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2024
Kehrli Janine, Husser Claire, Ryckelynck Michael
Fluorogenic RNA-Based Biosensors of Small Molecules: Current Developments, Uses, and Perspectives Journal Article
In: Biosensors (Basel), vol. 14, no. 8, 2024, ISSN: 2079-6374.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid39194605,
title = {Fluorogenic RNA-Based Biosensors of Small Molecules: Current Developments, Uses, and Perspectives},
author = {Janine Kehrli and Claire Husser and Michael Ryckelynck},
doi = {10.3390/bios14080376},
issn = {2079-6374},
year = {2024},
date = {2024-08-01},
urldate = {2024-08-01},
journal = {Biosensors (Basel)},
volume = {14},
number = {8},
abstract = {Small molecules are highly relevant targets for detection and quantification. They are also used to diagnose and monitor the progression of disease and infectious processes and track the presence of contaminants. Fluorogenic RNA-based biosensors (FRBs) represent an appealing solution to the problem of detecting these targets. They combine the portability of molecular systems with the sensitivity and multiplexing capacity of fluorescence, as well as the exquisite ligand selectivity of RNA aptamers. In this review, we first present the different sensing and reporting aptamer modules currently available to design an FRB, together with the main methodologies used to discover modules with new specificities. We next introduce and discuss how both modules can be functionally connected prior to exploring the main applications for which FRB have been used. Finally, we conclude by discussing how using alternative nucleotide chemistries may improve FRB properties and further widen their application scope.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Small molecules are highly relevant targets for detection and quantification. They are also used to diagnose and monitor the progression of disease and infectious processes and track the presence of contaminants. Fluorogenic RNA-based biosensors (FRBs) represent an appealing solution to the problem of detecting these targets. They combine the portability of molecular systems with the sensitivity and multiplexing capacity of fluorescence, as well as the exquisite ligand selectivity of RNA aptamers. In this review, we first present the different sensing and reporting aptamer modules currently available to design an FRB, together with the main methodologies used to discover modules with new specificities. We next introduce and discuss how both modules can be functionally connected prior to exploring the main applications for which FRB have been used. Finally, we conclude by discussing how using alternative nucleotide chemistries may improve FRB properties and further widen their application scope.
Pitolli Martina, Cela Marta, Kapps Delphine, Chicher Johana, Despons Laurence, Frugier Magali
Comparative proteomics uncovers low asparagine content in Plasmodium tRip-KO proteins Journal Article
In: IUBMB Life, 2024, ISSN: 1521-6551.
Abstract | Links | BibTeX | Tags: PPSE, RYCKELYNCK, Unité ARN
@article{pmid38963319,
title = {Comparative proteomics uncovers low asparagine content in Plasmodium tRip-KO proteins},
author = {Martina Pitolli and Marta Cela and Delphine Kapps and Johana Chicher and Laurence Despons and Magali Frugier},
doi = {10.1002/iub.2891},
issn = {1521-6551},
year = {2024},
date = {2024-07-01},
urldate = {2024-07-01},
journal = {IUBMB Life},
abstract = {tRNAs are not only essential for decoding the genetic code, but their abundance also has a strong impact on the rate of protein production, folding, and on the stability of the translated messenger RNAs. Plasmodium expresses a unique surface protein called tRip, involved in the import of exogenous tRNAs into the parasite. Comparative proteomic analysis of the blood stage of wild-type and tRip-KO variant of P. berghei parasites revealed that downregulated proteins in the mutant parasite are distinguished by a bias in their asparagine content. Furthermore, the demonstration of the possibility of charging host tRNAs with Plasmodium aminoacyl-tRNA synthetases led us to propose that imported host tRNAs participate in parasite protein synthesis. These results also suggest a novel mechanism of translational control in which import of host tRNAs emerge as regulators of gene expression in the Plasmodium developmental cycle and pathogenesis, by enabling the synthesis of asparagine-rich regulatory proteins that efficiently and selectively control the parasite infectivity.},
keywords = {PPSE, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
tRNAs are not only essential for decoding the genetic code, but their abundance also has a strong impact on the rate of protein production, folding, and on the stability of the translated messenger RNAs. Plasmodium expresses a unique surface protein called tRip, involved in the import of exogenous tRNAs into the parasite. Comparative proteomic analysis of the blood stage of wild-type and tRip-KO variant of P. berghei parasites revealed that downregulated proteins in the mutant parasite are distinguished by a bias in their asparagine content. Furthermore, the demonstration of the possibility of charging host tRNAs with Plasmodium aminoacyl-tRNA synthetases led us to propose that imported host tRNAs participate in parasite protein synthesis. These results also suggest a novel mechanism of translational control in which import of host tRNAs emerge as regulators of gene expression in the Plasmodium developmental cycle and pathogenesis, by enabling the synthesis of asparagine-rich regulatory proteins that efficiently and selectively control the parasite infectivity.
