Product Images from "Harnessing Peptide Nucleic Acids and the Eukaryotic Resolvase MOC1 for Programmable, Precise Generation of Double-Strand DNA Breaks"

    Article Title: Harnessing Peptide Nucleic Acids and the Eukaryotic Resolvase MOC1 for Programmable, Precise Generation of Double-Strand DNA Breaks

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.3c05133

    Proof of PNA-assisted AtMOC1-mediated cleavage of dsDNA. (a) Sketch providing an overview of PNA-guided AtMOC1-mediated double-strand break (DSB) formation. PNA invades the dsDNA in a sequence-specific manner and forms a loop. AtMOC1 recognizes and makes simultaneous nicks around the PNA invasion site, leading to the formation of DSBs. (b) Target regions 1 and 2 in pUC19 target-54 showing invasion of the top and bottom strands with γPNA1 and γPNA2 molecules, respectively. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of circular (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size controls included in Lanes 7 and 8. Lane 9 is the undigested plasmid control. XmnI was included in the circular plasmid cleavage reaction to release the fragment after AtMOC1-mediated DSB formation. A map of circular pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. (d) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. A map of XmnI-linearized pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. Lane M is the 1-kb plus DNA marker.
    Figure Legend Snippet: Proof of PNA-assisted AtMOC1-mediated cleavage of dsDNA. (a) Sketch providing an overview of PNA-guided AtMOC1-mediated double-strand break (DSB) formation. PNA invades the dsDNA in a sequence-specific manner and forms a loop. AtMOC1 recognizes and makes simultaneous nicks around the PNA invasion site, leading to the formation of DSBs. (b) Target regions 1 and 2 in pUC19 target-54 showing invasion of the top and bottom strands with γPNA1 and γPNA2 molecules, respectively. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of circular (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size controls included in Lanes 7 and 8. Lane 9 is the undigested plasmid control. XmnI was included in the circular plasmid cleavage reaction to release the fragment after AtMOC1-mediated DSB formation. A map of circular pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. (d) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. A map of XmnI-linearized pUC19 target-54 with γPNA binding regions and restriction enzyme sites is included above the gel image. Lane M is the 1-kb plus DNA marker.

    Techniques Used: Sequencing, Plasmid Preparation, Binding Assay, Marker

    AtMOC1-mediated cleavage of different XmnI-linearized targets with different nucleotide compositions around the γPNA1 and γPNA2 invasion sites. (a, b) Tables showing the nucleotide compositions at both ends of the γPNA1 and γPNA2 invasion sites in different pUC19 target plasmids. In the diagram, NN represents the nucleotides that are replaced with different nucleotides in different targets; the remaining pUC19 plasmid sequence is the same in all targets. (c, d) Gel images showing the AtMOC1-mediated cleavage of γPNA1- and γPNA2-invaded XmnI-linearized dsDNA, respectively (Lanes 1–8). Gel images also showing the cleavage of noninvaded XmnI-linearized targets (Lanes 9–16). Restriction enzyme size controls included in Lanes 17 in both gels. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1-mediated cleavage of different XmnI-linearized targets with different nucleotide compositions around the γPNA1 and γPNA2 invasion sites. (a, b) Tables showing the nucleotide compositions at both ends of the γPNA1 and γPNA2 invasion sites in different pUC19 target plasmids. In the diagram, NN represents the nucleotides that are replaced with different nucleotides in different targets; the remaining pUC19 plasmid sequence is the same in all targets. (c, d) Gel images showing the AtMOC1-mediated cleavage of γPNA1- and γPNA2-invaded XmnI-linearized dsDNA, respectively (Lanes 1–8). Gel images also showing the cleavage of noninvaded XmnI-linearized targets (Lanes 9–16). Restriction enzyme size controls included in Lanes 17 in both gels. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Plasmid Preparation, Sequencing, Marker

