dna substrates  (Integrated DNA Technologies)


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    Name:
    DNA oligo
    Description:
    Single stranded pooled or duplexed DNA synthesized to customer specifications Sspecialized platforms with industry leading synthesis capabilities deliver the purest primers for PCR dual labelled probes for qPCR indexed adapters and fusion primers for sequencing and a variety of advanced and custom products
    Catalog Number:
    do-577595
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    Nucleic acids
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    Structured Review

    Integrated DNA Technologies dna substrates
    Binding of HIV-1 IN to a <t>6-FAM-labeled,</t> recessed viral <t>DNA</t> oligo. (A), Illustration of the reaction. (B), Anisotropy measurements. Diamonds (dashed lines) show results with 6-His-tagged IN; solid lines are results with untagged IN. Solid symbols show binding in the presence of MgCl 2 ; open symbols, in the presence of MnCl 2 . (C), Structure of 6-carbofluorescein (6-FAM). Conjugation occurs through the terminal phosphate.
    Single stranded pooled or duplexed DNA synthesized to customer specifications Sspecialized platforms with industry leading synthesis capabilities deliver the purest primers for PCR dual labelled probes for qPCR indexed adapters and fusion primers for sequencing and a variety of advanced and custom products
    https://www.bioz.com/result/dna substrates/product/Integrated DNA Technologies
    Average 93 stars, based on 730 article reviews
    Price from $9.99 to $1999.99
    dna substrates - by Bioz Stars, 2020-09
    93/100 stars

    Images

    1) Product Images from "Oligonucleotide-based assays for integrase activity"

    Article Title: Oligonucleotide-based assays for integrase activity

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2008.10.024

    Binding of HIV-1 IN to a 6-FAM-labeled, recessed viral DNA oligo. (A), Illustration of the reaction. (B), Anisotropy measurements. Diamonds (dashed lines) show results with 6-His-tagged IN; solid lines are results with untagged IN. Solid symbols show binding in the presence of MgCl 2 ; open symbols, in the presence of MnCl 2 . (C), Structure of 6-carbofluorescein (6-FAM). Conjugation occurs through the terminal phosphate.
    Figure Legend Snippet: Binding of HIV-1 IN to a 6-FAM-labeled, recessed viral DNA oligo. (A), Illustration of the reaction. (B), Anisotropy measurements. Diamonds (dashed lines) show results with 6-His-tagged IN; solid lines are results with untagged IN. Solid symbols show binding in the presence of MgCl 2 ; open symbols, in the presence of MnCl 2 . (C), Structure of 6-carbofluorescein (6-FAM). Conjugation occurs through the terminal phosphate.

    Techniques Used: Binding Assay, Labeling, Conjugation Assay

    2) Product Images from "Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence"

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    Journal: bioRxiv

    doi: 10.1101/667758

    The CRISPR system of M. tuberculosis A. The CRISPR locus of M. tuberculosis includes genes encoding Cas6 (crRNA processing), Csm1-5 (type III-A interference complex), Csm6 (ancillary ribonuclease), Cas1 and Cas2 (Adaptation). Cas6 cleaves the CRISPR RNA at the base of a short hairpin to generate mature crRNA that is bound by the Csm complex. On target RNA binding, the Csm complex is expected to display three enzymatic activities: target RNA cleavage ( 1 ), DNA cleavage by the HD domain ( 2 ) and cOA production by the cyclase domain ( 3 ). B. Purified, recombinant CRISPR-associated proteins of M. tuberculosis . M: PageRuler Unstained (Thermo Scientific); 1: Csm1-5 interference complex; 2: Csm1-5, Csm1 D630A, D631A (Cy variant); 3: Csm1-5, Csm3 D35A (C3 variant); 4: Csm6; 5: Cas6.
    Figure Legend Snippet: The CRISPR system of M. tuberculosis A. The CRISPR locus of M. tuberculosis includes genes encoding Cas6 (crRNA processing), Csm1-5 (type III-A interference complex), Csm6 (ancillary ribonuclease), Cas1 and Cas2 (Adaptation). Cas6 cleaves the CRISPR RNA at the base of a short hairpin to generate mature crRNA that is bound by the Csm complex. On target RNA binding, the Csm complex is expected to display three enzymatic activities: target RNA cleavage ( 1 ), DNA cleavage by the HD domain ( 2 ) and cOA production by the cyclase domain ( 3 ). B. Purified, recombinant CRISPR-associated proteins of M. tuberculosis . M: PageRuler Unstained (Thermo Scientific); 1: Csm1-5 interference complex; 2: Csm1-5, Csm1 D630A, D631A (Cy variant); 3: Csm1-5, Csm3 D35A (C3 variant); 4: Csm6; 5: Cas6.

    Techniques Used: CRISPR, RNA Binding Assay, Purification, Recombinant, Variant Assay

    3) Product Images from "A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides"

    Article Title: A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

    Journal: Nanomaterials

    doi: 10.3390/nano9040615

    Multi-trigger systems can be composed in which each RNA/DNA hybrid contains a responsive DNA structural element. ( A ) A system comprising a 3-input AND gate and a NOT gate can be constructed by pairing sH ^CTGF.20/8 (activated by the connective tissue growth factor (CTGF) derived trigger) with aH ∨ KRAS (repressed by the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA derived trigger). Co-incubation of the two hybrids results in no interaction. Both hybrids and the CTGF trigger are required for dsRNA release, while the presence of the KRAS trigger will inhibit strand exchange. ( B ) The multi-trigger system was assessed by 10% acrylamide non-denaturing PAGE. The fraction of DsiRNA released is indicated in the gel depicted, in the presence of indicated trigger combinations following 30 min incubation at 37 °C. The sH and aH hybrid were present at equimolar concentration, while the triggers were added at a 2-fold or 3-fold excess, as indicated. In samples when both triggers are present, they were added to premixed hybrids sequentially (KRAS followed by CTGF). The antisense hybrid and DsiRNA control in were assembled using a 5′-AlexaFluor546 labeled antisense RNA strand for the purpose of visualization and quantification.
    Figure Legend Snippet: Multi-trigger systems can be composed in which each RNA/DNA hybrid contains a responsive DNA structural element. ( A ) A system comprising a 3-input AND gate and a NOT gate can be constructed by pairing sH ^CTGF.20/8 (activated by the connective tissue growth factor (CTGF) derived trigger) with aH ∨ KRAS (repressed by the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA derived trigger). Co-incubation of the two hybrids results in no interaction. Both hybrids and the CTGF trigger are required for dsRNA release, while the presence of the KRAS trigger will inhibit strand exchange. ( B ) The multi-trigger system was assessed by 10% acrylamide non-denaturing PAGE. The fraction of DsiRNA released is indicated in the gel depicted, in the presence of indicated trigger combinations following 30 min incubation at 37 °C. The sH and aH hybrid were present at equimolar concentration, while the triggers were added at a 2-fold or 3-fold excess, as indicated. In samples when both triggers are present, they were added to premixed hybrids sequentially (KRAS followed by CTGF). The antisense hybrid and DsiRNA control in were assembled using a 5′-AlexaFluor546 labeled antisense RNA strand for the purpose of visualization and quantification.

    Techniques Used: Construct, Derivative Assay, Incubation, Polyacrylamide Gel Electrophoresis, Concentration Assay, Labeling

    An RNA/DNA cognate pair system was designed to undergo conditional strand exchange by hybridizing to neighboring sites on an RNA trigger. ( A ) “Traditional” RNA/DNA hybrid pairs act as an 2-input AND gate. Hybridization between the single stranded toeholds of a sense hybrid ( sH ) and antisense hybrid ( aH ) initiates a thermodynamically driven strand exchange that generates a dsRNA duplex and DNA waste byproduct. ( B ) The “adjacent targeting” RNA/DNA hybrid system functions as a 3-input AND gate, requiring a hybrid pair as well as a specific RNA trigger sequence. The hybrid pair’s respective toeholds bind to regions of the trigger that are immediately upstream and downstream from one another. Anchoring the cognate hybrids in close proximity leads to initiation of the thermodynamically favorable strand exchange reaction and dsRNA release. ( C ) Five different cognate pairs of adjacent targeting hybrids were analyzed by 12% acrylamide non-denaturing PAGE for their ability to release a DsiRNA product. Each sense hybrid and the DsiRNA control assembly contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization. The pairs of constructs differ in the number of DNA nucleotides inserted between the single-strand toehold and the RNA/DNA hybrid duplex. These inserted nucleotides were complementary between cognate hybrids, resulting in either 0, +1, +2, +3 or +4 DNA bp that can seed the strand exchange (colored orange). The presence or absence of each component is indicated above each lane. The samples in the gel depicted were all incubated for 180 min at 37 °C. ( D ) Analysis of the fraction of dsRNA released by hybrid pairs in the presence and absence of the RNA trigger following 30, 90 or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.
    Figure Legend Snippet: An RNA/DNA cognate pair system was designed to undergo conditional strand exchange by hybridizing to neighboring sites on an RNA trigger. ( A ) “Traditional” RNA/DNA hybrid pairs act as an 2-input AND gate. Hybridization between the single stranded toeholds of a sense hybrid ( sH ) and antisense hybrid ( aH ) initiates a thermodynamically driven strand exchange that generates a dsRNA duplex and DNA waste byproduct. ( B ) The “adjacent targeting” RNA/DNA hybrid system functions as a 3-input AND gate, requiring a hybrid pair as well as a specific RNA trigger sequence. The hybrid pair’s respective toeholds bind to regions of the trigger that are immediately upstream and downstream from one another. Anchoring the cognate hybrids in close proximity leads to initiation of the thermodynamically favorable strand exchange reaction and dsRNA release. ( C ) Five different cognate pairs of adjacent targeting hybrids were analyzed by 12% acrylamide non-denaturing PAGE for their ability to release a DsiRNA product. Each sense hybrid and the DsiRNA control assembly contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization. The pairs of constructs differ in the number of DNA nucleotides inserted between the single-strand toehold and the RNA/DNA hybrid duplex. These inserted nucleotides were complementary between cognate hybrids, resulting in either 0, +1, +2, +3 or +4 DNA bp that can seed the strand exchange (colored orange). The presence or absence of each component is indicated above each lane. The samples in the gel depicted were all incubated for 180 min at 37 °C. ( D ) Analysis of the fraction of dsRNA released by hybrid pairs in the presence and absence of the RNA trigger following 30, 90 or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Techniques Used: Activated Clotting Time Assay, Hybridization, Sequencing, Polyacrylamide Gel Electrophoresis, Labeling, Construct, Incubation, Standard Deviation