Lim Shey-Li, Liu Jinhong, Dupouy Gilles, Singh Gaurav, Baudrey Stéphanie, Yang Lang, Zhong Jia Yi, Chabouté Marie-Edith, Lim Boon Leong
In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors Journal Article
In: Plant J, 2024, ISSN: 1365-313X.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid38761168,
title = {In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors},
author = {Shey-Li Lim and Jinhong Liu and Gilles Dupouy and Gaurav Singh and Stéphanie Baudrey and Lang Yang and Jia Yi Zhong and Marie-Edith Chabouté and Boon Leong Lim},
doi = {10.1111/tpj.16796},
issn = {1365-313X},
year = {2024},
date = {2024-05-01},
urldate = {2024-05-01},
journal = {Plant J},
abstract = {Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.
Nerantzaki Maria, Husser Claire, Ryckelynck Michael, Lutz Jean-François
Exchanging and Releasing Information in Synthetic Digital Polymers Using a Strand-Displacement Strategy Journal Article
In: J Am Chem Soc, 2024, ISSN: 1520-5126.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid38286022,
title = {Exchanging and Releasing Information in Synthetic Digital Polymers Using a Strand-Displacement Strategy},
author = {Maria Nerantzaki and Claire Husser and Michael Ryckelynck and Jean-François Lutz},
doi = {10.1021/jacs.3c13953},
issn = {1520-5126},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {J Am Chem Soc},
abstract = {Toehold-mediated strand displacement (TMSD) was tested as a tool to edit information in synthetic digital polymers. Uniform DNA-polymer biohybrid macromolecules were first synthesized by automated phosphoramidite chemistry and characterized by HPLC, mass spectrometry, and polyacrylamide gel electrophoresis (PAGE). These precursors were diblock structures containing a synthetic poly(phosphodiester) (PPDE) segment covalently attached to a single-stranded DNA sequence. Three types of biohybrids were prepared herein: a substrate containing an accessible toehold as well as input and output macromolecules. The substrate and the input macromolecules contained noncoded PPDE homopolymers, whereas the output macromolecule contained a digitally encoded segment. After hybridization of the substrate with the output, incubation in the presence of the input led to efficient TMSD and the release of the digital segment. TMSD can therefore be used to erase or rewrite information in self-assembled biohybrid superstructures. Furthermore, it was found in this work that the conjugation of DNA single strands to synthetic segments of chosen lengths greatly facilitates the characterization and PAGE visualization of the TMSD process.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Toehold-mediated strand displacement (TMSD) was tested as a tool to edit information in synthetic digital polymers. Uniform DNA-polymer biohybrid macromolecules were first synthesized by automated phosphoramidite chemistry and characterized by HPLC, mass spectrometry, and polyacrylamide gel electrophoresis (PAGE). These precursors were diblock structures containing a synthetic poly(phosphodiester) (PPDE) segment covalently attached to a single-stranded DNA sequence. Three types of biohybrids were prepared herein: a substrate containing an accessible toehold as well as input and output macromolecules. The substrate and the input macromolecules contained noncoded PPDE homopolymers, whereas the output macromolecule contained a digitally encoded segment. After hybridization of the substrate with the output, incubation in the presence of the input led to efficient TMSD and the release of the digital segment. TMSD can therefore be used to erase or rewrite information in self-assembled biohybrid superstructures. Furthermore, it was found in this work that the conjugation of DNA single strands to synthetic segments of chosen lengths greatly facilitates the characterization and PAGE visualization of the TMSD process.
Khodr Radi, Husser Claire, Ryckelynck Michael
Direct fluoride monitoring using a fluorogenic RNA-based biosensor Book Chapter
In: Stockbridge, Randy B. (Ed.): vol. 696, pp. 85–107, Randy B. Stockbridge, 2024, ISSN: 1557-7988.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@inbook{pmid38658090,
title = {Direct fluoride monitoring using a fluorogenic RNA-based biosensor},
author = {Radi Khodr and Claire Husser and Michael Ryckelynck},
editor = {Randy B. Stockbridge},
doi = {10.1016/bs.mie.2023.12.019},
issn = {1557-7988},
year = {2024},
date = {2024-01-01},
urldate = {2024-01-01},
journal = {Methods Enzymol},
volume = {696},
pages = {85--107},
publisher = {Randy B. Stockbridge},
abstract = {Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.
