xrn1  (New England Biolabs)


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    Structured Review

    New England Biolabs xrn1
    Anopheles -associated ISFs contain a single copy of xrRNA, which shares features of class 1a and class 1b xrRNAs. A Secondary structure of KRBV 3’UTR generated using SHAPE-assisted folding. Shading intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK -pseudoknot. B Secondary structure of KRBV xrRNA. Each element of the secondary structure is shown in colour. C In vitro <t>XRN1</t> resistance assay with WT and mutated KRBV 3’UTRs. PK1’ mutation was GUUGC - > CAACG change of PK-forming nucleotides in the L2 loop of the SLI. The experiment was repeated three times with similar results. D Sequence alignment of KRBV and AnFV1 xrRNAs. The colour coding matches ( B ) and shows conserved structural elements. E Predicted secondary structure of AnFV1 xrRNA. The conserved structural elements are shown in colours that match ( D ).
    Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinants of sfRNA biogenesis and provides new insights into flavivirus evolution"

    Article Title: Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinants of sfRNA biogenesis and provides new insights into flavivirus evolution

    Journal: Nature Communications

    doi: 10.1038/s41467-022-28977-3

    Anopheles -associated ISFs contain a single copy of xrRNA, which shares features of class 1a and class 1b xrRNAs. A Secondary structure of KRBV 3’UTR generated using SHAPE-assisted folding. Shading intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK -pseudoknot. B Secondary structure of KRBV xrRNA. Each element of the secondary structure is shown in colour. C In vitro XRN1 resistance assay with WT and mutated KRBV 3’UTRs. PK1’ mutation was GUUGC - > CAACG change of PK-forming nucleotides in the L2 loop of the SLI. The experiment was repeated three times with similar results. D Sequence alignment of KRBV and AnFV1 xrRNAs. The colour coding matches ( B ) and shows conserved structural elements. E Predicted secondary structure of AnFV1 xrRNA. The conserved structural elements are shown in colours that match ( D ).
    Figure Legend Snippet: Anopheles -associated ISFs contain a single copy of xrRNA, which shares features of class 1a and class 1b xrRNAs. A Secondary structure of KRBV 3’UTR generated using SHAPE-assisted folding. Shading intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK -pseudoknot. B Secondary structure of KRBV xrRNA. Each element of the secondary structure is shown in colour. C In vitro XRN1 resistance assay with WT and mutated KRBV 3’UTRs. PK1’ mutation was GUUGC - > CAACG change of PK-forming nucleotides in the L2 loop of the SLI. The experiment was repeated three times with similar results. D Sequence alignment of KRBV and AnFV1 xrRNAs. The colour coding matches ( B ) and shows conserved structural elements. E Predicted secondary structure of AnFV1 xrRNA. The conserved structural elements are shown in colours that match ( D ).

    Techniques Used: Generated, In Vitro, Mutagenesis, Sequencing

    Classical and dual host-associated ISFs produce sfRNAs by employing XRN1-resistance mechanism. A Northern blot detection of sfRNAs produced by ISFs. For PaRV, PCV, BinJV and HVV C6/36 cells were infected at MOI = 1. Total RNA was isolated at 5 dpi. For KRBV, total RNA was isolated from virus-positive and virus-negative (Mock) Anopheles mosquitoes. RNA was then used for Northern blotting with the probe complementary to the last 25nt of viral 3’UTRs. B The effect of XRN1 knock-down on the production of sfRNAs by ISFs. Aag2 cells were transfected with dsRNA against Aedes aegypti XRN1 (dsXRN1) or GFP (dsNC) and infected with respective viruses at MOI = 1 at 24hpt. At 48hpi, total RNA was isolated from the cells and used for Northern blotting as in ( A ). Bottom panels represent the Et-Br staining of the gels used for Northern transfer with 7SL cellular RNA visualised as a loading control. C In vitro XRN1 resistance assay with ISF 3’UTRs. RNA corresponding to 3’UTRs of ISFs was transcribed in vitro, briefly heated and then refolded by gradual cooling to 28 °C or placed on ice to preserve the denatured state. Samples were then treated with purified XRN1 and RppH (to convert 5’PPP into 5’P) and analysed by electrophoresis in denaturing PAAG. Gels were stained with ethidium bromide (Et-Br). All images are representative of at least two independent experiments that produced similar results.
    Figure Legend Snippet: Classical and dual host-associated ISFs produce sfRNAs by employing XRN1-resistance mechanism. A Northern blot detection of sfRNAs produced by ISFs. For PaRV, PCV, BinJV and HVV C6/36 cells were infected at MOI = 1. Total RNA was isolated at 5 dpi. For KRBV, total RNA was isolated from virus-positive and virus-negative (Mock) Anopheles mosquitoes. RNA was then used for Northern blotting with the probe complementary to the last 25nt of viral 3’UTRs. B The effect of XRN1 knock-down on the production of sfRNAs by ISFs. Aag2 cells were transfected with dsRNA against Aedes aegypti XRN1 (dsXRN1) or GFP (dsNC) and infected with respective viruses at MOI = 1 at 24hpt. At 48hpi, total RNA was isolated from the cells and used for Northern blotting as in ( A ). Bottom panels represent the Et-Br staining of the gels used for Northern transfer with 7SL cellular RNA visualised as a loading control. C In vitro XRN1 resistance assay with ISF 3’UTRs. RNA corresponding to 3’UTRs of ISFs was transcribed in vitro, briefly heated and then refolded by gradual cooling to 28 °C or placed on ice to preserve the denatured state. Samples were then treated with purified XRN1 and RppH (to convert 5’PPP into 5’P) and analysed by electrophoresis in denaturing PAAG. Gels were stained with ethidium bromide (Et-Br). All images are representative of at least two independent experiments that produced similar results.