    AtMOC1-mediated cleavage and cleavage site identification. (a, b) Sketches representing the separate invasions of γPNA1 and γPNA2 into the pUC19 target-54, respectively. The circular pUC19 target-54 was invaded by γPNA1 or γPNA2 and independently cleaved with AtMOC1 + XmnI. Both released fragments were sequenced separately with forward and reverse primers. The sequencing traces in the figures reveal precise AtMOC1-mediated cleavage at the 5′ ends and 2-nt away from the γPNA1 or γPNA2 invasion sites in the top or bottom strands.
    Figure Legend Snippet: AtMOC1-mediated cleavage and cleavage site identification. (a, b) Sketches representing the separate invasions of γPNA1 and γPNA2 into the pUC19 target-54, respectively. The circular pUC19 target-54 was invaded by γPNA1 or γPNA2 and independently cleaved with AtMOC1 + XmnI. Both released fragments were sequenced separately with forward and reverse primers. The sequencing traces in the figures reveal precise AtMOC1-mediated cleavage at the 5′ ends and 2-nt away from the γPNA1 or γPNA2 invasion sites in the top or bottom strands.

    Techniques Used: Sequencing

    AtMOC1-mediated cleavage of γtcPNA-invaded XmnI-linearized dsDNA. (a) Map of XmnI-linearized pUC19 target-50 showing γtcPNA binding regions and restriction enzyme sites. (b) Diagram showing γtcPNA binding regions 1 and 2 in the pUC19 target-50 plasmid. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γtcPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1-mediated cleavage of γtcPNA-invaded XmnI-linearized dsDNA. (a) Map of XmnI-linearized pUC19 target-50 showing γtcPNA binding regions and restriction enzyme sites. (b) Diagram showing γtcPNA binding regions 1 and 2 in the pUC19 target-50 plasmid. Arrows indicate the positions of AtMOC1 cleavage. (c) Gel image showing the AtMOC1-mediated cleavage of XmnI-linearized (γtcPNA-invaded, noninvaded, and nonspecific γPNA-invaded) dsDNA (Lanes 1–6). Restriction enzyme size control included in Lane 7. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Binding Assay, Plasmid Preparation, Marker

    AtMOC1-mediated cleavage of dsDNA targets invaded by different lengths of γPNA molecules. (a) Sketch showing the dsDNA target-containing complementary sequences for the different truncated γPNA1 and γPNA2 molecules. (b) Gel mobility shift assay of the invasion of different truncated γPNAs into the corresponding dsDNA targets. (c, d) Gel images showing AtMOC1-mediated cleavage of different circular and XmnI-linearized plasmids invaded independently by different lengths of truncated γPNA molecules (Lanes 1–8). Lane 9 in both the gels is the AtMOC1-mediated cleavage of noninvaded circular and linear targets. XmnI was added in all of the circular plasmid cleavage reactions to release the fragment after AtMOC1-mediated DSB formation. Lane 10 in both gels is the restriction enzyme size control. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1-mediated cleavage of dsDNA targets invaded by different lengths of γPNA molecules. (a) Sketch showing the dsDNA target-containing complementary sequences for the different truncated γPNA1 and γPNA2 molecules. (b) Gel mobility shift assay of the invasion of different truncated γPNAs into the corresponding dsDNA targets. (c, d) Gel images showing AtMOC1-mediated cleavage of different circular and XmnI-linearized plasmids invaded independently by different lengths of truncated γPNA molecules (Lanes 1–8). Lane 9 in both the gels is the AtMOC1-mediated cleavage of noninvaded circular and linear targets. XmnI was added in all of the circular plasmid cleavage reactions to release the fragment after AtMOC1-mediated DSB formation. Lane 10 in both gels is the restriction enzyme size control. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Mobility Shift, Plasmid Preparation, Marker

