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FluorMango, an RNA-based fluorogenic biosensor for the direct and specific detection of fluoride

Husser, Claire, Vuilleumier, Stéphane, Ryckelynck, Michael·2023·Small
DOI 10.1002/smll.202205232
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Abstract

Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)‐based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride‐specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light‐up RNA Mango‐III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh‐throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.

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Figure 1 Selection of an RNA-based fluoride biosensor from a randomized library using µ IVC-Seq. A) Secondary structure of the starting library. A crcB-derived RNA aptamer (black) was fused to the Mango-III aptamer (orange) by two stretches of four randomized nucleotides (red Ns), some of which were expected to behave as communication modules (CM). Base paires are represented according to Leontis-Westhof classification. [47] B) Impact of fluoride on the fluorescence of Mango-III and its derivatives. Purified RNAs were incubated with (white bars) or without (gray bars) 1 mm sodium fluoride (NaF) prior to measuring the fluorescence of the RNA/TO1-biotin complex and normalizing it to that of Mango-III/TO1-biotin in the absence of fluoride. Response efficiency (dots) was calculated as described in the Experimental Section. Values are the mean of three independent experiments, and error bars correspond to ± 1 standard deviation. C) µ IVC-seq screening strategy. The different libraries were screened in a four-step process as follows: C-i) genes diluted into a PCR mixture were individualized in water-in-oil droplets prior to PCR amplification. C-ii) An in vitro transcription medium (IVT) containing the fluorogen (TO1-biotin) and supplemented or not with NaF was added to each PCR-amplified droplet by droplet fusion. C-iii) Upon gene transcription, droplets of interest were sorted according to their fluorescence. C-iv) Last, the sequences recovered after each round of screening were determined by next generation sequencing (NGS) and bioinformatics. D) Functional validation of the rounds of screening. Libraries were in vitro transcribed in the presence of TO1-biotin and in the presence or not of NaF, and fluorescence was measured and normalized by that of Mango-III in the absence of fluoride. Response efficiency of each library (dots) was calculated. Values are the mean of three independent experiments, and error bars correspond to ± 1 standard deviation.
Figure 1 Selection of an RNA-based fluoride biosensor from a randomized library using µ IVC-Seq. A) Secondary structure of the starting library. A crcB-derived RNA aptamer (black) was fused to the Mango-III aptamer (orange) by two stretches of four randomized nucleotides (red Ns), some of which were expected to behave as communication modules (CM). Base paires are represented according to Leontis-Westhof classification. [47] B) Impact of fluoride on the fluorescence of Mango-III and its derivatives. Purified RNAs were incubated with (white bars) or without (gray bars) 1 mm sodium fluoride (NaF) prior to measuring the fluorescence of the RNA/TO1-biotin complex and normalizing it to that of Mango-III/TO1-biotin in the absence of fluoride. Response efficiency (dots) was calculated as described in the Experimental Section. Values are the mean of three independent experiments, and error bars correspond to ± 1 standard deviation. C) µ IVC-seq screening strategy. The different libraries were screened in a four-step process as follows: C-i) genes diluted into a PCR mixture were individualized in water-in-oil droplets prior to PCR amplification. C-ii) An in vitro transcription medium (IVT) containing the fluorogen (TO1-biotin) and supplemented or not with NaF was added to each PCR-amplified droplet by droplet fusion. C-iii) Upon gene transcription, droplets of interest were sorted according to their fluorescence. C-iv) Last, the sequences recovered after each round of screening were determined by next generation sequencing (NGS) and bioinformatics. D) Functional validation of the rounds of screening. Libraries were in vitro transcribed in the presence of TO1-biotin and in the presence or not of NaF, and fluorescence was measured and normalized by that of Mango-III in the absence of fluoride. Response efficiency of each library (dots) was calculated. Values are the mean of three independent experiments, and error bars correspond to ± 1 standard deviation.CC-BY · From the PDF