    Techniques Used: Northern Blot, Produced, Infection, Isolation, Transfection, Staining, In Vitro, Purification, Electrophoresis

    dISFs contain novel SL-PK elements in addition to canonical class 1a xrRNAs. A Secondary structure of BinJV 3’UTR generated by SHAPE. SL – stem-loop, DB – dumbbell, RCS3 – reverse conserved sequence 3, CS3 – conserved sequence 3, PK – pseudoknot. B Secondary structures of BinJV stem-loops. C Structure-based sequence alignment between BinJV and MBF xrRNAs. D XRN1 resistance assay with WT and mutated BinJV 3’UTRs. Mutations PK1’ (GAGAG- > CUCUC), PK2’ (UGGUUG- > ACCAAC), nPK1’ (UAGCG- > AUCGC) and nPK2’ (GCGUC- > CGCAG) were introduced into the terminal loop regions of the corresponding stem-loops. The image is representative of four independent experiments that produced similar results. E Densitometry analysis of ( D ). The values are the means of four independent experiments ± SD. Statistical analysis is two-sided one-way ANOVA. F Consensus structure of dISF xrRNAs built based on the covariance model. Covarying base pairs are highlighted in green ( G ) Secondary structure of HVV xrRNA generated by SHAPE.
    Figure Legend Snippet: dISFs contain novel SL-PK elements in addition to canonical class 1a xrRNAs. A Secondary structure of BinJV 3’UTR generated by SHAPE. SL – stem-loop, DB – dumbbell, RCS3 – reverse conserved sequence 3, CS3 – conserved sequence 3, PK – pseudoknot. B Secondary structures of BinJV stem-loops. C Structure-based sequence alignment between BinJV and MBF xrRNAs. D XRN1 resistance assay with WT and mutated BinJV 3’UTRs. Mutations PK1’ (GAGAG- > CUCUC), PK2’ (UGGUUG- > ACCAAC), nPK1’ (UAGCG- > AUCGC) and nPK2’ (GCGUC- > CGCAG) were introduced into the terminal loop regions of the corresponding stem-loops. The image is representative of four independent experiments that produced similar results. E Densitometry analysis of ( D ). The values are the means of four independent experiments ± SD. Statistical analysis is two-sided one-way ANOVA. F Consensus structure of dISF xrRNAs built based on the covariance model. Covarying base pairs are highlighted in green ( G ) Secondary structure of HVV xrRNA generated by SHAPE.

    Techniques Used: Generated, Sequencing, Produced

    cISFs PCV contains multiple copies of divergent class 1b xrRNAs. A Secondary structure of PCV 3’UTR generated using SHAPE-assisted folding. Shading intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK – pseudoknot. B Secondary structure of PCV xrRNAs. Each element of the secondary structure is shown in colour. C In vitro XRN1 resistance assay with WT and mutated PCV 3’UTRs. PK1’, PK2’ and PK3’ mutations were introduced into the terminal loop regions of SLI, SLII and SLIII, respectively, and represented the GAC - > CUG change in the PK-forming region. Image is representative from three independent experiments that showed similar results. D Alignment of sequence and structure of PCV xrRNAs was performed using LocARNA.
    Figure Legend Snippet: cISFs PCV contains multiple copies of divergent class 1b xrRNAs. A Secondary structure of PCV 3’UTR generated using SHAPE-assisted folding. Shading intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK – pseudoknot. B Secondary structure of PCV xrRNAs. Each element of the secondary structure is shown in colour. C In vitro XRN1 resistance assay with WT and mutated PCV 3’UTRs. PK1’, PK2’ and PK3’ mutations were introduced into the terminal loop regions of SLI, SLII and SLIII, respectively, and represented the GAC - > CUG change in the PK-forming region. Image is representative from three independent experiments that showed similar results. D Alignment of sequence and structure of PCV xrRNAs was performed using LocARNA.

