rnaseout™ recombinant ribonuclease inhibitor Search Results


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  • 90
    Thermo Fisher recombinant ribonuclease inhibitor rnaseout
    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of <t>RNaseOUT,</t> 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.
    Recombinant Ribonuclease Inhibitor Rnaseout, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega rnaseout recombinant ribonuclease inhibitor
    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of <t>RNaseOUT,</t> 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.
    Rnaseout Recombinant Ribonuclease Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific rnaseout recombinant ribonuclease inhibitor
    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of <t>RNaseOUT,</t> 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.
    Rnaseout Recombinant Ribonuclease Inhibitor, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnaseout recombinant rnase inhibitor
    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of <t>RNaseOUT,</t> 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.
    Rnaseout Recombinant Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 612 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Promega recombinant rnase inhibitor rnaseout
    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of <t>RNaseOUT,</t> 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.
    Recombinant Rnase Inhibitor Rnaseout, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore rnaseout recombinant ribonuclease inhibitor
    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of <t>RNaseOUT,</t> 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.
    Rnaseout Recombinant Ribonuclease Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher rnase a inhibitor
    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after <t>RNase</t> A treatment. “IB” indicates immunoblotting with the indicated antibody.
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    Thermo Fisher dnase rnase free water
    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after <t>RNase</t> A treatment. “IB” indicates immunoblotting with the indicated antibody.
    Dnase Rnase Free Water, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher recombinant rnase inhibitor
    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after <t>RNase</t> A treatment. “IB” indicates immunoblotting with the indicated antibody.
    Recombinant Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ribonuclease inhibitor
    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after <t>RNase</t> A treatment. “IB” indicates immunoblotting with the indicated antibody.
    Ribonuclease Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnaseout
    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after <t>RNase</t> A treatment. “IB” indicates immunoblotting with the indicated antibody.
    Rnaseout, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8699 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher verso cdna kit
    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after <t>RNase</t> A treatment. “IB” indicates immunoblotting with the indicated antibody.
    Verso Cdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2661 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lysis buffer
    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after <t>RNase</t> A treatment. “IB” indicates immunoblotting with the indicated antibody.
    Lysis Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 35040 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Kinetics and sensitivity of purified RNA CHA circuit. ( a ) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. ( b ) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT 17 . Circuits were executed in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. ( c ) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Purification, Amplification, Concentration Assay, Hybridization, Fluorescence

    Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Generated, Purification, Incubation, Sequencing, Binding Assay, Synthesized, Amplification

    Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Polymerase Chain Reaction, Generated, Electrophoresis, Purification, Incubation

    Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Generated, Sequencing, Amplification, Incubation, Purification, Negative Control, Transduction

    Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Filtration

    Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Gel Purification, Amplification, Clone Assay, Plasmid Preparation, Incubation

    An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).

    Journal: Nucleic Acids Research

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    doi: 10.1093/nar/gku074

    Figure Lengend Snippet: An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).

    Article Snippet: In vitro transcription In all, 100 pg to 1000 ng of double-stranded DNA transcription templates was transcribed using 100 U of T7 RNA polymerase (NEB) in 50 µl reactions containing 40 mM Tris–HCl, pH 7.9, 30 mM MgCl2 , 10 mM DTT, 2 mM spermidine, 4 mM ribonucleotide (rNTP) mix and 20 U of the recombinant ribonuclease inhibitor RNaseOUT (Life Technologies).

    Techniques: Polymerase Chain Reaction, Generated, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Incubation, Fluorescence, Purification, Concentration Assay

    Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Virion-Incorporated HA-A3G Associates with Viral Genomic RNA (A) Viral genomic RNA, detected by RT-PCR, was detected in virions and virus-producing cells but not in lysates of uninfected cells. Genomic RNA was also detected in the IVAC derived from virions (fraction 7) and coimmunoprecipitated with HA-A3G from both virions and producer cell lysates. RT was performed using RNA derived from either whole lysates (L) or anti-HA immunoprecipitates (IP). Control reactions were performed in the absence of RT (–RT). Control PCRs were performed using proviral plasmid DNA, in the absence or presence of Taq, as indicated. (B) Viral genomic RNA, detected by RT-PCR, was assessed from size-fractionated virion lysates that lacked (HA) or contained HA-A3G. Amplicons generated probed across the TAR/Gag region or Pol/Vpu regions, as indicated. (C) Incorporation of HA-A3G into virions enhances the recruitment of NC into the IVAC. (D) HA-A3G from virus-producing cells is HMM and is converted to LMM form after RNase A treatment. “IB” indicates immunoblotting with the indicated antibody.