    Effects of DNA structural alteration on the degree of trigger-inducible dsRNA release. ( A ) Four different sense hybrids that are responsive to the connective tissue growth factor (CTGF) trigger were designed, each having different features within the structured DNA hairpin. The hairpins differed in the size of their loop or the length of their stem. Two different cognate antisense hybrids were designed and differ in the length of their single-stranded toehold. Sequence regions are indicated by lowercase letters and different colors to convey sequence identity or sequence complementarity. ( B , D ) DsiRNA release in the presence and absence of trigger was assessed by 10% acrylamide non-denaturing PAGE for each sense hybrid paired with a cognate antisense hybrid exhibiting either ( B ) a 12 nt toehold ( aH ^CTGF-cgnt.12 ) or ( D ) a 16 nt toehold ( aH ^CTGF-cgnt.16 ). Each sense hybrid and the DsiRNA control contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization and quantification. Gels in both ( B ) and ( D ) depict samples that were incubated for 30 min at 37 °C. ( C , E ) Analysis of the fraction of dsRNA released by the four sense hybrids paired with ( C ) aH ^CTGF-cgnt.12 or ( E ) aH ^CTGF-cgnt.16 , in the presence and absence of the RNA trigger following 30, 90, or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.
    Figure Legend Snippet: Effects of DNA structural alteration on the degree of trigger-inducible dsRNA release. ( A ) Four different sense hybrids that are responsive to the connective tissue growth factor (CTGF) trigger were designed, each having different features within the structured DNA hairpin. The hairpins differed in the size of their loop or the length of their stem. Two different cognate antisense hybrids were designed and differ in the length of their single-stranded toehold. Sequence regions are indicated by lowercase letters and different colors to convey sequence identity or sequence complementarity. ( B , D ) DsiRNA release in the presence and absence of trigger was assessed by 10% acrylamide non-denaturing PAGE for each sense hybrid paired with a cognate antisense hybrid exhibiting either ( B ) a 12 nt toehold ( aH ^CTGF-cgnt.12 ) or ( D ) a 16 nt toehold ( aH ^CTGF-cgnt.16 ). Each sense hybrid and the DsiRNA control contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization and quantification. Gels in both ( B ) and ( D ) depict samples that were incubated for 30 min at 37 °C. ( C , E ) Analysis of the fraction of dsRNA released by the four sense hybrids paired with ( C ) aH ^CTGF-cgnt.12 or ( E ) aH ^CTGF-cgnt.16 , in the presence and absence of the RNA trigger following 30, 90, or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Techniques Used: Sequencing, Polyacrylamide Gel Electrophoresis, Labeling, Incubation, Standard Deviation

    Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.
    Figure Legend Snippet: Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.

    Techniques Used: Diagnostic Assay, Hybridization, Sequencing, Polyacrylamide Gel Electrophoresis, Staining, Incubation, Förster Resonance Energy Transfer, Labeling, Fluorescence

    4) Product Images from "Synthesis and Evaluation of a Rationally Designed Click-Based Library for G-Quadruplex Selective DNA Photocleavage"

    Article Title: Synthesis and Evaluation of a Rationally Designed Click-Based Library for G-Quadruplex Selective DNA Photocleavage

    Journal: Molecules

    doi: 10.3390/molecules200916446

    Photochemical cleavage of F21T by control compound 14 (black bars) compared with cleavage by TMPyP4 (gray bars) after 120 min of UVA irradiation.
    Figure Legend Snippet: Photochemical cleavage of F21T by control compound 14 (black bars) compared with cleavage by TMPyP4 (gray bars) after 120 min of UVA irradiation.

    Techniques Used: Irradiation

    Changes in T m upon formation of the DNA-compound complex ( A ) Average melt data for representative compounds incubated with FcMycT; ( B ) Average melt data for representative compounds incubated with F21T, Black, blue, green, and red bars represent positive control, benzophenone-incorporated, naphthalimide-incorporated, and anthraquinone-incorporated compounds respectively.
    Figure Legend Snippet: Changes in T m upon formation of the DNA-compound complex ( A ) Average melt data for representative compounds incubated with FcMycT; ( B ) Average melt data for representative compounds incubated with F21T, Black, blue, green, and red bars represent positive control, benzophenone-incorporated, naphthalimide-incorporated, and anthraquinone-incorporated compounds respectively.

    Techniques Used: Incubation, Positive Control

    ( A ) Effect of photoreactive group on the photochemical cleavage of G-quadruplex DNA by click-based compound library members. ( A ) Example polyacrylamide gel of photocleavage reactions of F21T after 30 min UV irradiation in the presence of 500 nM click-based compound library members 11a , b ; 12a – d ; and 13a , b ; ( B ) Quantification of G-quadruplex photochemical cleavage from gel electrophoresis analysis after irradiation and piperidine/heat treatment. Unless indicated, F21T was employed as the G-quadruplex substrate. Red, green, and blue bars correspond to compounds incorporating anthraquinone, naphthalimide, and benzophenone respectively. * FcMycT photocleavage data for comparison.
    Figure Legend Snippet: ( A ) Effect of photoreactive group on the photochemical cleavage of G-quadruplex DNA by click-based compound library members. ( A ) Example polyacrylamide gel of photocleavage reactions of F21T after 30 min UV irradiation in the presence of 500 nM click-based compound library members 11a , b ; 12a – d ; and 13a , b ; ( B ) Quantification of G-quadruplex photochemical cleavage from gel electrophoresis analysis after irradiation and piperidine/heat treatment. Unless indicated, F21T was employed as the G-quadruplex substrate. Red, green, and blue bars correspond to compounds incorporating anthraquinone, naphthalimide, and benzophenone respectively. * FcMycT photocleavage data for comparison.

    Techniques Used: Irradiation, Nucleic Acid Electrophoresis

    5) Product Images from "A Continuous Fluorometric Assay for the Assessment of MazF Ribonuclease Activity"

    Article Title: A Continuous Fluorometric Assay for the Assessment of MazF Ribonuclease Activity

    Journal:

    doi: 10.1016/j.ab.2007.07.017

    Substrate design. A chimeric DNA/RNA oligonucleotide (5′-AAGTCrGACATCAG-3′) previously shown to be cleaved by MazF was labeled with 6-carboxyfluorescein (6-FAM) on the 5′-end and with Black Hole Quencher 1 (BHQ1) on the 3′-end.
    Figure Legend Snippet: Substrate design. A chimeric DNA/RNA oligonucleotide (5′-AAGTCrGACATCAG-3′) previously shown to be cleaved by MazF was labeled with 6-carboxyfluorescein (6-FAM) on the 5′-end and with Black Hole Quencher 1 (BHQ1) on the 3′-end.

    Techniques Used: Labeling

    6) Product Images from "Rapid RNA Exchange in Aqueous Two-Phase System and Coacervate Droplets"

    Article Title: Rapid RNA Exchange in Aqueous Two-Phase System and Coacervate Droplets

    Journal: Origins of Life and Evolution of the Biosphere

    doi: 10.1007/s11084-014-9355-8

    Oleic acid vesicles do not exchange RNA with the surrounding fluid. Representative confocal microscope images of a sample (a) before photobleaching and (b) 590 s after photobleaching of the indicated non-gel-filtered oleic acid vesicle in 200 mM Bicine-NaOH pH 8.5 containing 5′-6-FAM labeled RNA 15-mer (5′-CCAGUCAGUCUACGC-3′) at room temperature ( Methods ). The vesicle samples were not gel filtered in order to maintain a high RNA concentration outside of the vesicles in order to simulate conditions similar to the ATPS and coacervate systems. After the entire window was photobleached, fluorescence outside of the vesicles recovered due to rapid RNA diffusion, but fluorescence inside vesicles did not recover due to lack of transport of RNA across the membrane. Scale bars, 10 μm. See Movie S5 for full movie of photobleaching and recovery
    Figure Legend Snippet: Oleic acid vesicles do not exchange RNA with the surrounding fluid. Representative confocal microscope images of a sample (a) before photobleaching and (b) 590 s after photobleaching of the indicated non-gel-filtered oleic acid vesicle in 200 mM Bicine-NaOH pH 8.5 containing 5′-6-FAM labeled RNA 15-mer (5′-CCAGUCAGUCUACGC-3′) at room temperature ( Methods ). The vesicle samples were not gel filtered in order to maintain a high RNA concentration outside of the vesicles in order to simulate conditions similar to the ATPS and coacervate systems. After the entire window was photobleached, fluorescence outside of the vesicles recovered due to rapid RNA diffusion, but fluorescence inside vesicles did not recover due to lack of transport of RNA across the membrane. Scale bars, 10 μm. See Movie S5 for full movie of photobleaching and recovery

    Techniques Used: Microscopy, Labeling, Concentration Assay, Fluorescence, Diffusion-based Assay