    Techniques Used: Generated, In Vitro, Sequencing

    cISFs PaRV contains multiple copies of divergent class 1b xrRNAs. A Secondary structure of PaRV 3’UTR generated using SHAPE-assisted folding. Colour intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK -pseudoknot. B Secondary structure of PaRV xrRNAs. Each structural element is shown in colour. C In vitro XRN1 resistance assay with WT and mutated PaRV 3’UTRs. PK1’, PK2’ and PK3’ mutations were introduced into the terminal loop regions of SLI, SLII and SLIII, respectively, and represented the CAC - > GUG change in the PK-forming regions. Image is representative from three independent experiments that showed similar results. D Structure-based alignment of PaRV xrRNAs was performed using LocARNA.
    Figure Legend Snippet: cISFs PaRV contains multiple copies of divergent class 1b xrRNAs. A Secondary structure of PaRV 3’UTR generated using SHAPE-assisted folding. Colour intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK -pseudoknot. B Secondary structure of PaRV xrRNAs. Each structural element is shown in colour. C In vitro XRN1 resistance assay with WT and mutated PaRV 3’UTRs. PK1’, PK2’ and PK3’ mutations were introduced into the terminal loop regions of SLI, SLII and SLIII, respectively, and represented the CAC - > GUG change in the PK-forming regions. Image is representative from three independent experiments that showed similar results. D Structure-based alignment of PaRV xrRNAs was performed using LocARNA.

    Techniques Used: Generated, In Vitro

    2) Product Images from "Molecular Determinants for RNA Release into Extracellular Vesicles"

    Article Title: Molecular Determinants for RNA Release into Extracellular Vesicles

    Journal: Cells

    doi: 10.3390/cells10102674

    EV-associated and cellular GAPDH mRNA differ in size and polyadenylation status. ( a ) Total RNA from ES-2 cells and their corresponding EVs were isolated, followed by RT-PCR. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used. Markers (200, 100 bp). ( b ) RNA was isolated from ES-2 cells and their corresponding EVs. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used, and GAPDH mRNA was quantified by RT-qPCR. ( c ) Detection of GAPDH mRNA in EVs by the glyoxal Northern blot analysis. Total cellular and EV-RNA (300 ng) were analyzed by glyoxal agarose gel electrophoresis and Northern blotting. Exposure time 3 min ( d ) Total RNA from ES-2-derived EVs was isolated and treated with Xrn1 exoribonuclease (+Xrn1). As control, no enzyme was added (−Xrn1). RT-PCR was performed using primers directed against transcripts indicated. Markers (200, 100 bp).
    Figure Legend Snippet: EV-associated and cellular GAPDH mRNA differ in size and polyadenylation status. ( a ) Total RNA from ES-2 cells and their corresponding EVs were isolated, followed by RT-PCR. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used. Markers (200, 100 bp). ( b ) RNA was isolated from ES-2 cells and their corresponding EVs. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used, and GAPDH mRNA was quantified by RT-qPCR. ( c ) Detection of GAPDH mRNA in EVs by the glyoxal Northern blot analysis. Total cellular and EV-RNA (300 ng) were analyzed by glyoxal agarose gel electrophoresis and Northern blotting. Exposure time 3 min ( d ) Total RNA from ES-2-derived EVs was isolated and treated with Xrn1 exoribonuclease (+Xrn1). As control, no enzyme was added (−Xrn1). RT-PCR was performed using primers directed against transcripts indicated. Markers (200, 100 bp).

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Northern Blot, Agarose Gel Electrophoresis, Derivative Assay

    3) Product Images from "Molecular Determinants for RNA Release into Extracellular Vesicles"

    Article Title: Molecular Determinants for RNA Release into Extracellular Vesicles

    Journal: Cells

    doi: 10.3390/cells10102674

    EV-associated and cellular GAPDH mRNA differ in size and polyadenylation status. ( a ) Total RNA from ES-2 cells and their corresponding EVs were isolated, followed by RT-PCR. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used. Markers (200, 100 bp). ( b ) RNA was isolated from ES-2 cells and their corresponding EVs. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used, and GAPDH mRNA was quantified by RT-qPCR. ( c ) Detection of GAPDH mRNA in EVs by the glyoxal Northern blot analysis. Total cellular and EV-RNA (300 ng) were analyzed by glyoxal agarose gel electrophoresis and Northern blotting. Exposure time 3 min ( d ) Total RNA from ES-2-derived EVs was isolated and treated with Xrn1 exoribonuclease (+Xrn1). As control, no enzyme was added (−Xrn1). RT-PCR was performed using primers directed against transcripts indicated. Markers (200, 100 bp).
    Figure Legend Snippet: EV-associated and cellular GAPDH mRNA differ in size and polyadenylation status. ( a ) Total RNA from ES-2 cells and their corresponding EVs were isolated, followed by RT-PCR. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used. Markers (200, 100 bp). ( b ) RNA was isolated from ES-2 cells and their corresponding EVs. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used, and GAPDH mRNA was quantified by RT-qPCR. ( c ) Detection of GAPDH mRNA in EVs by the glyoxal Northern blot analysis. Total cellular and EV-RNA (300 ng) were analyzed by glyoxal agarose gel electrophoresis and Northern blotting. Exposure time 3 min ( d ) Total RNA from ES-2-derived EVs was isolated and treated with Xrn1 exoribonuclease (+Xrn1). As control, no enzyme was added (−Xrn1). RT-PCR was performed using primers directed against transcripts indicated. Markers (200, 100 bp).