2023
Dufossez Robin, Krafft Marie-Pierre, Ursuegui Sylvain, Mosser Michel, Mouftakhir Safae, Pernod Ketty, Chaubet Guilhem, Ryckelynck Michael, Wagner Alain
Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface Journal Article
In: ACS Appl Mater Interfaces, vol. 15, iss. 38, pp. 45498-45505, 2023, ISSN: 1944-8252.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid37704020,
title = {Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface},
author = {Robin Dufossez and Marie-Pierre Krafft and Sylvain Ursuegui and Michel Mosser and Safae Mouftakhir and Ketty Pernod and Guilhem Chaubet and Michael Ryckelynck and Alain Wagner},
doi = {10.1021/acsami.3c10655},
issn = {1944-8252},
year = {2023},
date = {2023-09-01},
urldate = {2023-09-01},
journal = {ACS Appl Mater Interfaces},
volume = {15},
issue = {38},
pages = {45498-45505},
abstract = {Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.
Husser Claire, Vuilleumier Stéphane, Ryckelynck Michael
FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride Journal Article
In: Small, vol. 19, iss. 13, pp. e2205232, 2023, ISSN: 1613-6829.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid36436882,
title = {FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride},
author = {Claire Husser and Stéphane Vuilleumier and Michael Ryckelynck},
doi = {10.1002/smll.202205232},
issn = {1613-6829},
year = {2023},
date = {2023-03-29},
urldate = {2023-03-01},
journal = {Small},
volume = {19},
issue = {13},
pages = {e2205232},
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.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
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.
Dufossez Robin, Ursuegui Sylvain, Baudrey Stephanie, Pernod Ketty, Mouftakhir Safae, Oulad-Abdelghani Mustapha, Mosser Michel, Chaubet Guilhem, Ryckelynck Michael, Wagner Alain
Droplet Surface Immunoassay by Relocation (D-SIRe) for High-Throughput Analysis of Cytosolic Proteins at the Single-Cell Level Journal Article
In: Anal Chem, vol. 95, iss. 9, pp. 4470-4478, 2023, ISSN: 1520-6882.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid36821722,
title = {Droplet Surface Immunoassay by Relocation (D-SIRe) for High-Throughput Analysis of Cytosolic Proteins at the Single-Cell Level},
author = {Robin Dufossez and Sylvain Ursuegui and Stephanie Baudrey and Ketty Pernod and Safae Mouftakhir and Mustapha Oulad-Abdelghani and Michel Mosser and Guilhem Chaubet and Michael Ryckelynck and Alain Wagner},
doi = {10.1021/acs.analchem.2c05168},
issn = {1520-6882},
year = {2023},
date = {2023-02-01},
urldate = {2023-02-01},
journal = {Anal Chem},
volume = {95},
issue = {9},
pages = {4470-4478},
abstract = {Enzyme-linked immunosorbent assay (ELISA) is a central analytic method in biological science for the detection of proteins. Introduction of droplet-based microfluidics allowed the development of miniaturized, less-consuming, and more sensitive ELISA assays by coencapsulating the biological sample and antibody-functionalized particles. We report herein an alternative in-droplet immunoassay format, which avoids the use of particles. It exploits the oil/aqueous-phase interface as a protein capture and detection surface. This is achieved using tailored perfluorinated surfactants bearing azide-functionalized PEG-based polar headgroups, which spontaneously react when meeting at the droplet formation site, with strained alkyne-functionalized antibodies solubilized in the water phase. The resulting antibody-functionalized inner surface can then be used to capture a target protein. This surface capture process leads to concomitant relocation at the surface of a labeled detection antibody and in turn to a drastic change in the shape of the fluorescence signal from a convex shape (not captured) to a characteristic concave shape (captured). This novel droplet surface immunoassay by fluorescence relocation (D-SIRe) proved to be fast and sensitive at 2.3 attomoles of analyte per droplet. It was further demonstrated to allow detection of cytosolic proteins at the single bacteria level.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Enzyme-linked immunosorbent assay (ELISA) is a central analytic method in biological science for the detection of proteins. Introduction of droplet-based microfluidics allowed the development of miniaturized, less-consuming, and more sensitive ELISA assays by coencapsulating the biological sample and antibody-functionalized particles. We report herein an alternative in-droplet immunoassay format, which avoids the use of particles. It exploits the oil/aqueous-phase interface as a protein capture and detection surface. This is achieved using tailored perfluorinated surfactants bearing azide-functionalized PEG-based polar headgroups, which spontaneously react when meeting at the droplet formation site, with strained alkyne-functionalized antibodies solubilized in the water phase. The resulting antibody-functionalized inner surface can then be used to capture a target protein. This surface capture process leads to concomitant relocation at the surface of a labeled detection antibody and in turn to a drastic change in the shape of the fluorescence signal from a convex shape (not captured) to a characteristic concave shape (captured). This novel droplet surface immunoassay by fluorescence relocation (D-SIRe) proved to be fast and sensitive at 2.3 attomoles of analyte per droplet. It was further demonstrated to allow detection of cytosolic proteins at the single bacteria level.