    Rapid exchange of RNA oligomers between ATPS and coacervate droplets and the surrounding bulk phase. Representative confocal fluorescence images showing RNA enriched droplets ( green ) are shown at left. Normalized fluorescence recovery after photobleaching (FRAP) recovery curves are shown at right. All samples contained 5 μM 5′-6-FAM-labeled RNA 15-mer (5′-CCAGUCAGUCUACGC-3′) in: (a) 16 % w/v dextran 9-11 kDa/10 % w/v PEG 8 kDa in 50 mM Tris-Cl pH 8 and 100 mM NaCl (indicated droplet 25 μm diameter), (b) 25 % w/v DEAE-dextran > 500 kDa/25 % w/v PEG 8 kDa in 100 mM Tris-Cl pH 8 with the GODCAT (glucose oxidase/catalase) system ( Methods ) (indicated droplet 9.5 μm diameter), (c) 16 % w/v dextran-sulfate 9-20 kDa/10 % w/v PEG 8 kDa in 50 mM Tris-Cl pH 8 and 100 mM NaCl (indicated droplet 44 μm diameter), (d) 30 mM ATP/2 % w/v pLys 4-15 kDa in 100 mM Tris-Cl pH 8 with the GODCAT system ( Methods ) (indicated droplet 7.5 μm diameter). See Movies S1 - S4 for respective FRAP movies. Each curve was normalized to the intensities of a non-bleached droplet and the background within the same frame, to correct for photobleaching during sampling, as well as to its initial intensity, to account for variable photobleaching before the recovery step across runs ( Supplementary Information ). Data were fit to a single exponential to determine time constants (τ) and half-lives (t 1/2 ) for fluorescence recovery ( Supplementary Information ). Further details and data in Table S3 . Scale bars for (a) and (c) are 100 μm; scale bars for (b) and (d) are 10 μm. See Movies S1 - S4 for full movies of photobleaching and recovery for each of the indicated droplets in (a) - (d) , respectively
    Figure Legend Snippet: Rapid exchange of RNA oligomers between ATPS and coacervate droplets and the surrounding bulk phase. Representative confocal fluorescence images showing RNA enriched droplets ( green ) are shown at left. Normalized fluorescence recovery after photobleaching (FRAP) recovery curves are shown at right. All samples contained 5 μM 5′-6-FAM-labeled RNA 15-mer (5′-CCAGUCAGUCUACGC-3′) in: (a) 16 % w/v dextran 9-11 kDa/10 % w/v PEG 8 kDa in 50 mM Tris-Cl pH 8 and 100 mM NaCl (indicated droplet 25 μm diameter), (b) 25 % w/v DEAE-dextran > 500 kDa/25 % w/v PEG 8 kDa in 100 mM Tris-Cl pH 8 with the GODCAT (glucose oxidase/catalase) system ( Methods ) (indicated droplet 9.5 μm diameter), (c) 16 % w/v dextran-sulfate 9-20 kDa/10 % w/v PEG 8 kDa in 50 mM Tris-Cl pH 8 and 100 mM NaCl (indicated droplet 44 μm diameter), (d) 30 mM ATP/2 % w/v pLys 4-15 kDa in 100 mM Tris-Cl pH 8 with the GODCAT system ( Methods ) (indicated droplet 7.5 μm diameter). See Movies S1 - S4 for respective FRAP movies. Each curve was normalized to the intensities of a non-bleached droplet and the background within the same frame, to correct for photobleaching during sampling, as well as to its initial intensity, to account for variable photobleaching before the recovery step across runs ( Supplementary Information ). Data were fit to a single exponential to determine time constants (τ) and half-lives (t 1/2 ) for fluorescence recovery ( Supplementary Information ). Further details and data in Table S3 . Scale bars for (a) and (c) are 100 μm; scale bars for (b) and (d) are 10 μm. See Movies S1 - S4 for full movies of photobleaching and recovery for each of the indicated droplets in (a) - (d) , respectively

    Techniques Used: Fluorescence, Labeling, Sampling

    7) Product Images from "ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells"

    Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

    Journal: bioRxiv

    doi: 10.1101/2020.05.28.122184

    Intracellular DNAs modulate pluripotency gene expressions through ABCF1. (A) V5-ABCF1 D3 mouse ES cell WCEs are incubated with three different 5’ biotinylated 98mer oligonucleotides: single-stranded (ss), double-stranded (ds) with SOX2-binding motif (Matched, ds-M), or ds without the motif (Unmatched, ds-UM). These DNA sequences are derived from Listeria monocytogenes genome. Input WCEs (IN) and streptavidin-beads captured, DNA-bound ABCF1 proteins are analyzed by western blotting. α-tubulin (TUBA) is used as control for binding specificity. (B) Genomic DNA purified from nuclear extracts prepared from DMSO and etoposide-treated (ETO, 20 μM) V5-ABCF1 knock-in (KI) D3 ES cells were analyzed on agarose gel and stained with ethidium bromide. (C) WCEs prepared from ETO-treated (20 μM) V5-ABCF1 KI D3 ES cells are incubated with IgGs or anti-V5 antibodies. Co-purified nucleic acids are treated with RNase A, separated on urea-PAGE, and stained with SYBR Gold. Vertical bar denotes DNAs specifically bound by ABCF1. (D) DNA damage disrupts ABCF1-SOX2 interaction. Input (IN) and SOX2 IPs from WCEs of DMSO or ETO-treated (20 μM) V5-ABCF1 KI D3 ES cells are analyzed by western blotting. (E) MNase-ChIP of ABCF1 in DMSO and ETO-treated (80 μM) V5-ABCF1 KI D3 ES cells. Enrichment of ABCF1 on OCT4/SOX2-targeted regions of Oct4, Sox2 , and Nanog gene promoters is analyzed by qPCR as in Figure 4 . (F) Colony formation assays in control and ABCF1 gain-of-function D3 cells. 200 D3 ES cells stably expressing RFP or V5-ABCF1 are plated on 24-well plates, treated with DMSO (left) or ETO (1 μM, right) for indicated period of time (hr), and let recover for 6 days before staining for AP activity. AP-positive colonies are counted. Error bars represent SEM of three independent experiments. n = 3. (*) P
    Figure Legend Snippet: Intracellular DNAs modulate pluripotency gene expressions through ABCF1. (A) V5-ABCF1 D3 mouse ES cell WCEs are incubated with three different 5’ biotinylated 98mer oligonucleotides: single-stranded (ss), double-stranded (ds) with SOX2-binding motif (Matched, ds-M), or ds without the motif (Unmatched, ds-UM). These DNA sequences are derived from Listeria monocytogenes genome. Input WCEs (IN) and streptavidin-beads captured, DNA-bound ABCF1 proteins are analyzed by western blotting. α-tubulin (TUBA) is used as control for binding specificity. (B) Genomic DNA purified from nuclear extracts prepared from DMSO and etoposide-treated (ETO, 20 μM) V5-ABCF1 knock-in (KI) D3 ES cells were analyzed on agarose gel and stained with ethidium bromide. (C) WCEs prepared from ETO-treated (20 μM) V5-ABCF1 KI D3 ES cells are incubated with IgGs or anti-V5 antibodies. Co-purified nucleic acids are treated with RNase A, separated on urea-PAGE, and stained with SYBR Gold. Vertical bar denotes DNAs specifically bound by ABCF1. (D) DNA damage disrupts ABCF1-SOX2 interaction. Input (IN) and SOX2 IPs from WCEs of DMSO or ETO-treated (20 μM) V5-ABCF1 KI D3 ES cells are analyzed by western blotting. (E) MNase-ChIP of ABCF1 in DMSO and ETO-treated (80 μM) V5-ABCF1 KI D3 ES cells. Enrichment of ABCF1 on OCT4/SOX2-targeted regions of Oct4, Sox2 , and Nanog gene promoters is analyzed by qPCR as in Figure 4 . (F) Colony formation assays in control and ABCF1 gain-of-function D3 cells. 200 D3 ES cells stably expressing RFP or V5-ABCF1 are plated on 24-well plates, treated with DMSO (left) or ETO (1 μM, right) for indicated period of time (hr), and let recover for 6 days before staining for AP activity. AP-positive colonies are counted. Error bars represent SEM of three independent experiments. n = 3. (*) P

    Techniques Used: Incubation, Binding Assay, Derivative Assay, Western Blot, Purification, Knock-In, Agarose Gel Electrophoresis, Staining, Polyacrylamide Gel Electrophoresis, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Activity Assay

    8) Product Images from "A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides"

    Article Title: A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

    Journal: Nanomaterials

    doi: 10.3390/nano9040615

    Multi-trigger systems can be composed in which each RNA/DNA hybrid contains a responsive DNA structural element. ( A ) A system comprising a 3-input AND gate and a NOT gate can be constructed by pairing sH ^CTGF.20/8 (activated by the connective tissue growth factor (CTGF) derived trigger) with aH ∨ KRAS (repressed by the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA derived trigger). Co-incubation of the two hybrids results in no interaction. Both hybrids and the CTGF trigger are required for dsRNA release, while the presence of the KRAS trigger will inhibit strand exchange. ( B ) The multi-trigger system was assessed by 10% acrylamide non-denaturing PAGE. The fraction of DsiRNA released is indicated in the gel depicted, in the presence of indicated trigger combinations following 30 min incubation at 37 °C. The sH and aH hybrid were present at equimolar concentration, while the triggers were added at a 2-fold or 3-fold excess, as indicated. In samples when both triggers are present, they were added to premixed hybrids sequentially (KRAS followed by CTGF). The antisense hybrid and DsiRNA control in were assembled using a 5′-AlexaFluor546 labeled antisense RNA strand for the purpose of visualization and quantification.
    Figure Legend Snippet: Multi-trigger systems can be composed in which each RNA/DNA hybrid contains a responsive DNA structural element. ( A ) A system comprising a 3-input AND gate and a NOT gate can be constructed by pairing sH ^CTGF.20/8 (activated by the connective tissue growth factor (CTGF) derived trigger) with aH ∨ KRAS (repressed by the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA derived trigger). Co-incubation of the two hybrids results in no interaction. Both hybrids and the CTGF trigger are required for dsRNA release, while the presence of the KRAS trigger will inhibit strand exchange. ( B ) The multi-trigger system was assessed by 10% acrylamide non-denaturing PAGE. The fraction of DsiRNA released is indicated in the gel depicted, in the presence of indicated trigger combinations following 30 min incubation at 37 °C. The sH and aH hybrid were present at equimolar concentration, while the triggers were added at a 2-fold or 3-fold excess, as indicated. In samples when both triggers are present, they were added to premixed hybrids sequentially (KRAS followed by CTGF). The antisense hybrid and DsiRNA control in were assembled using a 5′-AlexaFluor546 labeled antisense RNA strand for the purpose of visualization and quantification.

    Techniques Used: Construct, Derivative Assay, Incubation, Polyacrylamide Gel Electrophoresis, Concentration Assay, Labeling

    Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.
    Figure Legend Snippet: Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.