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Northern Blot, Agarose Gel Electrophoresis, Derivative Assay

    4) Product Images from "Chimeric Flaviviral RNA−siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells"

    Article Title: Chimeric Flaviviral RNA−siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells

    Journal: Chembiochem

    doi: 10.1002/cbic.202100434

    (a) Chimeric xrRNA ΔP4−L4 −siRNA construct. The stem‐loop P2‐L2 of the subclass 1a xrRNA shown in Figure 1 was exchanged with an siRNA (blue). Antisense and sense sequences, strand 1 (S1) and strand 2 (S2), stems P1 and P3, loop L3, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. An adapter is yellow. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs with a two‐fold molar excess of S1 to S2 following incubation with Xrn1 at 37 °C for 20 min. The excess S1 is labelled as a single‐stranded RNA (ssRNA). (d) Non‐denaturing polyacrylamide gel mobilities of a control comprising DENV ΔP4−L4 −siREN S1 and siREN AS, which lacks the xrRNA 3WJ and pseudoknots, and a control siRNA (siREN) following incubation with Xrn1 at 37 °C for 20 min.
    Figure Legend Snippet: (a) Chimeric xrRNA ΔP4−L4 −siRNA construct. The stem‐loop P2‐L2 of the subclass 1a xrRNA shown in Figure 1 was exchanged with an siRNA (blue). Antisense and sense sequences, strand 1 (S1) and strand 2 (S2), stems P1 and P3, loop L3, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. An adapter is yellow. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs with a two‐fold molar excess of S1 to S2 following incubation with Xrn1 at 37 °C for 20 min. The excess S1 is labelled as a single‐stranded RNA (ssRNA). (d) Non‐denaturing polyacrylamide gel mobilities of a control comprising DENV ΔP4−L4 −siREN S1 and siREN AS, which lacks the xrRNA 3WJ and pseudoknots, and a control siRNA (siREN) following incubation with Xrn1 at 37 °C for 20 min.

    Techniques Used: Construct, Incubation

    (a) Subclass 1a (left) and 1b (right) xrRNAs. Stems P1 through P4, loops L2 through L4, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs and a single‐stranded RNA (ssRNA) following incubation with Xrn1 at 37 °C for 20 min. Constructs with stem‐loop deletions are denoted by ΔP4−L4. WT, wild type; DENV, Dengue virus; MECDV, Mercadeo virus; SLEV, Saint Louis encephalitis virus; ZIKV, Zika virus; TABV, Tamana bat virus; GBVB, GB virus B; APPV, atypical porcine pestivirus; SPgV, simian pegivirus.
    Figure Legend Snippet: (a) Subclass 1a (left) and 1b (right) xrRNAs. Stems P1 through P4, loops L2 through L4, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs and a single‐stranded RNA (ssRNA) following incubation with Xrn1 at 37 °C for 20 min. Constructs with stem‐loop deletions are denoted by ΔP4−L4. WT, wild type; DENV, Dengue virus; MECDV, Mercadeo virus; SLEV, Saint Louis encephalitis virus; ZIKV, Zika virus; TABV, Tamana bat virus; GBVB, GB virus B; APPV, atypical porcine pestivirus; SPgV, simian pegivirus.

    Techniques Used: Construct, Incubation

    5) Product Images from "Chimeric Flaviviral RNA−siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells"

    Article Title: Chimeric Flaviviral RNA−siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells

    Journal: Chembiochem

    doi: 10.1002/cbic.202100434

    (a) Chimeric xrRNA ΔP4−L4 −siRNA construct. The stem‐loop P2‐L2 of the subclass 1a xrRNA shown in Figure 1 was exchanged with an siRNA (blue). Antisense and sense sequences, strand 1 (S1) and strand 2 (S2), stems P1 and P3, loop L3, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. An adapter is yellow. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs with a two‐fold molar excess of S1 to S2 following incubation with Xrn1 at 37 °C for 20 min. The excess S1 is labelled as a single‐stranded RNA (ssRNA). (d) Non‐denaturing polyacrylamide gel mobilities of a control comprising DENV ΔP4−L4 −siREN S1 and siREN AS, which lacks the xrRNA 3WJ and pseudoknots, and a control siRNA (siREN) following incubation with Xrn1 at 37 °C for 20 min.
    Figure Legend Snippet: (a) Chimeric xrRNA ΔP4−L4 −siRNA construct. The stem‐loop P2‐L2 of the subclass 1a xrRNA shown in Figure 1 was exchanged with an siRNA (blue). Antisense and sense sequences, strand 1 (S1) and strand 2 (S2), stems P1 and P3, loop L3, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. An adapter is yellow. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs with a two‐fold molar excess of S1 to S2 following incubation with Xrn1 at 37 °C for 20 min. The excess S1 is labelled as a single‐stranded RNA (ssRNA). (d) Non‐denaturing polyacrylamide gel mobilities of a control comprising DENV ΔP4−L4 −siREN S1 and siREN AS, which lacks the xrRNA 3WJ and pseudoknots, and a control siRNA (siREN) following incubation with Xrn1 at 37 °C for 20 min.

