Please use this identifier to cite or link to this item: https://hdl.handle.net/11147/7566
Title: Sensitive and specific detection of ligands using engineered riboswitches
Authors: Morse, Daniel P.
Nevins, Colin E.
Aggrey-Fynn, Joana Efua
Bravo, Rick J.
Pfaeffle, Herman O.I.
Laney, Jess E.
Keywords: Biosensors
Specificity
Sensitivity
Gene expression
Ligands
Riboswitches
Issue Date: Apr-2018
Publisher: Elsevier Ltd.
Source: Morse, D. P., Nevins, C. E., Aggrey-Fynn, J. E., Bravo, R. J., Pfaeffle, H.O.I., and Laney, J. E. (2018). Sensitive and specific detection of ligands using engineered riboswitches. Journal of Biotechnology, 272-273, 22-32. doi:10.1016/j.jbiotec.2018.03.002
Abstract: Riboswitches are RNA elements found in non-coding regions of messenger RNAs that regulate gene expression through a ligand-triggered conformational change. Riboswitches typically bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. In B. subtilis and other gram-positive bacteria, purine nucleotide synthesis is regulated by riboswitches that bind to guanine. We modified the xpt-pbuX guanine riboswitch for use in a fluorescence quenching assay that allowed us to specifically detect and quantify guanine in vitro. Using this assay, we reproducibly detected as little as 5 nM guanine. We then produced sensors for 2′-deoxyguanosine and cyclic diguanylate (c-diGMP) by appending the P1 stem of the guanine riboswitch to the ligand-binding domains of a 2′-deoxyguanosine riboswitch and a c-diGMP riboswitch. These hybrid sensors could detect 15 nM 2′-deoxyguanosine and 3 nM c-diGMP, respectively. Each sensor retained the ligand specificity of its corresponding natural riboswitch. In order to extend the utility of our approach, we developed a strategy for the in vitro selection of sensors with novel ligand specificity. Here we report a proof-of-principle experiment that demonstrated the feasibility of our selection strategy.
URI: https://doi.org/10.1016/j.jbiotec.2018.03.002
https://hdl.handle.net/11147/7566
ISSN: 0168-1656
0168-1656
1873-4863
Appears in Collections:Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
PubMed İndeksli Yayınlar Koleksiyonu / PubMed Indexed Publications Collection
Scopus İndeksli Yayınlar Koleksiyonu / Scopus Indexed Publications Collection
WoS İndeksli Yayınlar Koleksiyonu / WoS Indexed Publications Collection

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