Fluoride-responsive riboswitches, fluoride transporters, and methods of use

{
  "publication_number": "US9580713B2",
  "country": "US",
  "kind": "B2",
  "title": "Fluoride-responsive riboswitches, fluoride transporters, and methods of use",
  "abstract": "Disclosed are compounds, compositions, and methods relating to fluoride aptamers, fluoride-responsive riboswitches, fluoride-regulated expression constructs, fluoride transporters, nucleic acids encoding fluoride transporters, expression constructs encoding fluoride transporters, and cells containing or including any combination of these.",
  "claims": [
    "1. A fluoride aptamer nucleic acid molecule comprising (a) a fluoride aptamer, wherein the fluoride aptamer comprises a nucleic acid structure that selectively binds a fluoride anion or fluoride with a counterion, and (b) (i) one or more nucleic acid sequences that are heterologous to the fluoride aptamer and linked to the nucleic acid molecule, (ii) a heterologous component that is conjugated to the nucleic acid molecule, or (iii) both.",
    "2. The fluoride aptamer nucleic acid molecule of claim 1, wherein the fluoride aptamer is derived from a naturally-occurring fluoride-responsive riboswitch.",
    "3. The fluoride aptamer nucleic acid molecule of claim 1 further comprising a sequestration tag, wherein the sequestration tag can be used to separate the fluoride aptamer nucleic acid molecule from a mixture.",
    "4. The fluoride aptamer nucleic acid molecule of claim 1 further comprising an expression platform domain operably linked to the fluoride aptamer, wherein the fluoride aptamer and the expression platform domain constitute a fluoride-responsive riboswitch, wherein the fluoride aptamer constitutes the aptamer domain of the fluoride-responsive riboswitch.",
    "5. The fluoride aptamer nucleic acid molecule of claim 4, wherein the expression platform domain is heterologous to the fluoride aptamer.",
    "6. The fluoride aptamer nucleic acid molecule of claim 1, wherein the fluoride aptamer is operably linked to a signal-generating component, wherein the signal-generating component generates a signal when the fluoride aptamer is bound by fluoride.",
    "7. The fluoride aptamer nucleic acid molecule of claim 6, wherein the fluoride aptamer and the signal-generating component are heterologous.",
    "8. A fluoride-regulated expression construct comprising a nucleic acid molecule encoding the fluoride aptamer nucleic acid molecule of claim 4, wherein the fluoride-responsive riboswitch is operably linked to a coding region, wherein expression of the coding region is regulated by the riboswitch.",
    "9. The fluoride-regulated expression construct of claim 8, wherein the riboswitch and coding region are heterologous.",
    "10. The construct of claim 8, wherein expression of the coding region induces or causes death of the cell in which it is expressed.",
    "11. A cell comprising the fluoride-regulated expression construct of claim 8, wherein the cell is a recombinant or isolated cell.",
    "12. The cell of claim 11, wherein expression of the coding region is regulated by fluoride.",
    "13. The cell of claim 11, wherein the coding region encodes an expression product, wherein production of the expression product by the cell is regulated by fluoride.",
    "14. A cell comprising the fluoride aptamer nucleic acid molecule of claim 1, wherein the cell is a recombinant or isolated cell.",
    "15. A method of sensing fluoride, the method comprising bringing into contact a sample or an environment to be assessed and the cell of claim 11, wherein expression of the coding region produces a signal, wherein the signal indicates the presence of fluoride in the sample or environment.",
    "16. A method of sensing fluoride, the method comprising bringing into contact a sample or an environment to be assessed and the cell of claim 14, wherein the fluoride aptamer is operably linked to a signal-generating component, wherein the signal-generating component generates a signal when the fluoride aptamer is bound by fluoride, wherein the signal indicates the presence of fluoride in the sample or environment.",
    "17. A method of sensing fluoride, the method comprising bringing into contact a sample or an environment to be assessed and the fluoride aptamer nucleic acid molecule of claim 6, wherein the signal indicates the presence of fluoride in the sample or environment.",
    "18. A method of separating fluoride from a mixture, the method comprising bringing into contact the mixture and the fluoride aptamer nucleic acid molecule of claim 3, and separating the fluoride aptamer nucleic acid molecule from the mixture via the sequestration tag, thereby separating fluoride from the mixture.",