    Techniques Used: Construct, Incubation

    (a) Subclass 1a (left) and 1b (right) xrRNAs. Stems P1 through P4, loops L2 through L4, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs and a single‐stranded RNA (ssRNA) following incubation with Xrn1 at 37 °C for 20 min. Constructs with stem‐loop deletions are denoted by ΔP4−L4. WT, wild type; DENV, Dengue virus; MECDV, Mercadeo virus; SLEV, Saint Louis encephalitis virus; ZIKV, Zika virus; TABV, Tamana bat virus; GBVB, GB virus B; APPV, atypical porcine pestivirus; SPgV, simian pegivirus.
    Figure Legend Snippet: (a) Subclass 1a (left) and 1b (right) xrRNAs. Stems P1 through P4, loops L2 through L4, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs and a single‐stranded RNA (ssRNA) following incubation with Xrn1 at 37 °C for 20 min. Constructs with stem‐loop deletions are denoted by ΔP4−L4. WT, wild type; DENV, Dengue virus; MECDV, Mercadeo virus; SLEV, Saint Louis encephalitis virus; ZIKV, Zika virus; TABV, Tamana bat virus; GBVB, GB virus B; APPV, atypical porcine pestivirus; SPgV, simian pegivirus.

    Techniques Used: Construct, Incubation

    6) Product Images from "Chimeric Flaviviral RNA−siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells"

    Article Title: Chimeric Flaviviral RNA−siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells

    Journal: Chembiochem

    doi: 10.1002/cbic.202100434

    (a) Chimeric xrRNA ΔP4−L4 −siRNA construct. The stem‐loop P2‐L2 of the subclass 1a xrRNA shown in Figure 1 was exchanged with an siRNA (blue). Antisense and sense sequences, strand 1 (S1) and strand 2 (S2), stems P1 and P3, loop L3, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. An adapter is yellow. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs with a two‐fold molar excess of S1 to S2 following incubation with Xrn1 at 37 °C for 20 min. The excess S1 is labelled as a single‐stranded RNA (ssRNA). (d) Non‐denaturing polyacrylamide gel mobilities of a control comprising DENV ΔP4−L4 −siREN S1 and siREN AS, which lacks the xrRNA 3WJ and pseudoknots, and a control siRNA (siREN) following incubation with Xrn1 at 37 °C for 20 min.
    Figure Legend Snippet: (a) Chimeric xrRNA ΔP4−L4 −siRNA construct. The stem‐loop P2‐L2 of the subclass 1a xrRNA shown in Figure 1 was exchanged with an siRNA (blue). Antisense and sense sequences, strand 1 (S1) and strand 2 (S2), stems P1 and P3, loop L3, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. An adapter is yellow. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs with a two‐fold molar excess of S1 to S2 following incubation with Xrn1 at 37 °C for 20 min. The excess S1 is labelled as a single‐stranded RNA (ssRNA). (d) Non‐denaturing polyacrylamide gel mobilities of a control comprising DENV ΔP4−L4 −siREN S1 and siREN AS, which lacks the xrRNA 3WJ and pseudoknots, and a control siRNA (siREN) following incubation with Xrn1 at 37 °C for 20 min.

    Techniques Used: Construct, Incubation

    (a) Subclass 1a (left) and 1b (right) xrRNAs. Stems P1 through P4, loops L2 through L4, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs and a single‐stranded RNA (ssRNA) following incubation with Xrn1 at 37 °C for 20 min. Constructs with stem‐loop deletions are denoted by ΔP4−L4. WT, wild type; DENV, Dengue virus; MECDV, Mercadeo virus; SLEV, Saint Louis encephalitis virus; ZIKV, Zika virus; TABV, Tamana bat virus; GBVB, GB virus B; APPV, atypical porcine pestivirus; SPgV, simian pegivirus.
    Figure Legend Snippet: (a) Subclass 1a (left) and 1b (right) xrRNAs. Stems P1 through P4, loops L2 through L4, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs and a single‐stranded RNA (ssRNA) following incubation with Xrn1 at 37 °C for 20 min. Constructs with stem‐loop deletions are denoted by ΔP4−L4. WT, wild type; DENV, Dengue virus; MECDV, Mercadeo virus; SLEV, Saint Louis encephalitis virus; ZIKV, Zika virus; TABV, Tamana bat virus; GBVB, GB virus B; APPV, atypical porcine pestivirus; SPgV, simian pegivirus.