Image 2
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Figure 2 Identification and functional characterization of fluoride biosensors. A) Response of purified RNAs to fluoride. The most promising RNAs identified by bioinformatic analysis were produced by in vitro transcription and purified. 400 nm of RNA was then incubated with 700 nm TO1-biotin in the presence (white bars) or absence (gray bars) of 1 mm sodium fluoride (NaF). After 60 min, green fluorescence of the RNA/TO1-biotin complex was measured and normalized to that of Mango-III/TO1-biotin in the absence of fluoride. Response efficiency was calculated as described in the Experimental Section. Values are the mean of three independent experiments, and error bars correspond to ± 1 standard deviation. Sequences of the communication modules of tested variants are provided in Table S1, Supporting Information. B) Determination of the apparent dissociation constant ( K D) of three RNA candidates. Purified RNAs were incubated with a range of NaF concentrations, and fluorescence of the RNA/TO1-biotin complex was measured. The K D value was calculated by fitting the data to a Hill equation when possible. Non-measurable (n.m.) values are also indicated. C) Secondary structure of the fluoride RNA-based biosensor FluorMango (CM8). The selected communication module (CM) sequence is shown in red, the reporter module Mango-III in orange, and the sensor module crcB aptamer in black. Positions mutated to improve the transcription rate that preserves the formation of the pseudoknot are shown in blue. D) Fluoride detection by FluorMango. Limit of detection (LOD), limit of quantification (LOQ), and linear range of fluoride detection by FluorMango were determined as described elsewhere. [36] 400 nm of RNA was incubated in the presence of a range of NaF concentrations prior to measuring fluorescence of RNA/TO1-biotin complex. Values are the mean of seven (no NaF) or three (other NaF concentrations) independent experiments, and error bars correspond to ± 1 standard deviation. LOD was derived from three times the standard deviation, and LOQ from ten times the standard deviation. E) Specificity of FluorMango. 400 nm of purified RNAs was incubated with 1 mm of the specified halide, and fluorescence of the FluorMango/TO1-biotin complex was measured. Values are the mean of three independent experiments, and error bars correspond to ± 1 standard deviation.
Figure 2 Identification and functional characterization of fluoride biosensors. A) Response of purified RNAs to fluoride. The most promising RNAs identified by bioinformatic analysis were produced by in vitro transcription and purified. 400 nm of RNA was then incubated with 700 nm TO1-biotin in the presence (white bars) or absence (gray bars) of 1 mm sodium fluoride (NaF). After 60 min, green fluorescence of the RNA/TO1-biotin complex was measured and normalized to that of Mango-III/TO1-biotin in the absence of fluoride. Response efficiency was calculated as described in the Experimental Section. Values are the mean of three independent experiments, and error bars correspond to ± 1 standard deviation. Sequences of the communication modules of tested variants are provided in Table S1, Supporting Information. B) Determination of the apparent dissociation constant ( K D) of three RNA candidates. Purified RNAs were incubated with a range of NaF concentrations, and fluorescence of the RNA/TO1-biotin complex was measured. The K D value was calculated by fitting the data to a Hill equation when possible. Non-measurable (n.m.) values are also indicated. C) Secondary structure of the fluoride RNA-based biosensor FluorMango (CM8). The selected communication module (CM) sequence is shown in red, the reporter module Mango-III in orange, and the sensor module crcB aptamer in black. Positions mutated to improve the transcription rate that preserves the formation of the pseudoknot are shown in blue. D) Fluoride detection by FluorMango. Limit of detection (LOD), limit of quantification (LOQ), and linear range of fluoride detection by FluorMango were determined as described elsewhere. [36] 400 nm of RNA was incubated in the presence of a range of NaF concentrations prior to measuring fluorescence of RNA/TO1-biotin complex. Values are the mean of seven (no NaF) or three (other NaF concentrations) independent experiments, and error bars correspond to ± 1 standard deviation. LOD was derived from three times the standard deviation, and LOQ from ten times the standard deviation. E) Specificity of FluorMango. 