Husser Claire, Baudrey Stéphanie, Ryckelynck Michael
In: Günter Mayer, Marcus M. Menger (Ed.): vol. 2570, pp. 243–269, Günter Mayer, Marcus M. Menger, 2023, ISSN: 1940-6029.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@inbook{pmid36156788,
title = {High-Throughput Development and Optimization of RNA-Based Fluorogenic Biosensors of Small Molecules Using Droplet-Based Microfluidics},
author = {Claire Husser and Stéphanie Baudrey and Michael Ryckelynck},
editor = {Günter Mayer, Marcus M. Menger},
doi = {10.1007/978-1-0716-2695-5_19},
issn = {1940-6029},
year = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {Methods Mol Biol},
volume = {2570},
pages = {243--269},
publisher = {Günter Mayer, Marcus M. Menger},
series = {Nucleic Acid Aptamers Selection, Characterization, and Application},
abstract = {Small-molecule sensing is a major issue as they can serve both in fundamental science and as makers of various diseases, contaminations, or even environment pollution. RNA aptamers are single-stranded nucleic acids that can adopt different conformations and specifically recognize a wide range of ligands, making them good candidates to develop biosensors of small molecules. Recently, light-up RNA aptamers have been introduced and used as starting building blocks of RNA-based fluorogenic biosensors. They are typically made of three domains: a reporter domain (a light-up aptamer), connected to a sensor domain (another aptamer) via a communication module. The latter is instrumental as being in charge of information transmission between the sensor and the reporting domains. Here we present an ultrahigh-throughput screening procedure to develop RNA-based fluorogenic biosensors by selecting optimized communication modules through an exhaustive functional exploration of every possible sequence permutation using droplet-based microfluidics and next-generation sequencing.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Small-molecule sensing is a major issue as they can serve both in fundamental science and as makers of various diseases, contaminations, or even environment pollution. RNA aptamers are single-stranded nucleic acids that can adopt different conformations and specifically recognize a wide range of ligands, making them good candidates to develop biosensors of small molecules. Recently, light-up RNA aptamers have been introduced and used as starting building blocks of RNA-based fluorogenic biosensors. They are typically made of three domains: a reporter domain (a light-up aptamer), connected to a sensor domain (another aptamer) via a communication module. The latter is instrumental as being in charge of information transmission between the sensor and the reporting domains. Here we present an ultrahigh-throughput screening procedure to develop RNA-based fluorogenic biosensors by selecting optimized communication modules through an exhaustive functional exploration of every possible sequence permutation using droplet-based microfluidics and next-generation sequencing.
2022
Geraci I., Autour A., Pietruschka G., Shiian A., Borisova M., Mayer C., Ryckelynck M., Mayer G.