    Techniques Used: Construct, Incubation

    7) Product Images from "Chimeric Flaviviral RNA−siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells"

    Article Title: Chimeric Flaviviral RNA−siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells

    Journal: Chembiochem

    doi: 10.1002/cbic.202100434

    (a) Chimeric xrRNA ΔP4−L4 −siRNA construct. The stem‐loop P2‐L2 of the subclass 1a xrRNA shown in Figure 1 was exchanged with an siRNA (blue). Antisense and sense sequences, strand 1 (S1) and strand 2 (S2), stems P1 and P3, loop L3, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. An adapter is yellow. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs with a two‐fold molar excess of S1 to S2 following incubation with Xrn1 at 37 °C for 20 min. The excess S1 is labelled as a single‐stranded RNA (ssRNA). (d) Non‐denaturing polyacrylamide gel mobilities of a control comprising DENV ΔP4−L4 −siREN S1 and siREN AS, which lacks the xrRNA 3WJ and pseudoknots, and a control siRNA (siREN) following incubation with Xrn1 at 37 °C for 20 min.
    Figure Legend Snippet: (a) Chimeric xrRNA ΔP4−L4 −siRNA construct. The stem‐loop P2‐L2 of the subclass 1a xrRNA shown in Figure 1 was exchanged with an siRNA (blue). Antisense and sense sequences, strand 1 (S1) and strand 2 (S2), stems P1 and P3, loop L3, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. An adapter is yellow. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA ΔP4−L4 −siRNA constructs with a two‐fold molar excess of S1 to S2 following incubation with Xrn1 at 37 °C for 20 min. The excess S1 is labelled as a single‐stranded RNA (ssRNA). (d) Non‐denaturing polyacrylamide gel mobilities of a control comprising DENV ΔP4−L4 −siREN S1 and siREN AS, which lacks the xrRNA 3WJ and pseudoknots, and a control siRNA (siREN) following incubation with Xrn1 at 37 °C for 20 min.

    Techniques Used: Construct, Incubation

    (a) Subclass 1a (left) and 1b (right) xrRNAs. Stems P1 through P4, loops L2 through L4, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs and a single‐stranded RNA (ssRNA) following incubation with Xrn1 at 37 °C for 20 min. Constructs with stem‐loop deletions are denoted by ΔP4−L4. WT, wild type; DENV, Dengue virus; MECDV, Mercadeo virus; SLEV, Saint Louis encephalitis virus; ZIKV, Zika virus; TABV, Tamana bat virus; GBVB, GB virus B; APPV, atypical porcine pestivirus; SPgV, simian pegivirus.
    Figure Legend Snippet: (a) Subclass 1a (left) and 1b (right) xrRNAs. Stems P1 through P4, loops L2 through L4, pseudoknots Pk1 and Pk2, and a base triple (BT) are labelled. Base‐pairing interactions are indicated with dashed lines. (b) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs. Reference bands in the ladder are single‐stranded RNA. (c) Non‐denaturing polyacrylamide gel mobilities of xrRNA constructs and a single‐stranded RNA (ssRNA) following incubation with Xrn1 at 37 °C for 20 min. Constructs with stem‐loop deletions are denoted by ΔP4−L4. WT, wild type; DENV, Dengue virus; MECDV, Mercadeo virus; SLEV, Saint Louis encephalitis virus; ZIKV, Zika virus; TABV, Tamana bat virus; GBVB, GB virus B; APPV, atypical porcine pestivirus; SPgV, simian pegivirus.

    Techniques Used: Construct, Incubation

    8) Product Images from "Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution"

    Article Title: Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution

    Journal: bioRxiv

    doi: 10.1101/2021.06.23.449515

    Classical and dual host associated ISFs produce sfRNAs employing the XRN1-resistance mechanism. (A) Northern blot detection of sfRNAs produced by ISFs. For PaRV, PCV, BinJV and HVV C6/36 cells were infected at MOI=1. Total RNA was isolated at 5 dpi. For KRBV, total RNA was isolated from virus-positive and virus-negative (Mock) Anopheles mosquitoes. RNA was then used for Northern blotting with the probe complementary to the last 25nt of viral 3’UTRs. (B) The effect of XRN-1 knock-down on the production of sfRNAs by ISFs. Aag2 cells were transfected with dsRNA against Aedes aegypti XRN-1 (dsXRN1) or GFP (dsNC) and infected with respective viruses at MOI=1 at 24hpt. At 48hpi, total RNA was isolated from the cells and used for Northern blotting as in (A). Bottom panels represent the Et-Br staining of the gels used for Northern transfer with 5.8S rRNA visualised as an equal loading control. (C) In vitro XRN-1 resistance assay with ISF 3’UTRs. RNA corresponding to 3’UTRs of ISFs was in vitro transcribed, briefly heated and then either refolded by gradual cooling to 28C or placed on ice to preserve the denatured state. Samples were then treated purified XRN1 and RppH (to convert 5’PPP into 5’P) and analysed by electrophoresis in denaturing PAAG. Gels were stained with Et-Br. All images are representative of at least two independent experiments that produced similar results.
    Figure Legend Snippet: Classical and dual host associated ISFs produce sfRNAs employing the XRN1-resistance mechanism. (A) Northern blot detection of sfRNAs produced by ISFs. For PaRV, PCV, BinJV and HVV C6/36 cells were infected at MOI=1. Total RNA was isolated at 5 dpi. For KRBV, total RNA was isolated from virus-positive and virus-negative (Mock) Anopheles mosquitoes. RNA was then used for Northern blotting with the probe complementary to the last 25nt of viral 3’UTRs. (B) The effect of XRN-1 knock-down on the production of sfRNAs by ISFs. Aag2 cells were transfected with dsRNA against Aedes aegypti XRN-1 (dsXRN1) or GFP (dsNC) and infected with respective viruses at MOI=1 at 24hpt. At 48hpi, total RNA was isolated from the cells and used for Northern blotting as in (A). Bottom panels represent the Et-Br staining of the gels used for Northern transfer with 5.8S rRNA visualised as an equal loading control. (C) In vitro XRN-1 resistance assay with ISF 3’UTRs. RNA corresponding to 3’UTRs of ISFs was in vitro transcribed, briefly heated and then either refolded by gradual cooling to 28C or placed on ice to preserve the denatured state. Samples were then treated purified XRN1 and RppH (to convert 5’PPP into 5’P) and analysed by electrophoresis in denaturing PAAG. Gels were stained with Et-Br. All images are representative of at least two independent experiments that produced similar results.

