16
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: not found
      • Article: not found

      Facile Expansion of the Variety of Orthogonal Ligand/Aptamer Pairs for Artificial Riboswitches

      Read this article at

      ScienceOpenPublisherPubMed
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="first" dir="auto" id="d3124748e77">An RNA aptamer that induces suitable conformational changes upon binding to a user-defined ligand allows us to artificially construct a riboswitch, a ligand-dependent and cis-acting gene regulatory RNA. Although such an aptamer can be obtained through in vitro selection, it is still challenging to rationally expand the variety of orthogonal ligand/aptamer (ligand/riboswitch) pairs. To achieve this in a facile, selection-free way, we herein focused on a specific type of ligand, 6-nt nanosized DNA (nDNA) and its aptamer that was previously selected to construct a eukaryotic artificial riboswitch. Specifically, we merely mutated one or more possible Watson-Crick base pairs in the nDNA/aptamer (nDNA/riboswitch) interactions into another base pair or pairs. Using two sets that each had 16 comprehensive mutations, we obtained three groups of several orthogonal nDNA/riboswitch pairs. These pairs could be used to create complex gene circuits, including multiple simultaneous and/or multistep cascading regulations in synthetic biology. </p>

          Related collections

          Most cited references38

          • Record: found
          • Abstract: found
          • Article: not found

          Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase

          High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            In vitro selection of RNA molecules that bind specific ligands.

            Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

              Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.
                Bookmark

                Author and article information

                Contributors
                (View ORCID Profile)
                Journal
                ACS Synthetic Biology
                ACS Synth. Biol.
                American Chemical Society (ACS)
                2161-5063
                2161-5063
                January 20 2023
                December 25 2022
                January 20 2023
                : 12
                : 1
                : 35-42
                Affiliations
                [1 ]Proteo-Science Center, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan
                Article
                10.1021/acssynbio.2c00475
                36566430
                afa235be-b527-4188-b66a-45b2b1c39008
                © 2023

                https://doi.org/10.15223/policy-029

                https://doi.org/10.15223/policy-037

                https://doi.org/10.15223/policy-045

                History

                Comments

                Comment on this article

                scite_
                0
                0
                0
                0
                Smart Citations
                0
                0
                0
                0
                Citing PublicationsSupportingMentioningContrasting
                View Citations

                See how this article has been cited at scite.ai

                scite shows how a scientific paper has been cited by providing the context of the citation, a classification describing whether it supports, mentions, or contrasts the cited claim, and a label indicating in which section the citation was made.

                Similar content1,729

                Cited by3

                Most referenced authors788