400 nm of purified RNAs was incubated with 1 mm of the specified halide, and fluorescence of the FluorMango/TO1-biotin complex was measured. Values are the mean of three independent experiments, and error bars correspond to ± 1 standard deviation.CC-BY · From the PDF
Image 4
CC-BY · From the PDF
Image 5
CC-BY · From the PDF
Figure 4 Screening of bacteria for defluorinase activity. A) Microfluidic screening strategy. A mixed bacterial population (1 FAcD for 99 DhlA producing strains) was screened in a three-step process as follows: A-i) bacteria were diluted into a refolding buffer supplemented with 1 mg mL -1 sodium fluoroacetate, purified RNA FluorMango, TO1-biotin, and proteinase K individualized in water-in-oil droplets. A-ii) The emulsion was incubated off-chip at 30 ° C prior to A-iii) sorting droplets according to their fluorescence. B) Fluorescence profile of droplets analyzed during the screening. Green fluorescence of RNA/TO1-biotin complex and Cy5 red fluorescence (droplet tracer) were measured, and droplets containing elevated green fluorescence (so high fluoride concentration) were sorted (red box). C) Validation of the round of screening. Plasmids were extracted from sorted and unsorted bacteria and used to transform E. coli . The same number of cells was incubated in PBS supplemented with refolding buffer in the presence of FluorMango, TO1biotin, proteinase K, and 1 mg mL -1 fluoroacetate. Fluoride production was monitored in real time for 12 h at 28 ° C in a microtiter plate. The fluorescence appearance rate of the RNA/TO1-biotin complex was normalized to that of a control condition with a strain transformed by pure FAcD-coding plasmid.
Figure 4 Screening of bacteria for defluorinase activity. A) Microfluidic screening strategy. A mixed bacterial population (1 FAcD for 99 DhlA producing strains) was screened in a three-step process as follows: A-i) bacteria were diluted into a refolding buffer supplemented with 1 mg mL -1 sodium fluoroacetate, purified RNA FluorMango, TO1-biotin, and proteinase K individualized in water-in-oil droplets. A-ii) The emulsion was incubated off-chip at 30 ° C prior to A-iii) sorting droplets according to their fluorescence. B) Fluorescence profile of droplets analyzed during the screening. Green fluorescence of RNA/TO1-biotin complex and Cy5 red fluorescence (droplet tracer) were measured, and droplets containing elevated green fluorescence (so high fluoride concentration) were sorted (red box). C) Validation of the round of screening. Plasmids were extracted from sorted and unsorted bacteria and used to transform E. coli . The same number of cells was incubated in PBS supplemented with refolding buffer in the presence of FluorMango, TO1biotin, proteinase K, and 1 mg mL -1 fluoroacetate. Fluoride production was monitored in real time for 12 h at 28 ° C in a microtiter plate. The fluorescence appearance rate of the RNA/TO1-biotin complex was normalized to that of a control condition with a strain transformed by pure FAcD-coding plasmid.CC-BY · From the PDF
Image 7
CC-BY · From the PDF

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Metadata

Authors
Husser, C., Vuilleumier, S., & Ryckelynck, M.
Affiliations
  • CNRS, Architecture et Réactivité de l'ARN Université de Strasbourg UPR 9002, 2 allée Konrad Roentgen Strasbourg 67000 France
  • CNRS, Génétique Moléculaire, Génomique, Microbiologie Université de Strasbourg UMR 7156, 4 allée Konrad Roentgen Strasbourg 67000 France
ORCID
Venue
Small
Publisher
Wiley
Published
2022-11-27
Volume
19
Issue
13
ISSN
  • 1613-6810
  • 1613-6829
DOI
10.1002/smll.202205232
Cited by
30
References
0
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • Agence Nationale de la Recherche (ANR‐17‐CE07‐0009)
  • Centre National de la Recherche Scientifique
  • Université de Strasbourg

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