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Yi Xiao - One of the best experts on this subject based on the ideXlab platform.

  • sensitive detection of small molecule targets using Cooperative Binding split aptamers and enzyme assisted target recycling
    Analytical Chemistry, 2018
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Obtin Alkhamis, Yi Xiao
    Abstract:

    Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on Cooperative Binding split aptamers (CBSAs). CBSAs contain two target-Binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two Binding domains, enabling EATR signal amplification through exonuclease III’s apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine....

  • Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling
    2018
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Obtin Alkhamis, Yi Xiao
    Abstract:

    Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on Cooperative Binding split aptamers (CBSAs). CBSAs contain two target-Binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two Binding domains, enabling EATR signal amplification through exonuclease III’s apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine. We further developed an instrument-free colorimetric assay employing EATR-mediated aggregation of CBSA-modified gold nanoparticles for the visual detection of low-micromolar concentrations of cocaine. On the basis of the generalizability of CBSA engineering and the robust performance of EATR in complex samples, we believe that such assays should prove valuable for detecting small-molecule targets in diverse fields

  • a Cooperative Binding split aptamer assay for rapid specific and ultra sensitive fluorescence detection of cocaine in saliva
    Chemical Science, 2017
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Xinhui Lou, Yi Xiao
    Abstract:

    Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-Binding affinity through Cooperative Binding. We have generated a split cocaine-Binding aptamer that incorporates two Binding domains, such that target Binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting Cooperative-Binding split aptamer (CBSA) exhibits higher target Binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-Binding affinity of our CBSA can be affected by the cooperativity of its Binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

Juan Canoura - One of the best experts on this subject based on the ideXlab platform.

  • sensitive detection of small molecule targets using Cooperative Binding split aptamers and enzyme assisted target recycling
    Analytical Chemistry, 2018
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Obtin Alkhamis, Yi Xiao
    Abstract:

    Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on Cooperative Binding split aptamers (CBSAs). CBSAs contain two target-Binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two Binding domains, enabling EATR signal amplification through exonuclease III’s apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine....

  • Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling
    2018
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Obtin Alkhamis, Yi Xiao
    Abstract:

    Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on Cooperative Binding split aptamers (CBSAs). CBSAs contain two target-Binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two Binding domains, enabling EATR signal amplification through exonuclease III’s apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine. We further developed an instrument-free colorimetric assay employing EATR-mediated aggregation of CBSA-modified gold nanoparticles for the visual detection of low-micromolar concentrations of cocaine. On the basis of the generalizability of CBSA engineering and the robust performance of EATR in complex samples, we believe that such assays should prove valuable for detecting small-molecule targets in diverse fields

  • a Cooperative Binding split aptamer assay for rapid specific and ultra sensitive fluorescence detection of cocaine in saliva
    Chemical Science, 2017
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Xinhui Lou, Yi Xiao
    Abstract:

    Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-Binding affinity through Cooperative Binding. We have generated a split cocaine-Binding aptamer that incorporates two Binding domains, such that target Binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting Cooperative-Binding split aptamer (CBSA) exhibits higher target Binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-Binding affinity of our CBSA can be affected by the cooperativity of its Binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

Bhargav Guntupalli - One of the best experts on this subject based on the ideXlab platform.

  • sensitive detection of small molecule targets using Cooperative Binding split aptamers and enzyme assisted target recycling
    Analytical Chemistry, 2018
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Obtin Alkhamis, Yi Xiao
    Abstract:

    Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on Cooperative Binding split aptamers (CBSAs). CBSAs contain two target-Binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two Binding domains, enabling EATR signal amplification through exonuclease III’s apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine....

  • Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling
    2018
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Obtin Alkhamis, Yi Xiao
    Abstract:

    Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on Cooperative Binding split aptamers (CBSAs). CBSAs contain two target-Binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two Binding domains, enabling EATR signal amplification through exonuclease III’s apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine. We further developed an instrument-free colorimetric assay employing EATR-mediated aggregation of CBSA-modified gold nanoparticles for the visual detection of low-micromolar concentrations of cocaine. On the basis of the generalizability of CBSA engineering and the robust performance of EATR in complex samples, we believe that such assays should prove valuable for detecting small-molecule targets in diverse fields

  • a Cooperative Binding split aptamer assay for rapid specific and ultra sensitive fluorescence detection of cocaine in saliva
    Chemical Science, 2017
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Xinhui Lou, Yi Xiao
    Abstract:

    Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-Binding affinity through Cooperative Binding. We have generated a split cocaine-Binding aptamer that incorporates two Binding domains, such that target Binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting Cooperative-Binding split aptamer (CBSA) exhibits higher target Binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-Binding affinity of our CBSA can be affected by the cooperativity of its Binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

Obtin Alkhamis - One of the best experts on this subject based on the ideXlab platform.

  • sensitive detection of small molecule targets using Cooperative Binding split aptamers and enzyme assisted target recycling
    Analytical Chemistry, 2018
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Obtin Alkhamis, Yi Xiao
    Abstract:

    Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on Cooperative Binding split aptamers (CBSAs). CBSAs contain two target-Binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two Binding domains, enabling EATR signal amplification through exonuclease III’s apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine....

  • Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling
    2018
    Co-Authors: Juan Canoura, Bhargav Guntupalli, Obtin Alkhamis, Yi Xiao
    Abstract:

    Signal amplification via enzyme-assisted target recycling (EATR) offers a powerful means for improving the sensitivity of DNA detection assays, but it has proven challenging to employ EATR with aptamer-based assays for small-molecule detection due to insensitive target response of aptamers. Here, we describe a general approach for the development of rapid and sensitive EATR-amplified small-molecule sensors based on Cooperative Binding split aptamers (CBSAs). CBSAs contain two target-Binding domains and exhibit enhanced target response compared with single-domain split aptamers. We introduced a duplexed C3 spacer abasic site between the two Binding domains, enabling EATR signal amplification through exonuclease III’s apurinic endonuclease activity. As a demonstration, we engineered a CBSA-based EATR-amplified fluorescence assay to detect dehydroisoandrosterone-3-sulfate. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine. We further developed an instrument-free colorimetric assay employing EATR-mediated aggregation of CBSA-modified gold nanoparticles for the visual detection of low-micromolar concentrations of cocaine. On the basis of the generalizability of CBSA engineering and the robust performance of EATR in complex samples, we believe that such assays should prove valuable for detecting small-molecule targets in diverse fields

Tahir H. Tahirov - One of the best experts on this subject based on the ideXlab platform.

  • Structural basis of Ets1 Cooperative Binding to palindromic sequences on stromelysin-1 promoter DNA
    Cell Cycle, 2010
    Co-Authors: Nigar D. Babayeva, Koshiki Mino, Phillip J. Wilder, Masaaki Shiina, Michelle Desler, Kazuhiro Ogata, A Angie Rizzino, Tahir H. Tahirov
    Abstract:

    Ets1 is a member of the Ets family of transcription factors. Ets1 is autoinhibited and its activation requires heterodimerization with a partner protein or DNA-mediated homodimerization for Cooperative DNA Binding. In the latter case, Ets1 molecules bind to palindromic sequences in which two Ets-Binding sites (EBS) are separated by four base pairs, for example in the promoters of stromelysin-1 and p53. Interestingly, counteraction of autoinhibition requires the autoinhibitory region encoded by exon VII of the gene. The structural basis for the requirement of autoinhibitory sequences for Ets1 Binding to palindromic EBS still remains unresolved. Here we report the crystal structure of two Ets1 molecules bound to an EBS palindrome of the stromelysin-1 promoter DNA, providing a plausible explanation for the requirement of exon VII-encoded sequences for Ets1 Cooperative DNA Binding. The proposed mechanism was verified both in vitro by surface plasmon resonance and in vivo by transcription-based assays.