    Techniques Used: Diagnostic Assay, Hybridization, Sequencing, Polyacrylamide Gel Electrophoresis, Staining, Incubation, Förster Resonance Energy Transfer, Labeling, Fluorescence

    9) Product Images from "Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target for Antiviral Drug Development"

    Article Title: Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target for Antiviral Drug Development

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01879-18

    Establishment of a 6-carboxyfluorescein (FAM)-based in vitro nicking assay. (A) Diagram of the FAM-labeled oligonucleotides. The sequences of Ori20 are shown with FAM and the Iowa Black FQ quencher (Q) at the 5′ and 3′ ends, respectively. After incubation with NS1N, Ori20 is cleaved into two shorter oligonucleotides, and then a FAM-linked short oligonucleotide of 9 nt is released for fluorescence detection. (B) FAM Ori20 Q -based nicking assay. FAM Ori20 Q (200 nM) was incubated with 2 µM NS1N protein in the nicking buffer. The fluorescence intensity of each sample was detected on a microplate reader. FAM Ori20 Q without NS1N and FAM Ori20 without a quencher were used as controls. (C) Optimization of the probe concentration. Various concentrations of the FAM Ori20 Q probe were used in the nicking assay. Fluorescence intensity was determined with or without NS1N, as indicated. The fold changes in fluorescence intensity in the presence of NS1N from the fluorescence intensity with no NS1N are shown.
    Figure Legend Snippet: Establishment of a 6-carboxyfluorescein (FAM)-based in vitro nicking assay. (A) Diagram of the FAM-labeled oligonucleotides. The sequences of Ori20 are shown with FAM and the Iowa Black FQ quencher (Q) at the 5′ and 3′ ends, respectively. After incubation with NS1N, Ori20 is cleaved into two shorter oligonucleotides, and then a FAM-linked short oligonucleotide of 9 nt is released for fluorescence detection. (B) FAM Ori20 Q -based nicking assay. FAM Ori20 Q (200 nM) was incubated with 2 µM NS1N protein in the nicking buffer. The fluorescence intensity of each sample was detected on a microplate reader. FAM Ori20 Q without NS1N and FAM Ori20 without a quencher were used as controls. (C) Optimization of the probe concentration. Various concentrations of the FAM Ori20 Q probe were used in the nicking assay. Fluorescence intensity was determined with or without NS1N, as indicated. The fold changes in fluorescence intensity in the presence of NS1N from the fluorescence intensity with no NS1N are shown.

    Techniques Used: In Vitro, Labeling, Incubation, Fluorescence, Concentration Assay

    10) Product Images from "Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence"

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    Journal: bioRxiv

    doi: 10.1101/667758

    The CRISPR system of M. tuberculosis A. The CRISPR locus of M. tuberculosis includes genes encoding Cas6 (crRNA processing), Csm1-5 (type III-A interference complex), Csm6 (ancillary ribonuclease), Cas1 and Cas2 (Adaptation). Cas6 cleaves the CRISPR RNA at the base of a short hairpin to generate mature crRNA that is bound by the Csm complex. On target RNA binding, the Csm complex is expected to display three enzymatic activities: target RNA cleavage ( 1 ), DNA cleavage by the HD domain ( 2 ) and cOA production by the cyclase domain ( 3 ). B. Purified, recombinant CRISPR-associated proteins of M. tuberculosis . M: PageRuler Unstained (Thermo Scientific); 1: Csm1-5 interference complex; 2: Csm1-5, Csm1 D630A, D631A (Cy variant); 3: Csm1-5, Csm3 D35A (C3 variant); 4: Csm6; 5: Cas6.
    Figure Legend Snippet: The CRISPR system of M. tuberculosis A. The CRISPR locus of M. tuberculosis includes genes encoding Cas6 (crRNA processing), Csm1-5 (type III-A interference complex), Csm6 (ancillary ribonuclease), Cas1 and Cas2 (Adaptation). Cas6 cleaves the CRISPR RNA at the base of a short hairpin to generate mature crRNA that is bound by the Csm complex. On target RNA binding, the Csm complex is expected to display three enzymatic activities: target RNA cleavage ( 1 ), DNA cleavage by the HD domain ( 2 ) and cOA production by the cyclase domain ( 3 ). B. Purified, recombinant CRISPR-associated proteins of M. tuberculosis . M: PageRuler Unstained (Thermo Scientific); 1: Csm1-5 interference complex; 2: Csm1-5, Csm1 D630A, D631A (Cy variant); 3: Csm1-5, Csm3 D35A (C3 variant); 4: Csm6; 5: Cas6.

    Techniques Used: CRISPR, RNA Binding Assay, Purification, Recombinant, Variant Assay

    The Cas6 ribonuclease cleaves the Mtb CRISPR repeat sequence to generate crRNA. ( A ) Cas6 (0.5 µM) was incubated with 50 nM 5’-FAM CRISPR repeat RNA at 37 °C for 5, 15, 45 min in 20 mM Tris, 100 mM potassium glutamate, pH 7.5 in the absence or presence of 5 mM divalent metal ions as indicated. Reactions were stopped by phenol-chloroform extraction. ( B ) Cas6 cleavage leaves a 3’-(cyclic) phosphate group. CRISPR repeat RNA (crRepeat, 5’-FAM labeled, 400 nM) was digested with 2 µM Cas6 for 1 h in the presence of Mg 2+ using the same reaction conditions as before. Phenol-chloroform followed by chloroform extraction provided the substrate for the E. coli Poly(A) polymerase (PAP, New England Biolabs) reaction. Polyadenylation was performed according to the manufacturer’s instructions. In a parallel experiment, Cas6 was omitted. The CRISPR repeat RNA but not the Cas6 product can be 3’-polyadenylated by PAP. This suggests that the reaction product has a cyclic 2’,3’-phosphate, as observed for other Cas6 enzymes. This observation, together with the observation that calcium supports enhanced cleavage of the CRISPR repeat, suggests that the metal ion does not participate directly in catalysis but rather plays a role in stabilisation of the RNA substrate or RNA:protein complex.
    Figure Legend Snippet: The Cas6 ribonuclease cleaves the Mtb CRISPR repeat sequence to generate crRNA. ( A ) Cas6 (0.5 µM) was incubated with 50 nM 5’-FAM CRISPR repeat RNA at 37 °C for 5, 15, 45 min in 20 mM Tris, 100 mM potassium glutamate, pH 7.5 in the absence or presence of 5 mM divalent metal ions as indicated. Reactions were stopped by phenol-chloroform extraction. ( B ) Cas6 cleavage leaves a 3’-(cyclic) phosphate group. CRISPR repeat RNA (crRepeat, 5’-FAM labeled, 400 nM) was digested with 2 µM Cas6 for 1 h in the presence of Mg 2+ using the same reaction conditions as before. Phenol-chloroform followed by chloroform extraction provided the substrate for the E. coli Poly(A) polymerase (PAP, New England Biolabs) reaction. Polyadenylation was performed according to the manufacturer’s instructions. In a parallel experiment, Cas6 was omitted. The CRISPR repeat RNA but not the Cas6 product can be 3’-polyadenylated by PAP. This suggests that the reaction product has a cyclic 2’,3’-phosphate, as observed for other Cas6 enzymes. This observation, together with the observation that calcium supports enhanced cleavage of the CRISPR repeat, suggests that the metal ion does not participate directly in catalysis but rather plays a role in stabilisation of the RNA substrate or RNA:protein complex.

    Techniques Used: CRISPR, Sequencing, Incubation, Labeling

    11) Product Images from "FRET-Enabled Optical modulation for High Sensitivity Fluorescence Imaging"

    Article Title: FRET-Enabled Optical modulation for High Sensitivity Fluorescence Imaging

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja100175r

    A. Schematic model enabling donor fluorescence enhancement and modulation through dual laser excitation. Bold arrows show the transitions giving enhanced fluorescence. The arrow connecting D*A d to DA d is the primary enhancement pathway in the Cy3-Cy5
    Figure Legend Snippet: A. Schematic model enabling donor fluorescence enhancement and modulation through dual laser excitation. Bold arrows show the transitions giving enhanced fluorescence. The arrow connecting D*A d to DA d is the primary enhancement pathway in the Cy3-Cy5

    Techniques Used: Fluorescence

    A. Average frame of Cy3-Cy5 hairpin structure in solution with 496 nm cw defocused excitation (700 W/cm 2 ) and more tightly focused 633 nm cw laser (10 kW/cm 2 ). Secondary excitation was chopped at 4 Hz; synchronous ccd detection was at 40 Hz. B. Whole
    Figure Legend Snippet: A. Average frame of Cy3-Cy5 hairpin structure in solution with 496 nm cw defocused excitation (700 W/cm 2 ) and more tightly focused 633 nm cw laser (10 kW/cm 2 ). Secondary excitation was chopped at 4 Hz; synchronous ccd detection was at 40 Hz. B. Whole

    Techniques Used:

    A. Cy3 fluorescence enhancement within the Cy3-Cy5 hairpin (1μM, aqueous), calculated from the 50-μs-binned time trace modulation depth, versus modulation frequency (red ▲, varied from 500 Hz to 3900 Hz). Cy3 emission was excited
    Figure Legend Snippet: A. Cy3 fluorescence enhancement within the Cy3-Cy5 hairpin (1μM, aqueous), calculated from the 50-μs-binned time trace modulation depth, versus modulation frequency (red ▲, varied from 500 Hz to 3900 Hz). Cy3 emission was excited

    Techniques Used: Fluorescence

    12) Product Images from "Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation"

    Article Title: Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation

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

    doi: 10.1073/pnas.1504232112

    ( A ) Distribution profiles for the Smchd1 hinge domain R1867G mutant with unmethylated 20-mer dsDNA (annealed with ssDNA containing the Ctcf consensus sequence in sense and antisense orientations) containing either one or two 5′ 6-FAM molecules.
    Figure Legend Snippet: ( A ) Distribution profiles for the Smchd1 hinge domain R1867G mutant with unmethylated 20-mer dsDNA (annealed with ssDNA containing the Ctcf consensus sequence in sense and antisense orientations) containing either one or two 5′ 6-FAM molecules.