    Article Snippet: Unless otherwise indicated, 0.1 U of RNase A inhibitor (RNaseOUT; Invitrogen, http://www.invitrogen.com ) was added to virion pellets, which were then immediately lysed or flash-frozen on liquid nitrogen and stored at −80 °C until lysis.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Generated

    Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Intravirion A3G Enzymatic Activity Is Negatively Regulated by Binding to Genomic HIV RNA (A) HA-A3G was immunoprecipitated from IVAC fraction 7 (F7) of virion lysates ( Figure 3 A) or from a lower fraction, F17, generated by treatment of the virion lysates with RNase A ( Figure 3 B). Immunoprecipitates (IPs) were tested for enzymatic activity in an in vitro deoxycytidine deaminase assay with or without RNase A addition and contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot. The generation of a shorter cleavage product from the input ssDNA substrate reveals A3G deoxycytidine deaminase activity. Data shown are representative of multiple experiments. (B) Lysates of virions containing or lacking A3G were assessed in the deaminase assay, with or without RNase A treatment. (C) Lysates of virions containing increasing amounts of HA-A3G (as shown in the corresponding immunoblot) were assessed in the deaminase assay, with or without RNase A treatment. The asterisk marks bleed-through of marker loaded to the left of the samples. The triangles represent the increasing dose of A3G relative to provirus and correspond to the sample numbers presented in Figure 1 A. (A–C) All deaminase reactions were carried out in 50 mM Tris (pH 7.4) with (+) or without (−) RNase A, as indicated. (D) IPs of HMM or LMM HA-A3G from producer cell lysates were similarly assessed in the deaminase assay, with (+) or without (−) added RNase A. The IPs contained equivalent amounts of HA-A3G as shown in the corresponding immunoblot (IB).

    Article Snippet: Unless otherwise indicated, 0.1 U of RNase A inhibitor (RNaseOUT; Invitrogen, http://www.invitrogen.com ) was added to virion pellets, which were then immediately lysed or flash-frozen on liquid nitrogen and stored at −80 °C until lysis.

    Techniques: Activity Assay, Binding Assay, Immunoprecipitation, Generated, In Vitro, Marker

    Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Enzymatically Inactive Virion-Incorporated HA-A3G Is Activated by Viral RNase H (A) Recombinant RTs containing either a WT or mutant (E478Q) RNase H catalytic domain were assessed for RNase H activity in vitro in the absence or presence of the RNase H inhibitor Compound I (final concentration of 1, 10, or 100 μM). The RNA of an RNA–DNA hybrid remains intact unless RNase H digests the RNA into a smaller cleavage product that is distinguishable from the more complete cleavage product generated by RNase A. WT RNase H cannot digest ssDNA or DNA of an RNA–DNA hybrid, or RNA–RNA hybrids (data not shown). RNase H assays were performed in RNase H buffer (50 mM Tris [pH 8.0], 60 mM KCl) with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (B) Viruses bearing the RNase H E478Q mutation are compromised for in vitro RNase H activity. RNase H assays were performed in RNase H buffer with (+) or without (−) 5 mM MgCl 2 or RNase A, as indicated. (C) Virion lysates were subjected to endogenous reverse transcription (enRT) conditions with or without Compound I (final concentration of 0.1, 1, 10, or 100 μM), and A3G activity in these samples assessed in the in vitro deoxycytidine deaminase assay. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (D) Compound I does not inhibit the intrinsic deoxycytidine deaminase activity of A3G. HA-A3G from RNase A–treated virion lysates was assessed for in vitro deaminase activity in the presence of increasing doses of Compound I (0.1, 1, 10, and 100 μM). Deaminase assay was performed in RNase H buffer supplemented with RNase A only. (E) Virions containing WT RNase H or the E478Q mutation in the RNase H catalytic domain were subjected to the enRT reaction followed by assessment of A3G enzymatic activity. Deaminase assays were performed in RNase H buffer either supplemented (enRT:+) or not (enRT:−) with 4 mM MgCl 2 and 1 mM dNTPs. (F) WT and RNase H–compromised ΔVif virions containing WT or mutant RNase H displayed equivalent A3G activity when RNase A was added to the virion lysate. Deaminase assay was performed in RNase H buffer with (+) or without (−) RNase A, as indicated. All data are representative of multiple experiments.

    Article Snippet: Unless otherwise indicated, 0.1 U of RNase A inhibitor (RNaseOUT; Invitrogen, http://www.invitrogen.com ) was added to virion pellets, which were then immediately lysed or flash-frozen on liquid nitrogen and stored at −80 °C until lysis.

    Techniques: Recombinant, Mutagenesis, Activity Assay, In Vitro, Concentration Assay, Generated

    Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.

    Journal: PLoS Pathogens

    Article Title: Newly Synthesized APOBEC3G Is Incorporated into HIV Virions, Inhibited by HIV RNA, and Subsequently Activated by RNase H

    doi: 10.1371/journal.ppat.0030015

    Figure Lengend Snippet: Virion-Incorporated HA-A3G Resides in a Large RNase A–Sensitive Complex and Biochemically Fractionates with Viral RNP Proteins (A) Virions collected from cells expressing HIV-1ΔVif contain HA-A3G that predominantly fractionates in a large complex (fractions 6 to 8) as assessed by gel filtration. (B) The IVAC is sensitive to RNase A treatment which shifts HA-A3G into lower fractions (fractions 15 to 19). (C) Virion cores obtained in Figure 1 were subjected to further biochemical fractionation to generate viral RNPs. Shown are the viral RNPs from virions either lacking or containing A3G, as indicated, and containing viral RT, IN, and NC but not p24-CA, as detected by immunoblotting (IB). The triangles represent the increasing dose of A3G relative to provirus and correspond exactly to the sample numbers in Figure 1 A.

    Article Snippet: Unless otherwise indicated, 0.1 U of RNase A inhibitor (RNaseOUT; Invitrogen, http://www.invitrogen.com ) was added to virion pellets, which were then immediately lysed or flash-frozen on liquid nitrogen and stored at −80 °C until lysis.

    Techniques: Expressing, Filtration, Fractionation