    "19. A method of separating fluoride from a mixture, the method comprising bringing into contact the mixture and a solid support, wherein the solid support comprises the fluoride aptamer nucleic acid molecule of claim 1, and separating the mixture from the solid support, thereby separating fluoride from the mixture.",
    "20. A method of separating fluoride from a mixture, the method comprising bringing into contact the mixture and the cell of claim 14, wherein the fluoride aptamer nucleic acid molecule is sequestered in an inclusion body, thereby separating fluoride from the mixture.",
    "21. The method of claim 20, wherein the fluoride aptamer nucleic acid molecule is sequestered in an inclusion body via the sequestration tag.",
    "22. The method of claim 20 further comprising separating the cell from the mixture.",
    "23. The fluoride aptamer nucleic acid molecule of claim 1, wherein the fluoride aptamer comprises a crcB motif, or an eriC F motif, or both a crcB motif and an eriC F motif.",
    "24. The fluoride aptamer nucleic acid molecule of claim 1, wherein the fluoride aptamer is selected from the group consisting of SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:350, SEQ ID NO:351, SEQ ID NO:352, and SEQ ID NO:353.",
    "25. The fluoride aptamer nucleic acid molecule of claim 1, wherein the fluoride aptamer is SEQ ID NO:349.",
    "26. The fluoride aptamer nucleic acid molecule of claim 1, wherein the fluoride aptamer is SEQ ID NO:348.",
    "27. The fluoride aptamer nucleic acid molecule of claim 1, wherein the fluoride aptamer binds fluoride anions with a dissociation constant of between 50 μM and 60 μM, inclusive.",
    "28. The fluoride aptamer nucleic acid molecule of claim 1, wherein the fluoride aptamer does not bind to chloride, bromide, and iodine anions.",
    "29. The fluoride aptamer nucleic acid molecule of claim 1, wherein the heterologous nucleic acid sequence comprises a heterologous open reading frame.",
    "30. The fluoride aptamer nucleic acid molecule of claim 29, wherein the heterologous open reading frame is operable linked to the fluoride aptamer."
  ],
  "description_excerpt": "The disclosed invention is generally in the field of riboswitches, ion transporters, and regulation of gene expression.\n\nFluoride can be both beneficial and toxic. For years fluoride has been used in oral hygiene for its beneficial effects in products such as toothpaste and mouthwash. On the other hand, a build up of fluoride in a cell can be toxic. Therefore, appropriate amounts of fluoride and careful regulation of fluoride in cells is essential.\n\nA key biochemical component of a bacterial cell's fluoride surveillance and response system is the new-found fluoride-responsive riboswitch class based on the crcB motif (Weinberg et al. 2010. Comparative genomics reveals 104 candidate structured RNAs from bacteria, archaeal, and their metagenomes. Genome Biol 11:R31). The general architectures and functional mechanisms for fluoride riboswitches are similar to many other known riboswitch classes. Members of each riboswitch class carry at least one ligand-binding “aptamer” domain and one adjoining “expression platform” domain that together control expression of the downstream gene(s) by one of several known mechanisms (Wickiser et al. 2005. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol Cell 18:49-60; Barrick and Breaker, 2007. The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol 8:R239). The most common mechanisms used by bacteria include transcription termination and translation initiation, although some bacterial riboswitch classes exploit other mechanisms such as allosteric ribozyme-mediated splicing (Lee et al. 2010.",
  "cpc": [
    "C12N 15/115",
    "A23L 33/13",
    "A61K 31/166",
    "A61K 31/381",
    "A61K 31/4436",
    "A61K 31/55",
    "A61K 45/06",
    "B01D 15/38",
    "C11D 3/48",
    "C12N 2310/16",
    "C12N 2320/12",
    "C12N 2320/30",
    "C12Q 1/18"
  ],
  "ipc": [
    "A23L 1/30",
    "A61K 31/166",
    "A61K 31/381",
    "A61K 31/4436",
    "A61K 31/55",
    "A61K 45/06",
    "B01D 15/38",
    "C11D 3/48",
    "C12N 15/115",
    "C12Q 1/18"
  ],
  "assignees": [
    "Yale University"
  ],
  "inventors": [
    "Ronald Breaker",
    "Jenny Baker",
    "Narasimhan Sudarsan",
    "Zasha Weinberg",
    "Adam Roth",
    "Tyler Ames",
    "James Nelson"
  ],
  "filing_date": "2012-09-17",
  "publication_date": "2017-02-28",
  "grant_date": "2017-02-28",
  "priority_date": "2011-09-17",
  "application_number": "US-201214344006-A",
  "family_id": "46964074",
  "cited_by_count": 9
}