Riboswitch-based fluoride sensing in cell-free extract
Inventors
LUCKS, Julius B. • Thavarajah, Walter M. • Silverman, Adam D. • Jewett, Michael C.
Assignees
Publication Number
US-12325884-B2
Publication Date
2025-06-10
Expiration Date
2040-03-03
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Disclosed are methods and systems for detecting fluoride as a target molecule in a test sample which utilize a cell-protein synthesis (CFPS) reaction and an engineered fluoride-sensing riboswitch. The components used in the disclosed methods and systems may be dried or lyophilized and may be present or immobilized on a paper substrate.
Core Innovation
The invention provides a method and system for detecting fluoride as a target molecule in a test sample using a cell-free protein synthesis (CFPS) reaction and an engineered fluoride-sensing riboswitch. The method utilizes a genetic construct where the presence of fluoride in a sample activates the riboswitch, thereby enabling the transcription and translation of a reporter gene that produces a detectable output, such as colorimetric or fluorescent signal. Importantly, all necessary components can be dried or lyophilized and preserved for long-term storage, and can be incorporated into paper substrates for portability and ease of use.
The patent addresses the pressing problem of detecting fluoride in environmental, industrial, or biological samples, especially in resource-limited settings where traditional analytical methods are expensive, require complex equipment, or are not field-deployable. The disclosed system leverages the modularity and tunability of CFPS platforms, coupled with a fluoride-specific riboswitch that provides a genetically encoded regulatory element for sensing fluoride and generating a rapid, visible output.
Distinct from whole-cell biosensors or systems dependent on transcription factors, the disclosed approach relies solely on a cis-acting RNA riboswitch responsive to fluoride, thereby simplifying optimization and removing concerns related to biocontainment or cytotoxicity. The system's output can be visual, electronic, or optical, and its preservation through lyophilization ensures stability over time, enabling real-time, on-demand detection of fluoride at concentrations relevant to environmental safety standards.
Claims Coverage
The patent contains several independent claims encompassing methods, devices, kits, and platforms related to the detection of fluoride using cell-free protein synthesis systems and riboswitch-regulated reporter expression.
Method for detecting fluoride in a sample with a cell-free protein synthesis reaction and fluoride-sensing riboswitch
A method comprising obtaining a sample (which may or may not contain fluoride), adding it to a cell-free protein synthesis (CFPS) reaction comprising a fluoride-sensing riboswitch, and generating a detectable output within less than about 300 minutes if fluoride is present at a concentration of about 0.05 mM or higher. Key features: - Use of CFPS reaction incorporating a fluoride-sensing riboswitch. - Detection of fluoride at or above 0.05 mM in a sample. - Detectable output produced in less than about 300 minutes.
Expression cassette incorporating fluoride-sensing riboswitch and reporter for CFPS detection
An expression cassette for CFPS reactions comprising: - A promoter selected from a synthetic constitutive E. coli Promoter BBa_J23119_Spe1 or a T7 promoter. - A fluoride-sensing riboswitch comprising a Bacillus cereus fluoride riboswitch sequence. - A transcription terminator comprising the T1/TE double transcriptional terminator. - An encoded reporter: either a coding sequence for catechol 2,3-dioxygenase, a coding sequence for GFP or its derivatives, with a ribosome binding sequence, or an RNA aptamer.
Device or kit with preserved CFPS components for fluoride detection in liquid samples
A device or kit comprising preserved cell-free protein synthesis reaction components that include a fluoride-sensing riboswitch, configured to generate a detectable output within less than about 300 minutes if fluoride is present in a sample at a concentration of about 0.05 mM or higher. The preserved components may be freeze-dried and supported on a substrate, optionally a paper substrate, and may include components for reading the detectable output.
Platform for fluoride detection in environmental or biological samples using engineered expression cassette
A platform comprising components for performing a CFPS reaction and an expression cassette consisting of: - A promoter (synthetic constitutive E. coli Promoter BBa_J23119_Spe1 or T7 promoter). - Bacillus cereus fluoride riboswitch sequence. - T1/TE double transcriptional terminator. - Reporter gene (catechol 2,3-dioxygenase, GFP or derivative with a ribosome binding sequence, or an RNA aptamer). The platform enables detection of fluoride in environmental or biological samples.
In summary, the independent claims cover core inventions around methods and compositions for detecting fluoride using a fluoride-sensing riboswitch in a CFPS platform, including modular expression cassettes, preserved and field-deployable devices or kits, and platforms enabling the detection of fluoride with a quick, tunable, and visually detectable output.
Stated Advantages
Faster, more portable, and less expensive than existing chemical, electrochemical, or analytical processes for detecting environmental contaminants or analytes.
Enables rapid visible detection of target molecules with colorimetric measurement, allowing for straightforward readout.
Requires little-to-no concern for biocontainment, regulations on transgenic organisms, ligand permeability, degradation inside cells, or cytotoxicity, compared to whole-cell biosensors.
Simplifies optimization since it consists of one regulatory component (riboswitch), not multiple components such as transcription factors and operator sites.
Capable of sensing targets, such as fluoride, that lack well-characterized transcription factors.
Documented Applications
On-demand biosensing of molecules using colorimetric reporters.
Lyophilization and reconstitution of these biosensors and/or immobilization on a fibrous substrate.
Real-time detection of toxins and/or contaminants in biological or environmental samples.
Point-of-care diagnostics in biological samples.
Interested in licensing this patent?