    AtMOC1 activity assay using the γPNA1-invaded target at different temperatures and time points. (a, b) Gel images showing the activity of AtMOC1 on γPNA1-invaded circular and XmnI-linearized pUC19 target-54 at temperatures ranging from 4 to 90 °C (Lanes 1–11). Lane 12 in (a) and (b) gels showing AtMOC1-mediated cleavage of noninvaded circular and XmnI-linearized targets at 37 °C. XmnI was added to all of the circular plasmid cleavage reactions to release a fragment after AtMOC1-mediated cleavage. Restriction enzyme size control included in lane 13 in (a) and (b) gels. (c, d) Gel images showing AtMOC1 activity on γPNA1-invaded circular and XmnI-linearized pUC19 target-54 at different time points, independently (Lanes 1–7). Lane 8 in (c) and (d) gels showing AtMOC1-mediated cleavage of noninvaded circular and XmnI-linearized targets after 30 min. XmnI was added to all of the circular plasmid cleavage reactions to release a fragment after AtMOC1-mediated cleavage. Restriction enzyme size control included in lane 9 in (c) and (d) gels. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1 activity assay using the γPNA1-invaded target at different temperatures and time points. (a, b) Gel images showing the activity of AtMOC1 on γPNA1-invaded circular and XmnI-linearized pUC19 target-54 at temperatures ranging from 4 to 90 °C (Lanes 1–11). Lane 12 in (a) and (b) gels showing AtMOC1-mediated cleavage of noninvaded circular and XmnI-linearized targets at 37 °C. XmnI was added to all of the circular plasmid cleavage reactions to release a fragment after AtMOC1-mediated cleavage. Restriction enzyme size control included in lane 13 in (a) and (b) gels. (c, d) Gel images showing AtMOC1 activity on γPNA1-invaded circular and XmnI-linearized pUC19 target-54 at different time points, independently (Lanes 1–7). Lane 8 in (c) and (d) gels showing AtMOC1-mediated cleavage of noninvaded circular and XmnI-linearized targets after 30 min. XmnI was added to all of the circular plasmid cleavage reactions to release a fragment after AtMOC1-mediated cleavage. Restriction enzyme size control included in lane 9 in (c) and (d) gels. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Activity Assay, Plasmid Preparation, Marker

    AtMOC1-mediated multiplex cleavage of γPNA-invaded circular and linear plasmids. (a) Circular and XmnI-linearized pUC19 multiplex target-151 containing γPNA1 and γPNA4 binding regions. Restriction sites and the fragments released after multiplex cleavage are indicated in the plasmid maps. Target regions 1 and 2 are the sequences that can be invaded by γPNA1 and γPNA4 molecules, respectively. AtMOC1 cleavage sites are indicated by arrows. (b, c) Gel images showing the AtMOC1-mediated multiplex cleavage of circular and XmnI-linearized plasmids, respectively. Lanes 1–3 in both gels are the only invasion of targets with γPNA1, γPNA4, and γPNA1+γPNA4, respectively. Lanes 4–6 in both gels are the AtMOC1-mediated cleavage of γPNA1, γPNA4, and γPNA1+γPNA4 invaded targets, respectively. Lane 7 in both gels shows the AtMOC1-mediated cleavage of noninvaded targets. Lanes 8–11 in both the gels are restriction digestions as the size controls for AtMOC1-mediated multiplex cleavage. Lane M shows the 1-kb plus DNA marker.
    Figure Legend Snippet: AtMOC1-mediated multiplex cleavage of γPNA-invaded circular and linear plasmids. (a) Circular and XmnI-linearized pUC19 multiplex target-151 containing γPNA1 and γPNA4 binding regions. Restriction sites and the fragments released after multiplex cleavage are indicated in the plasmid maps. Target regions 1 and 2 are the sequences that can be invaded by γPNA1 and γPNA4 molecules, respectively. AtMOC1 cleavage sites are indicated by arrows. (b, c) Gel images showing the AtMOC1-mediated multiplex cleavage of circular and XmnI-linearized plasmids, respectively. Lanes 1–3 in both gels are the only invasion of targets with γPNA1, γPNA4, and γPNA1+γPNA4, respectively. Lanes 4–6 in both gels are the AtMOC1-mediated cleavage of γPNA1, γPNA4, and γPNA1+γPNA4 invaded targets, respectively. Lane 7 in both gels shows the AtMOC1-mediated cleavage of noninvaded targets. Lanes 8–11 in both the gels are restriction digestions as the size controls for AtMOC1-mediated multiplex cleavage. Lane M shows the 1-kb plus DNA marker.

    Techniques Used: Multiplex Assay, Binding Assay, Plasmid Preparation, Marker

    xmni  (New England Biolabs)


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