    Techniques Used: Northern Blot, Produced, Infection, Isolation, Transfection, Staining, In Vitro, Purification, Electrophoresis

    9) Product Images from "Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing"

    Article Title: Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab001

    Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.
    Figure Legend Snippet: Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.

    Techniques Used: Activation Assay, Incubation

    Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.
    Figure Legend Snippet: Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.

    Techniques Used: Fluorescence

    Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.
    Figure Legend Snippet: Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.

    Techniques Used: Labeling, Fluorescence

    Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.
    Figure Legend Snippet: Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.

    Techniques Used: Methylation, Negative Control

    Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).
    Figure Legend Snippet: Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).

    Techniques Used: Fluorescence, Labeling

    Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.
    Figure Legend Snippet: Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.

    Techniques Used: Modification, Software

    10) Product Images from "Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing"

    Article Title: Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab001

    Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.
    Figure Legend Snippet: Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.

    Techniques Used: Activation Assay, Incubation

    Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.
    Figure Legend Snippet: Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.

    Techniques Used: Fluorescence

    Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.
    Figure Legend Snippet: Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.

    Techniques Used: Labeling, Fluorescence

    Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.
    Figure Legend Snippet: Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.

    Techniques Used: Methylation, Negative Control

    Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).
    Figure Legend Snippet: Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).

    Techniques Used: Fluorescence, Labeling

    Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.
    Figure Legend Snippet: Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.

    Techniques Used: Modification, Software

    11) Product Images from "Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing"

    Article Title: Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab001

    Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.
    Figure Legend Snippet: Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.

    Techniques Used: Activation Assay, Incubation

    Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.
    Figure Legend Snippet: Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.

    Techniques Used: Fluorescence

    Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.
    Figure Legend Snippet: Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.

    Techniques Used: Labeling, Fluorescence

    Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.
    Figure Legend Snippet: Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.

    Techniques Used: Methylation, Negative Control

    Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).
    Figure Legend Snippet: Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).

    Techniques Used: Fluorescence, Labeling

    Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.
    Figure Legend Snippet: Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.

    Techniques Used: Modification, Software

    12) Product Images from "Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing"

    Article Title: Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab001

    Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.
    Figure Legend Snippet: Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.

    Techniques Used: Activation Assay, Incubation

    Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.
    Figure Legend Snippet: Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.

    Techniques Used: Fluorescence

    Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.
    Figure Legend Snippet: Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.

    Techniques Used: Labeling, Fluorescence

    Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.
    Figure Legend Snippet: Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.

    Techniques Used: Methylation, Negative Control

    Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).
    Figure Legend Snippet: Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).

    Techniques Used: Fluorescence, Labeling

    Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.
    Figure Legend Snippet: Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.

    Techniques Used: Modification, Software

    13) Product Images from "Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing"

    Article Title: Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab001

    Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.
    Figure Legend Snippet: Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.

    Techniques Used: Activation Assay, Incubation

    Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.
    Figure Legend Snippet: Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.

    Techniques Used: Fluorescence

    Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.
    Figure Legend Snippet: Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.

    Techniques Used: Labeling, Fluorescence

    Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.
    Figure Legend Snippet: Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.

    Techniques Used: Methylation, Negative Control

    Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).
    Figure Legend Snippet: Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).

    Techniques Used: Fluorescence, Labeling

    Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.
    Figure Legend Snippet: Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.

    Techniques Used: Modification, Software

    14) Product Images from "Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing"

    Article Title: Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab001

    Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.
    Figure Legend Snippet: Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.

    Techniques Used: Activation Assay, Incubation

    Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.
    Figure Legend Snippet: Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.

    Techniques Used: Fluorescence

    Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.
    Figure Legend Snippet: Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.

    Techniques Used: Labeling, Fluorescence

    Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.
    Figure Legend Snippet: Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.

    Techniques Used: Methylation, Negative Control

    Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).
    Figure Legend Snippet: Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).

    Techniques Used: Fluorescence, Labeling

    Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.
    Figure Legend Snippet: Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.

    Techniques Used: Modification, Software

    15) Product Images from "Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing"

    Article Title: Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab001

    Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.
    Figure Legend Snippet: Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.

    Techniques Used: Activation Assay, Incubation

    Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.
    Figure Legend Snippet: Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.