    Techniques Used: Mutagenesis, Sequencing

    13) Product Images from "A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides"

    Article Title: A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

    Journal: Nanomaterials

    doi: 10.3390/nano9040615

    Multi-trigger systems can be composed in which each RNA/DNA hybrid contains a responsive DNA structural element. ( A ) A system comprising a 3-input AND gate and a NOT gate can be constructed by pairing sH ^CTGF.20/8 (activated by the connective tissue growth factor (CTGF) derived trigger) with aH ∨ KRAS (repressed by the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA derived trigger). Co-incubation of the two hybrids results in no interaction. Both hybrids and the CTGF trigger are required for dsRNA release, while the presence of the KRAS trigger will inhibit strand exchange. ( B ) The multi-trigger system was assessed by 10% acrylamide non-denaturing PAGE. The fraction of DsiRNA released is indicated in the gel depicted, in the presence of indicated trigger combinations following 30 min incubation at 37 °C. The sH and aH hybrid were present at equimolar concentration, while the triggers were added at a 2-fold or 3-fold excess, as indicated. In samples when both triggers are present, they were added to premixed hybrids sequentially (KRAS followed by CTGF). The antisense hybrid and DsiRNA control in were assembled using a 5′-AlexaFluor546 labeled antisense RNA strand for the purpose of visualization and quantification.
    Figure Legend Snippet: Multi-trigger systems can be composed in which each RNA/DNA hybrid contains a responsive DNA structural element. ( A ) A system comprising a 3-input AND gate and a NOT gate can be constructed by pairing sH ^CTGF.20/8 (activated by the connective tissue growth factor (CTGF) derived trigger) with aH ∨ KRAS (repressed by the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA derived trigger). Co-incubation of the two hybrids results in no interaction. Both hybrids and the CTGF trigger are required for dsRNA release, while the presence of the KRAS trigger will inhibit strand exchange. ( B ) The multi-trigger system was assessed by 10% acrylamide non-denaturing PAGE. The fraction of DsiRNA released is indicated in the gel depicted, in the presence of indicated trigger combinations following 30 min incubation at 37 °C. The sH and aH hybrid were present at equimolar concentration, while the triggers were added at a 2-fold or 3-fold excess, as indicated. In samples when both triggers are present, they were added to premixed hybrids sequentially (KRAS followed by CTGF). The antisense hybrid and DsiRNA control in were assembled using a 5′-AlexaFluor546 labeled antisense RNA strand for the purpose of visualization and quantification.

    Techniques Used: Construct, Derivative Assay, Incubation, Polyacrylamide Gel Electrophoresis, Concentration Assay, Labeling

    An RNA/DNA cognate pair system was designed to undergo conditional strand exchange by hybridizing to neighboring sites on an RNA trigger. ( A ) “Traditional” RNA/DNA hybrid pairs act as an 2-input AND gate. Hybridization between the single stranded toeholds of a sense hybrid ( sH ) and antisense hybrid ( aH ) initiates a thermodynamically driven strand exchange that generates a dsRNA duplex and DNA waste byproduct. ( B ) The “adjacent targeting” RNA/DNA hybrid system functions as a 3-input AND gate, requiring a hybrid pair as well as a specific RNA trigger sequence. The hybrid pair’s respective toeholds bind to regions of the trigger that are immediately upstream and downstream from one another. Anchoring the cognate hybrids in close proximity leads to initiation of the thermodynamically favorable strand exchange reaction and dsRNA release. ( C ) Five different cognate pairs of adjacent targeting hybrids were analyzed by 12% acrylamide non-denaturing PAGE for their ability to release a DsiRNA product. Each sense hybrid and the DsiRNA control assembly contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization. The pairs of constructs differ in the number of DNA nucleotides inserted between the single-strand toehold and the RNA/DNA hybrid duplex. These inserted nucleotides were complementary between cognate hybrids, resulting in either 0, +1, +2, +3 or +4 DNA bp that can seed the strand exchange (colored orange). The presence or absence of each component is indicated above each lane. The samples in the gel depicted were all incubated for 180 min at 37 °C. ( D ) Analysis of the fraction of dsRNA released by hybrid pairs in the presence and absence of the RNA trigger following 30, 90 or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.
    Figure Legend Snippet: An RNA/DNA cognate pair system was designed to undergo conditional strand exchange by hybridizing to neighboring sites on an RNA trigger. ( A ) “Traditional” RNA/DNA hybrid pairs act as an 2-input AND gate. Hybridization between the single stranded toeholds of a sense hybrid ( sH ) and antisense hybrid ( aH ) initiates a thermodynamically driven strand exchange that generates a dsRNA duplex and DNA waste byproduct. ( B ) The “adjacent targeting” RNA/DNA hybrid system functions as a 3-input AND gate, requiring a hybrid pair as well as a specific RNA trigger sequence. The hybrid pair’s respective toeholds bind to regions of the trigger that are immediately upstream and downstream from one another. Anchoring the cognate hybrids in close proximity leads to initiation of the thermodynamically favorable strand exchange reaction and dsRNA release. ( C ) Five different cognate pairs of adjacent targeting hybrids were analyzed by 12% acrylamide non-denaturing PAGE for their ability to release a DsiRNA product. Each sense hybrid and the DsiRNA control assembly contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization. The pairs of constructs differ in the number of DNA nucleotides inserted between the single-strand toehold and the RNA/DNA hybrid duplex. These inserted nucleotides were complementary between cognate hybrids, resulting in either 0, +1, +2, +3 or +4 DNA bp that can seed the strand exchange (colored orange). The presence or absence of each component is indicated above each lane. The samples in the gel depicted were all incubated for 180 min at 37 °C. ( D ) Analysis of the fraction of dsRNA released by hybrid pairs in the presence and absence of the RNA trigger following 30, 90 or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Techniques Used: Activated Clotting Time Assay, Hybridization, Sequencing, Polyacrylamide Gel Electrophoresis, Labeling, Construct, Incubation, Standard Deviation

    Effects of DNA structural alteration on the degree of trigger-inducible dsRNA release. ( A ) Four different sense hybrids that are responsive to the connective tissue growth factor (CTGF) trigger were designed, each having different features within the structured DNA hairpin. The hairpins differed in the size of their loop or the length of their stem. Two different cognate antisense hybrids were designed and differ in the length of their single-stranded toehold. Sequence regions are indicated by lowercase letters and different colors to convey sequence identity or sequence complementarity. ( B , D ) DsiRNA release in the presence and absence of trigger was assessed by 10% acrylamide non-denaturing PAGE for each sense hybrid paired with a cognate antisense hybrid exhibiting either ( B ) a 12 nt toehold ( aH ^CTGF-cgnt.12 ) or ( D ) a 16 nt toehold ( aH ^CTGF-cgnt.16 ). Each sense hybrid and the DsiRNA control contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization and quantification. Gels in both ( B ) and ( D ) depict samples that were incubated for 30 min at 37 °C. ( C , E ) Analysis of the fraction of dsRNA released by the four sense hybrids paired with ( C ) aH ^CTGF-cgnt.12 or ( E ) aH ^CTGF-cgnt.16 , in the presence and absence of the RNA trigger following 30, 90, or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.
    Figure Legend Snippet: Effects of DNA structural alteration on the degree of trigger-inducible dsRNA release. ( A ) Four different sense hybrids that are responsive to the connective tissue growth factor (CTGF) trigger were designed, each having different features within the structured DNA hairpin. The hairpins differed in the size of their loop or the length of their stem. Two different cognate antisense hybrids were designed and differ in the length of their single-stranded toehold. Sequence regions are indicated by lowercase letters and different colors to convey sequence identity or sequence complementarity. ( B , D ) DsiRNA release in the presence and absence of trigger was assessed by 10% acrylamide non-denaturing PAGE for each sense hybrid paired with a cognate antisense hybrid exhibiting either ( B ) a 12 nt toehold ( aH ^CTGF-cgnt.12 ) or ( D ) a 16 nt toehold ( aH ^CTGF-cgnt.16 ). Each sense hybrid and the DsiRNA control contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization and quantification. Gels in both ( B ) and ( D ) depict samples that were incubated for 30 min at 37 °C. ( C , E ) Analysis of the fraction of dsRNA released by the four sense hybrids paired with ( C ) aH ^CTGF-cgnt.12 or ( E ) aH ^CTGF-cgnt.16 , in the presence and absence of the RNA trigger following 30, 90, or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Techniques Used: Sequencing, Polyacrylamide Gel Electrophoresis, Labeling, Incubation, Standard Deviation

    Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.
    Figure Legend Snippet: Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.

    Techniques Used: Diagnostic Assay, Hybridization, Sequencing, Polyacrylamide Gel Electrophoresis, Staining, Incubation, Förster Resonance Energy Transfer, Labeling, Fluorescence

    14) Product Images from "A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides"

    Article Title: A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

    Journal: Nanomaterials

    doi: 10.3390/nano9040615

    An RNA/DNA cognate pair system was designed to undergo conditional strand exchange by hybridizing to neighboring sites on an RNA trigger. ( A ) “Traditional” RNA/DNA hybrid pairs act as an 2-input AND gate. Hybridization between the single stranded toeholds of a sense hybrid ( sH ) and antisense hybrid ( aH ) initiates a thermodynamically driven strand exchange that generates a dsRNA duplex and DNA waste byproduct. ( B ) The “adjacent targeting” RNA/DNA hybrid system functions as a 3-input AND gate, requiring a hybrid pair as well as a specific RNA trigger sequence. The hybrid pair’s respective toeholds bind to regions of the trigger that are immediately upstream and downstream from one another. Anchoring the cognate hybrids in close proximity leads to initiation of the thermodynamically favorable strand exchange reaction and dsRNA release. ( C ) Five different cognate pairs of adjacent targeting hybrids were analyzed by 12% acrylamide non-denaturing PAGE for their ability to release a DsiRNA product. Each sense hybrid and the DsiRNA control assembly contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization. The pairs of constructs differ in the number of DNA nucleotides inserted between the single-strand toehold and the RNA/DNA hybrid duplex. These inserted nucleotides were complementary between cognate hybrids, resulting in either 0, +1, +2, +3 or +4 DNA bp that can seed the strand exchange (colored orange). The presence or absence of each component is indicated above each lane. The samples in the gel depicted were all incubated for 180 min at 37 °C. ( D ) Analysis of the fraction of dsRNA released by hybrid pairs in the presence and absence of the RNA trigger following 30, 90 or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.
    Figure Legend Snippet: An RNA/DNA cognate pair system was designed to undergo conditional strand exchange by hybridizing to neighboring sites on an RNA trigger. ( A ) “Traditional” RNA/DNA hybrid pairs act as an 2-input AND gate. Hybridization between the single stranded toeholds of a sense hybrid ( sH ) and antisense hybrid ( aH ) initiates a thermodynamically driven strand exchange that generates a dsRNA duplex and DNA waste byproduct. ( B ) The “adjacent targeting” RNA/DNA hybrid system functions as a 3-input AND gate, requiring a hybrid pair as well as a specific RNA trigger sequence. The hybrid pair’s respective toeholds bind to regions of the trigger that are immediately upstream and downstream from one another. Anchoring the cognate hybrids in close proximity leads to initiation of the thermodynamically favorable strand exchange reaction and dsRNA release. ( C ) Five different cognate pairs of adjacent targeting hybrids were analyzed by 12% acrylamide non-denaturing PAGE for their ability to release a DsiRNA product. Each sense hybrid and the DsiRNA control assembly contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization. The pairs of constructs differ in the number of DNA nucleotides inserted between the single-strand toehold and the RNA/DNA hybrid duplex. These inserted nucleotides were complementary between cognate hybrids, resulting in either 0, +1, +2, +3 or +4 DNA bp that can seed the strand exchange (colored orange). The presence or absence of each component is indicated above each lane. The samples in the gel depicted were all incubated for 180 min at 37 °C. ( D ) Analysis of the fraction of dsRNA released by hybrid pairs in the presence and absence of the RNA trigger following 30, 90 or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Techniques Used: Activated Clotting Time Assay, Hybridization, Sequencing, Polyacrylamide Gel Electrophoresis, Labeling, Construct, Incubation, Standard Deviation

    Effects of DNA structural alteration on the degree of trigger-inducible dsRNA release. ( A ) Four different sense hybrids that are responsive to the connective tissue growth factor (CTGF) trigger were designed, each having different features within the structured DNA hairpin. The hairpins differed in the size of their loop or the length of their stem. Two different cognate antisense hybrids were designed and differ in the length of their single-stranded toehold. Sequence regions are indicated by lowercase letters and different colors to convey sequence identity or sequence complementarity. ( B , D ) DsiRNA release in the presence and absence of trigger was assessed by 10% acrylamide non-denaturing PAGE for each sense hybrid paired with a cognate antisense hybrid exhibiting either ( B ) a 12 nt toehold ( aH ^CTGF-cgnt.12 ) or ( D ) a 16 nt toehold ( aH ^CTGF-cgnt.16 ). Each sense hybrid and the DsiRNA control contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization and quantification. Gels in both ( B ) and ( D ) depict samples that were incubated for 30 min at 37 °C. ( C , E ) Analysis of the fraction of dsRNA released by the four sense hybrids paired with ( C ) aH ^CTGF-cgnt.12 or ( E ) aH ^CTGF-cgnt.16 , in the presence and absence of the RNA trigger following 30, 90, or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.
    Figure Legend Snippet: Effects of DNA structural alteration on the degree of trigger-inducible dsRNA release. ( A ) Four different sense hybrids that are responsive to the connective tissue growth factor (CTGF) trigger were designed, each having different features within the structured DNA hairpin. The hairpins differed in the size of their loop or the length of their stem. Two different cognate antisense hybrids were designed and differ in the length of their single-stranded toehold. Sequence regions are indicated by lowercase letters and different colors to convey sequence identity or sequence complementarity. ( B , D ) DsiRNA release in the presence and absence of trigger was assessed by 10% acrylamide non-denaturing PAGE for each sense hybrid paired with a cognate antisense hybrid exhibiting either ( B ) a 12 nt toehold ( aH ^CTGF-cgnt.12 ) or ( D ) a 16 nt toehold ( aH ^CTGF-cgnt.16 ). Each sense hybrid and the DsiRNA control contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization and quantification. Gels in both ( B ) and ( D ) depict samples that were incubated for 30 min at 37 °C. ( C , E ) Analysis of the fraction of dsRNA released by the four sense hybrids paired with ( C ) aH ^CTGF-cgnt.12 or ( E ) aH ^CTGF-cgnt.16 , in the presence and absence of the RNA trigger following 30, 90, or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Techniques Used: Sequencing, Polyacrylamide Gel Electrophoresis, Labeling, Incubation, Standard Deviation

    Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.
    Figure Legend Snippet: Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.

    Techniques Used: Diagnostic Assay, Hybridization, Sequencing, Polyacrylamide Gel Electrophoresis, Staining, Incubation, Förster Resonance Energy Transfer, Labeling, Fluorescence

    15) Product Images from "A Continuous Fluorometric Assay for the Assessment of MazF Ribonuclease Activity"

    Article Title: A Continuous Fluorometric Assay for the Assessment of MazF Ribonuclease Activity

    Journal:

    doi: 10.1016/j.ab.2007.07.017

    Substrate design. A chimeric DNA/RNA oligonucleotide (5′-AAGTCrGACATCAG-3′) previously shown to be cleaved by MazF was labeled with 6-carboxyfluorescein (6-FAM) on the 5′-end and with Black Hole Quencher 1 (BHQ1) on the 3′-end.
    Figure Legend Snippet: Substrate design. A chimeric DNA/RNA oligonucleotide (5′-AAGTCrGACATCAG-3′) previously shown to be cleaved by MazF was labeled with 6-carboxyfluorescein (6-FAM) on the 5′-end and with Black Hole Quencher 1 (BHQ1) on the 3′-end.

    Techniques Used: Labeling

    16) Product Images from "FRET-Enabled Optical modulation for High Sensitivity Fluorescence Imaging"

    Article Title: FRET-Enabled Optical modulation for High Sensitivity Fluorescence Imaging

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja100175r

    A. Schematic model enabling donor fluorescence enhancement and modulation through dual laser excitation. Bold arrows show the transitions giving enhanced fluorescence. The arrow connecting D*A d to DA d is the primary enhancement pathway in the Cy3-Cy5
    Figure Legend Snippet: A. Schematic model enabling donor fluorescence enhancement and modulation through dual laser excitation. Bold arrows show the transitions giving enhanced fluorescence. The arrow connecting D*A d to DA d is the primary enhancement pathway in the Cy3-Cy5

    Techniques Used: Fluorescence

    A. Average frame of Cy3-Cy5 hairpin structure in solution with 496 nm cw defocused excitation (700 W/cm 2 ) and more tightly focused 633 nm cw laser (10 kW/cm 2 ). Secondary excitation was chopped at 4 Hz; synchronous ccd detection was at 40 Hz. B. Whole
    Figure Legend Snippet: A. Average frame of Cy3-Cy5 hairpin structure in solution with 496 nm cw defocused excitation (700 W/cm 2 ) and more tightly focused 633 nm cw laser (10 kW/cm 2 ). Secondary excitation was chopped at 4 Hz; synchronous ccd detection was at 40 Hz. B. Whole

    Techniques Used:

    A. Cy3 fluorescence enhancement within the Cy3-Cy5 hairpin (1μM, aqueous), calculated from the 50-μs-binned time trace modulation depth, versus modulation frequency (red ▲, varied from 500 Hz to 3900 Hz). Cy3 emission was excited
    Figure Legend Snippet: A. Cy3 fluorescence enhancement within the Cy3-Cy5 hairpin (1μM, aqueous), calculated from the 50-μs-binned time trace modulation depth, versus modulation frequency (red ▲, varied from 500 Hz to 3900 Hz). Cy3 emission was excited

    Techniques Used: Fluorescence

    17) Product Images from "Homogeneous Polymerase Chain Reaction Nucleobase Quenching Assay to Detect the 1-kbp Deletion in CLN3 That Causes Batten Disease"

    Article Title: Homogeneous Polymerase Chain Reaction Nucleobase Quenching Assay to Detect the 1-kbp Deletion in CLN3 That Causes Batten Disease

    Journal: The Journal of molecular diagnostics : JMD

    doi:

    Strategy. A: The positions of the primers ( arrows ) and probe on the wild-type and mutant CLN3 gene are shown. The fluorophore on the probe is indicated with an asterisk ; not drawn to scale. B: The sequence of the probe is shown in between the complementary sequences of the wild-type and deletion alleles, with vertical lines connecting the base-paired residues. The –F on the 3′ end of the probe indicates the 6-FAM fluorophore and shows its position with respect to the G residues on the opposite strand. The probe is fully base-paired with the mutant sequence, but has three unmatched nucleotides at the 5′ end when annealed to a wild-type amplicon. The G residues that contribute to the quenching of the fluorescent signal are underlined in the normal and mutant sequences.
    Figure Legend Snippet: Strategy. A: The positions of the primers ( arrows ) and probe on the wild-type and mutant CLN3 gene are shown. The fluorophore on the probe is indicated with an asterisk ; not drawn to scale. B: The sequence of the probe is shown in between the complementary sequences of the wild-type and deletion alleles, with vertical lines connecting the base-paired residues. The –F on the 3′ end of the probe indicates the 6-FAM fluorophore and shows its position with respect to the G residues on the opposite strand. The probe is fully base-paired with the mutant sequence, but has three unmatched nucleotides at the 5′ end when annealed to a wild-type amplicon. The G residues that contribute to the quenching of the fluorescent signal are underlined in the normal and mutant sequences.

    Techniques Used: Mutagenesis, Sequencing, Amplification

    18) Product Images from "Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target for Antiviral Drug Development"

    Article Title: Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target for Antiviral Drug Development

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01879-18

    Establishment of a 6-carboxyfluorescein (FAM)-based in vitro nicking assay. (A) Diagram of the FAM-labeled oligonucleotides. The sequences of Ori20 are shown with FAM and the Iowa Black FQ quencher (Q) at the 5′ and 3′ ends, respectively. After incubation with NS1N, Ori20 is cleaved into two shorter oligonucleotides, and then a FAM-linked short oligonucleotide of 9 nt is released for fluorescence detection. (B) FAM Ori20 Q -based nicking assay. FAM Ori20 Q (200 nM) was incubated with 2 µM NS1N protein in the nicking buffer. The fluorescence intensity of each sample was detected on a microplate reader. FAM Ori20 Q without NS1N and FAM Ori20 without a quencher were used as controls. (C) Optimization of the probe concentration. Various concentrations of the FAM Ori20 Q probe were used in the nicking assay. Fluorescence intensity was determined with or without NS1N, as indicated. The fold changes in fluorescence intensity in the presence of NS1N from the fluorescence intensity with no NS1N are shown.
    Figure Legend Snippet: Establishment of a 6-carboxyfluorescein (FAM)-based in vitro nicking assay. (A) Diagram of the FAM-labeled oligonucleotides. The sequences of Ori20 are shown with FAM and the Iowa Black FQ quencher (Q) at the 5′ and 3′ ends, respectively. After incubation with NS1N, Ori20 is cleaved into two shorter oligonucleotides, and then a FAM-linked short oligonucleotide of 9 nt is released for fluorescence detection. (B) FAM Ori20 Q -based nicking assay. FAM Ori20 Q (200 nM) was incubated with 2 µM NS1N protein in the nicking buffer. The fluorescence intensity of each sample was detected on a microplate reader. FAM Ori20 Q without NS1N and FAM Ori20 without a quencher were used as controls. (C) Optimization of the probe concentration. Various concentrations of the FAM Ori20 Q probe were used in the nicking assay. Fluorescence intensity was determined with or without NS1N, as indicated. The fold changes in fluorescence intensity in the presence of NS1N from the fluorescence intensity with no NS1N are shown.