    Techniques Used: Fluorescence

    Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.
    Figure Legend Snippet: Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.

    Techniques Used: Labeling, Fluorescence

    Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.
    Figure Legend Snippet: Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.

    Techniques Used: Methylation, Negative Control

    Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).
    Figure Legend Snippet: Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).

    Techniques Used: Fluorescence, Labeling

    Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.
    Figure Legend Snippet: Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.

    Techniques Used: Modification, Software

    16) Product Images from "Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing"

    Article Title: Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab001

    Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.
    Figure Legend Snippet: Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.

    Techniques Used: Activation Assay, Incubation

    Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.
    Figure Legend Snippet: Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.

    Techniques Used: Fluorescence

    Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.
    Figure Legend Snippet: Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.

    Techniques Used: Labeling, Fluorescence

    Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.
    Figure Legend Snippet: Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.

    Techniques Used: Methylation, Negative Control

    Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).
    Figure Legend Snippet: Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).

    Techniques Used: Fluorescence, Labeling

    Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.
    Figure Legend Snippet: Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.

    Techniques Used: Modification, Software

    17) Product Images from "Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing"

    Article Title: Solid-phase XRN1 reactions for RNA cleavage: application in single-molecule sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab001

    Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.
    Figure Legend Snippet: Covalent attachment of XRN1 onto UV/O 3 activated PMMA. ( A ) Schematic representation of the process of covalent attachment of XRN1 onto PMMA surface by EDC/NHS coupling reaction. 5 μm × 5 μm AFM image of PMMA surface after UV/O 3 activation, and incubation with 40 nM XRN1 enzyme ( B ) without ( C ) with EDC/NHS coupling reagents. ( D ) Height distribution of surface features present on (C). The average height of a surface structure is 8.4 ± 0.5 nm.

    Techniques Used: Activation Assay, Incubation

    Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.
    Figure Legend Snippet: Solution phase clipping rate and processivity of XRN1. ( A ) Schematic representation of the reaction procedure. ( B ) Fluorescence intensity of RiboGreen labelled FLuc RNA with time. According to the average length of FLuc RNA fragment remaining after the reaction (Δ nt ave ), the processivity of XRN1 in solution phase is 1113 ± 132 nucleotides. ( C ) Clipping rate calculated using the fluorescence decay portion from 5A. According to the slope of the graph ( R 2 = 0.99121), the average clipping rate of XRN1 in solution is 3.06 ± 0.11 nt s –1 at 25°C.

    Techniques Used: Fluorescence

    Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.
    Figure Legend Snippet: Digestion of SYTO 82 labeled DMD RNA by immobilized XRN1. ( A ) Fluorescence still images and corresponding intensity plot profiles of labeled DMD RNA-immobilized XRN1 complex acquired at different times, after introduction of Mg 2+ to initiate digestion. ( B ) Relative fluorescence intensity of RNA–enzyme complexes with time. The black spectrum depicts the intensity of the complex in the absence of the cofactor Mg 2+ . The dark cyan, dark yellow and magenta spectra illustrate the fluorescence intensity of the complexes when Mg 2+ is introduced. The average fluorescence intensity becomes indistinguishable from the background intensity ∼400 s.

    Techniques Used: Labeling, Fluorescence

    Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.
    Figure Legend Snippet: Digestion of methylated RNA sequences. Chemical structures of ( A ) m6A and ( B ) m5C. Digestion of methylated RNA sequences by ( C ) solution phase and ( D ) immobilized XRN1. (1) Ladder (L). negative control for (2) unmethylated (c–) (4) m6A-methylated (m6A–) and (6) m5C-methylated (m5C–) RNA. Digestion results for (3) unmethylated (c+) (5) m6A-methylated (m6A+) and (7) m5C-methylated (m5C+) RNA by XRN1.

    Techniques Used: Methylation, Negative Control

    Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).
    Figure Legend Snippet: Solid-phase digestion reactions of XRN1. ( A ) Top down view of the pillared IMER channel. ( B ) Schematic representation of the covalently attached enzyme on the micropillars of the device. Fluorescence emission spectra of SYTO RNASelect Green labeled monophosphorylated RNA solutions digested by XRN1 in ( C ) free solution and ( D ) Immobilized state. The reaction time was 60 s and 2.32 pmol of enzyme was used in both free solution and immobilized digestion. SYTO RNASelect Green was added after digestion and fluorescence emission spectra were taken from 495 to 700 nm with 480 nm excitation. ( E ) Percentage digestion and relative fluorescence intensity of digested RNA with varied reaction time and constant surface enzyme density. The XRN1 reactions were all performed at room temperature. The error bars represent standard deviations in the measurements ( n ≥ 3).

    Techniques Used: Fluorescence, Labeling

    Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.
    Figure Legend Snippet: Front and back view of XRN1 with lysine groups highlighted in red. The lysine residues on the surface of the enzyme indicate potential attachment sites to PMMA surface. Structure of XRN1 was obtained from RCSB protein data bank and modified using PYMOL v2.1.1 software.

    Techniques Used: Modification, Software

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