    Techniques Used: In Vitro, Labeling, Incubation, Fluorescence, Concentration Assay

    19) Product Images from "Human DNA polymerase α in binary complex with a DNA:DNA template-primer"

    Article Title: Human DNA polymerase α in binary complex with a DNA:DNA template-primer

    Journal: Scientific Reports

    doi: 10.1038/srep23784

    Polα/primase mediated assembly of RNA-DNA primers during DNA replication. ( A ) Because DNA synthesis proceeds in the 5′ to 3, direction, the replication fork is asymmetrical, with continuous DNA synthesis on the leading strand and discontinuous DNA synthesis (via Okazaki fragments) on the lagging strand. ( B ) The Polα/primase complex is composed of four subunits (p180, p70, p49 and p58 in humans). ( C ) The Polα/primase complex assembles RNA-DNA primers required to initiate DNA synthesis on leading and lagging strands in eukaryotes.
    Figure Legend Snippet: Polα/primase mediated assembly of RNA-DNA primers during DNA replication. ( A ) Because DNA synthesis proceeds in the 5′ to 3, direction, the replication fork is asymmetrical, with continuous DNA synthesis on the leading strand and discontinuous DNA synthesis (via Okazaki fragments) on the lagging strand. ( B ) The Polα/primase complex is composed of four subunits (p180, p70, p49 and p58 in humans). ( C ) The Polα/primase complex assembles RNA-DNA primers required to initiate DNA synthesis on leading and lagging strands in eukaryotes.

    Techniques Used: DNA Synthesis

    Comparison between binary and ternary structures of hPolα. ( A ) The hPolα binary complex bound to DNA:DNA is colored yellow and the ternary complex bound to RNA:DNA (PDB code 4 QCL) is colored gray. The palm, fingers, thumb, exonuclease and N-terminal domains are labeled. ( B ) Close-up view of the hPolα active site. Side chains for residues Arg784, Asp860 and Asp1004 are shown with oxygen atoms in red and nitrogen atoms in blue. The incoming nucleotide (dCTP) from the ternary structure is shown in red.
    Figure Legend Snippet: Comparison between binary and ternary structures of hPolα. ( A ) The hPolα binary complex bound to DNA:DNA is colored yellow and the ternary complex bound to RNA:DNA (PDB code 4 QCL) is colored gray. The palm, fingers, thumb, exonuclease and N-terminal domains are labeled. ( B ) Close-up view of the hPolα active site. Side chains for residues Arg784, Asp860 and Asp1004 are shown with oxygen atoms in red and nitrogen atoms in blue. The incoming nucleotide (dCTP) from the ternary structure is shown in red.

    Techniques Used: Labeling

    Conformation of nucleic acid. ( A ) Comparison of the DNA conformations of the hPolα binary complex (yellow) and the hPolα ternary complex (gray). The bound and unbound regions are marked in the figure. ( B ) Scatter plot of Z p , the mean z-coordinate of the backbone phosphorous atoms with respect to individual dinucleotide reference frames, against the mean value for the four χ torsion angles at each dinucleotide step. The values for 7 DNA:DNA base steps bound to hPolα in the binary complex are shown as yellow circles. The 7 RNA:DNA base steps bound to hPolα in the ternary complex (PDB code 4 QCL) are shown as gray triangles. The values for RNA:DNA steps bound to yPolα (red squares), DNA:DNA steps bound to yPolδ (blue diamonds) and DNA:DNA steps bound to yPolε (orange diamonds) are also plotted. ( C ) Binding affinities of hPolα for DNA/DNA and RNA/DNA measured by a fluorescence anisotropy assay. The fraction of DNA/DNA or RNA/DNA bound is plotted versus hPolα concentration (logarithmic scale) in order to determine the dissociation constants. ( D ) Distribution of the pseudorotational phase angle P and puckering amplitude v max of the base steps in contact with hPolα in the binary complex (yellow circles), with hPolα in the ternary complex (gray triangles) and with yPolα in the ternary complex (red squares).
    Figure Legend Snippet: Conformation of nucleic acid. ( A ) Comparison of the DNA conformations of the hPolα binary complex (yellow) and the hPolα ternary complex (gray). The bound and unbound regions are marked in the figure. ( B ) Scatter plot of Z p , the mean z-coordinate of the backbone phosphorous atoms with respect to individual dinucleotide reference frames, against the mean value for the four χ torsion angles at each dinucleotide step. The values for 7 DNA:DNA base steps bound to hPolα in the binary complex are shown as yellow circles. The 7 RNA:DNA base steps bound to hPolα in the ternary complex (PDB code 4 QCL) are shown as gray triangles. The values for RNA:DNA steps bound to yPolα (red squares), DNA:DNA steps bound to yPolδ (blue diamonds) and DNA:DNA steps bound to yPolε (orange diamonds) are also plotted. ( C ) Binding affinities of hPolα for DNA/DNA and RNA/DNA measured by a fluorescence anisotropy assay. The fraction of DNA/DNA or RNA/DNA bound is plotted versus hPolα concentration (logarithmic scale) in order to determine the dissociation constants. ( D ) Distribution of the pseudorotational phase angle P and puckering amplitude v max of the base steps in contact with hPolα in the binary complex (yellow circles), with hPolα in the ternary complex (gray triangles) and with yPolα in the ternary complex (red squares).

    Techniques Used: Binding Assay, Fluorescence, Concentration Assay

    20) Product Images from "Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence"

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    Journal: bioRxiv

    doi: 10.1101/667758

    The CRISPR system of M. tuberculosis A. The CRISPR locus of M. tuberculosis includes genes encoding Cas6 (crRNA processing), Csm1-5 (type III-A interference complex), Csm6 (ancillary ribonuclease), Cas1 and Cas2 (Adaptation). Cas6 cleaves the CRISPR RNA at the base of a short hairpin to generate mature crRNA that is bound by the Csm complex. On target RNA binding, the Csm complex is expected to display three enzymatic activities: target RNA cleavage ( 1 ), DNA cleavage by the HD domain ( 2 ) and cOA production by the cyclase domain ( 3 ). B. Purified, recombinant CRISPR-associated proteins of M. tuberculosis . M: PageRuler Unstained (Thermo Scientific); 1: Csm1-5 interference complex; 2: Csm1-5, Csm1 D630A, D631A (Cy variant); 3: Csm1-5, Csm3 D35A (C3 variant); 4: Csm6; 5: Cas6.
    Figure Legend Snippet: The CRISPR system of M. tuberculosis A. The CRISPR locus of M. tuberculosis includes genes encoding Cas6 (crRNA processing), Csm1-5 (type III-A interference complex), Csm6 (ancillary ribonuclease), Cas1 and Cas2 (Adaptation). Cas6 cleaves the CRISPR RNA at the base of a short hairpin to generate mature crRNA that is bound by the Csm complex. On target RNA binding, the Csm complex is expected to display three enzymatic activities: target RNA cleavage ( 1 ), DNA cleavage by the HD domain ( 2 ) and cOA production by the cyclase domain ( 3 ). B. Purified, recombinant CRISPR-associated proteins of M. tuberculosis . M: PageRuler Unstained (Thermo Scientific); 1: Csm1-5 interference complex; 2: Csm1-5, Csm1 D630A, D631A (Cy variant); 3: Csm1-5, Csm3 D35A (C3 variant); 4: Csm6; 5: Cas6.

    Techniques Used: CRISPR, RNA Binding Assay, Purification, Recombinant, Variant Assay

    The Cas6 ribonuclease cleaves the Mtb CRISPR repeat sequence to generate crRNA. ( A ) Cas6 (0.5 µM) was incubated with 50 nM 5’-FAM CRISPR repeat RNA at 37 °C for 5, 15, 45 min in 20 mM Tris, 100 mM potassium glutamate, pH 7.5 in the absence or presence of 5 mM divalent metal ions as indicated. Reactions were stopped by phenol-chloroform extraction. ( B ) Cas6 cleavage leaves a 3’-(cyclic) phosphate group. CRISPR repeat RNA (crRepeat, 5’-FAM labeled, 400 nM) was digested with 2 µM Cas6 for 1 h in the presence of Mg 2+ using the same reaction conditions as before. Phenol-chloroform followed by chloroform extraction provided the substrate for the E. coli Poly(A) polymerase (PAP, New England Biolabs) reaction. Polyadenylation was performed according to the manufacturer’s instructions. In a parallel experiment, Cas6 was omitted. The CRISPR repeat RNA but not the Cas6 product can be 3’-polyadenylated by PAP. This suggests that the reaction product has a cyclic 2’,3’-phosphate, as observed for other Cas6 enzymes. This observation, together with the observation that calcium supports enhanced cleavage of the CRISPR repeat, suggests that the metal ion does not participate directly in catalysis but rather plays a role in stabilisation of the RNA substrate or RNA:protein complex.
    Figure Legend Snippet: The Cas6 ribonuclease cleaves the Mtb CRISPR repeat sequence to generate crRNA. ( A ) Cas6 (0.5 µM) was incubated with 50 nM 5’-FAM CRISPR repeat RNA at 37 °C for 5, 15, 45 min in 20 mM Tris, 100 mM potassium glutamate, pH 7.5 in the absence or presence of 5 mM divalent metal ions as indicated. Reactions were stopped by phenol-chloroform extraction. ( B ) Cas6 cleavage leaves a 3’-(cyclic) phosphate group. CRISPR repeat RNA (crRepeat, 5’-FAM labeled, 400 nM) was digested with 2 µM Cas6 for 1 h in the presence of Mg 2+ using the same reaction conditions as before. Phenol-chloroform followed by chloroform extraction provided the substrate for the E. coli Poly(A) polymerase (PAP, New England Biolabs) reaction. Polyadenylation was performed according to the manufacturer’s instructions. In a parallel experiment, Cas6 was omitted. The CRISPR repeat RNA but not the Cas6 product can be 3’-polyadenylated by PAP. This suggests that the reaction product has a cyclic 2’,3’-phosphate, as observed for other Cas6 enzymes. This observation, together with the observation that calcium supports enhanced cleavage of the CRISPR repeat, suggests that the metal ion does not participate directly in catalysis but rather plays a role in stabilisation of the RNA substrate or RNA:protein complex.

    Techniques Used: CRISPR, Sequencing, Incubation, Labeling

    21) Product Images from "Inhibition of HIV-1 by Octadecyloxyethyl Esters of (S)-[3-Hydroxy-2-(Phosphonomethoxy)Propyl] Nucleosides and Evaluation of Their Mechanism of Action ▿"

    Article Title: Inhibition of HIV-1 by Octadecyloxyethyl Esters of (S)-[3-Hydroxy-2-(Phosphonomethoxy)Propyl] Nucleosides and Evaluation of Their Mechanism of Action ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.05161-11

    Effects of increasing concentrations of HPMPCpp or HPMPApp on HIV-1 RT activity. (A) Primer extension reactions using RNA or DNA templates directing the incorporation of a single HPMPC molecule. Each 10-μl reaction mixture contained FAM-labeled
    Figure Legend Snippet: Effects of increasing concentrations of HPMPCpp or HPMPApp on HIV-1 RT activity. (A) Primer extension reactions using RNA or DNA templates directing the incorporation of a single HPMPC molecule. Each 10-μl reaction mixture contained FAM-labeled

    Techniques Used: Activity Assay, Labeling

    HIV-1 RT can use HPMPCpp or HPMPApp to support DNA synthesis. (A and B) Primer extension reactions examining the ability of HIV-1 RT to replace dCTP with HPMPCpp. FAM-labeled primer P1 was annealed to template T9 RNA or T9 (A) or to template T2 RNA or T2
    Figure Legend Snippet: HIV-1 RT can use HPMPCpp or HPMPApp to support DNA synthesis. (A and B) Primer extension reactions examining the ability of HIV-1 RT to replace dCTP with HPMPCpp. FAM-labeled primer P1 was annealed to template T9 RNA or T9 (A) or to template T2 RNA or T2

    Techniques Used: DNA Synthesis, Labeling

    22) Product Images from "Solution NMR Structure of the C-terminal DNA Binding Domain of Mcm10 Reveals a Conserved MCM Motif *"

    Article Title: Solution NMR Structure of the C-terminal DNA Binding Domain of Mcm10 Reveals a Conserved MCM Motif *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.131276

    ssDNA binding to the CCCH zinc motif. A , overlays of a representative region of the 15 N- 1 H HSQC spectra of 15 N-XMcm10 755–842 in the absence ( black ) and presence of 0.5 ( gray ), 1 ( green ), 2 ( red ), and 4 ( blue ) fold molar excess of ssDNA. B , residues perturbed by ssDNA binding from the HSQC titration are colored orange against the molecular surface of XMcm10 755–842 . The asterisk denotes the position of Phe776. C , ssDNA binding to CTD deletion constructs was monitored by the change in fluorescence polarization as protein was added to fluorescein ( FAM )-labeled d(ATGGTAGGCAACCAT). Addition of buffer only to FAM-DNA is shown as gray Xs. Data shown are from one representative experiment and were reproduced in triplicate. D , schematic representation of the data shown in panel C .
    Figure Legend Snippet: ssDNA binding to the CCCH zinc motif. A , overlays of a representative region of the 15 N- 1 H HSQC spectra of 15 N-XMcm10 755–842 in the absence ( black ) and presence of 0.5 ( gray ), 1 ( green ), 2 ( red ), and 4 ( blue ) fold molar excess of ssDNA. B , residues perturbed by ssDNA binding from the HSQC titration are colored orange against the molecular surface of XMcm10 755–842 . The asterisk denotes the position of Phe776. C , ssDNA binding to CTD deletion constructs was monitored by the change in fluorescence polarization as protein was added to fluorescein ( FAM )-labeled d(ATGGTAGGCAACCAT). Addition of buffer only to FAM-DNA is shown as gray Xs. Data shown are from one representative experiment and were reproduced in triplicate. D , schematic representation of the data shown in panel C .

    Techniques Used: Binding Assay, Titration, Construct, Fluorescence, Labeling

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    Purification:

    Article Title: A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production
    Article Snippet: .. 2.1 Generation and Validation of Genome Engineered Strains using MAGE All multiplex allele-specific PCR (MASC-PCR), Sanger Sequencing oligos, and recombineering oligos were created manually and ordered from IDT (Coralville, IA) with standard purification. ..

    Polymerase Chain Reaction:

    Article Title: A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production
    Article Snippet: .. 2.1 Generation and Validation of Genome Engineered Strains using MAGE All multiplex allele-specific PCR (MASC-PCR), Sanger Sequencing oligos, and recombineering oligos were created manually and ordered from IDT (Coralville, IA) with standard purification. ..

    other:

    Article Title: Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity
    Article Snippet: DNA oligonucleotides and tracrRNA were purchased from Integrated DNA Technologies (IDT).

    Article Title: CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks
    Article Snippet: Additional files Sequences of plasmids, DNA oligonucleotides, and RNA oligonucleotides used in this work.

    Article Title: RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements
    Article Snippet: DNA oligonucleotides and synthetic dsDNA were obtained from Integrated DNA Technologies (Coralville, IA, USA).

    Sequencing:

    Article Title: A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production
    Article Snippet: .. 2.1 Generation and Validation of Genome Engineered Strains using MAGE All multiplex allele-specific PCR (MASC-PCR), Sanger Sequencing oligos, and recombineering oligos were created manually and ordered from IDT (Coralville, IA) with standard purification. ..

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    Integrated DNA Technologies 6 fam labeled dna oligos
    6 Fam Labeled Dna Oligos, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/6 fam labeled dna oligos/product/Integrated DNA Technologies
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    6 fam labeled dna oligos - by Bioz Stars, 2020-09
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    85
    Integrated DNA Technologies 6 fam labeled oligos
    Strategy. A: The positions of the primers ( arrows ) and probe on the wild-type and mutant CLN3 gene are shown. The fluorophore on the probe is indicated with an asterisk ; not drawn to scale. B: The sequence of the probe is shown in between the complementary sequences of the wild-type and deletion alleles, with vertical lines connecting the base-paired residues. The –F on the 3′ end of the probe indicates the <t>6-FAM</t> fluorophore and shows its position with respect to the G residues on the opposite strand. The probe is fully base-paired with the mutant sequence, but has three unmatched nucleotides at the 5′ end when annealed to a wild-type amplicon. The G residues that contribute to the quenching of the fluorescent signal are underlined in the normal and mutant sequences.
    6 Fam Labeled Oligos, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/6 fam labeled oligos/product/Integrated DNA Technologies
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    6 fam labeled oligos - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    93
    Integrated DNA Technologies 6 carboxyfluorescein fam labeled primers
    Strategy. A: The positions of the primers ( arrows ) and probe on the wild-type and mutant CLN3 gene are shown. The fluorophore on the probe is indicated with an asterisk ; not drawn to scale. B: The sequence of the probe is shown in between the complementary sequences of the wild-type and deletion alleles, with vertical lines connecting the base-paired residues. The –F on the 3′ end of the probe indicates the <t>6-FAM</t> fluorophore and shows its position with respect to the G residues on the opposite strand. The probe is fully base-paired with the mutant sequence, but has three unmatched nucleotides at the 5′ end when annealed to a wild-type amplicon. The G residues that contribute to the quenching of the fluorescent signal are underlined in the normal and mutant sequences.
    6 Carboxyfluorescein Fam Labeled Primers, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/6 carboxyfluorescein fam labeled primers/product/Integrated DNA Technologies
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    6 carboxyfluorescein fam labeled primers - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

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    Strategy. A: The positions of the primers ( arrows ) and probe on the wild-type and mutant CLN3 gene are shown. The fluorophore on the probe is indicated with an asterisk ; not drawn to scale. B: The sequence of the probe is shown in between the complementary sequences of the wild-type and deletion alleles, with vertical lines connecting the base-paired residues. The –F on the 3′ end of the probe indicates the 6-FAM fluorophore and shows its position with respect to the G residues on the opposite strand. The probe is fully base-paired with the mutant sequence, but has three unmatched nucleotides at the 5′ end when annealed to a wild-type amplicon. The G residues that contribute to the quenching of the fluorescent signal are underlined in the normal and mutant sequences.

    Journal: The Journal of molecular diagnostics : JMD

    Article Title: Homogeneous Polymerase Chain Reaction Nucleobase Quenching Assay to Detect the 1-kbp Deletion in CLN3 That Causes Batten Disease

    doi:

    Figure Lengend Snippet: Strategy. A: The positions of the primers ( arrows ) and probe on the wild-type and mutant CLN3 gene are shown. The fluorophore on the probe is indicated with an asterisk ; not drawn to scale. B: The sequence of the probe is shown in between the complementary sequences of the wild-type and deletion alleles, with vertical lines connecting the base-paired residues. The –F on the 3′ end of the probe indicates the 6-FAM fluorophore and shows its position with respect to the G residues on the opposite strand. The probe is fully base-paired with the mutant sequence, but has three unmatched nucleotides at the 5′ end when annealed to a wild-type amplicon. The G residues that contribute to the quenching of the fluorescent signal are underlined in the normal and mutant sequences.

    Article Snippet: A single probe with only a single fluorescent label is less expensive than two dual-labeled probes used for 5′ nuclease assays, and also less expensive than the two labeled probes used for fluorescence resonance energy transfer assays., In contrast to Crockett and Wittwer we used 6-FAM (6-carboxyfluorescein) instead of fluorescein (fluorescein isothiocyanate) because the excitation and emission wavelengths are very similar, the 6-FAM fluorescence is quenched by G residues, and 6-FAM-labeled oligos are less expensive because they do not require additional purification before use (Integrated DNA Technologies, personal communication).

    Techniques: Mutagenesis, Sequencing, Amplification