Fluorogenic RNA-Based Biosensor to Sense the Glycolytic Flux in Mammalian Cells Journal Article
In: ACS Chem Biol, vol. 17, iss. 5, pp. 1164-1173, 2022, ISBN: 35427113, (1554-8937 (Electronic) 1554-8929 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Labex, RYCKELYNCK, Unité ARN
@article{nokey,
title = {Fluorogenic RNA-Based Biosensor to Sense the Glycolytic Flux in Mammalian Cells},
author = {I. Geraci and A. Autour and G. Pietruschka and A. Shiian and M. Borisova and C. Mayer and M. Ryckelynck and G. Mayer},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35427113},
doi = {10.1021/acschembio.2c00100},
isbn = {35427113},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {ACS Chem Biol},
volume = {17},
issue = {5},
pages = {1164-1173},
abstract = {The visualization of metabolic flux in real time requires sensor molecules that transduce variations of metabolite concentrations into an appropriate output signal. In this regard, fluorogenic RNA-based biosensors are promising molecular tools as they fluoresce only upon binding to another molecule. However, to date no such sensor is available that enables the direct observation of key metabolites in mammalian cells. Toward this direction, we selected and characterized an RNA light-up sensor designed to respond to fructose 1,6-bisphosphate and applied it to probe glycolytic flux variation in mammal cells.},
note = {1554-8937 (Electronic)
1554-8929 (Linking)
Journal Article},
keywords = {Labex, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The visualization of metabolic flux in real time requires sensor molecules that transduce variations of metabolite concentrations into an appropriate output signal. In this regard, fluorogenic RNA-based biosensors are promising molecular tools as they fluoresce only upon binding to another molecule. However, to date no such sensor is available that enables the direct observation of key metabolites in mammalian cells. Toward this direction, we selected and characterized an RNA light-up sensor designed to respond to fructose 1,6-bisphosphate and applied it to probe glycolytic flux variation in mammal cells.
Fam K. T., Pelletier R., Bouhedda F., Ryckelynck M., Collot M., Klymchenko A. S.
Rational Design of Self-Quenched Rhodamine Dimers as Fluorogenic Aptamer Probes for Live-Cell RNA Imaging Journal Article
In: Anal Chem, vol. 94, iss. 18, pp. 6657-6664, 2022, ISBN: 35486532, (1520-6882 (Electronic) 0003-2700 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Labex, RYCKELYNCK, Unité ARN
@article{nokey,
title = {Rational Design of Self-Quenched Rhodamine Dimers as Fluorogenic Aptamer Probes for Live-Cell RNA Imaging},
author = {K. T. Fam and R. Pelletier and F. Bouhedda and M. Ryckelynck and M. Collot and A. S. Klymchenko},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35486532},
doi = {10.1021/acs.analchem.1c04556},
isbn = {35486532},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Anal Chem},
volume = {94},
issue = {18},
pages = {6657-6664},
abstract = {With the growing interest in the understanding of the importance of RNAs in health and disease, detection of RNAs in living cells is of high importance. Fluorogenic dyes that light up specifically selected RNA aptamers constitute an attractive direction in the design of RNA imaging probes. In this work, based on our recently proposed concept of a fluorogenic dimer, we aim to develop a robust molecular tool for intracellular RNA imaging. We rationally designed a fluorogenic self-quenched dimer (orange Gemini, o-Gemini) based on rhodamine and evaluated its capacity to light up its cognate aptamer o-Coral in solution and live cells. We found that the removal of biotin from the dimer slightly improved the fluorogenic response without losing the affinity to the cognate aptamer (o-Coral). On the other hand, replacing sulforhodamine with a carboxyrhodamine produced drastic improvement of the affinity and the turn-on response to o-Coral and, thus, a better limit of detection. In live cells expressing o-Coral-tagged RNAs, the carboxyrhodamine analogue of o-Gemini without a biotin unit displayed a higher signal as well as faster internalization into the cells. We suppose that less hydrophilic carboxyrhodamine compared to sulforhodamine can more readily penetrate through the cell plasma membrane and, together with its higher affinity to o-Coral, provide the observed improvement in the imaging experiments. The promiscuity of the o-Coral RNA aptamer to the fluorogenic dimer allowed us to tune a fluorogen chemical structure and thus drastically improve the fluorescence response of the probe to o-Coral-tagged RNAs.},
note = {1520-6882 (Electronic)
0003-2700 (Linking)
Journal Article},
keywords = {Labex, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
With the growing interest in the understanding of the importance of RNAs in health and disease, detection of RNAs in living cells is of high importance. Fluorogenic dyes that light up specifically selected RNA aptamers constitute an attractive direction in the design of RNA imaging probes. In this work, based on our recently proposed concept of a fluorogenic dimer, we aim to develop a robust molecular tool for intracellular RNA imaging. We rationally designed a fluorogenic self-quenched dimer (orange Gemini, o-Gemini) based on rhodamine and evaluated its capacity to light up its cognate aptamer o-Coral in solution and live cells. We found that the removal of biotin from the dimer slightly improved the fluorogenic response without losing the affinity to the cognate aptamer (o-Coral). On the other hand, replacing sulforhodamine with a carboxyrhodamine produced drastic improvement of the affinity and the turn-on response to o-Coral and, thus, a better limit of detection. In live cells expressing o-Coral-tagged RNAs, the carboxyrhodamine analogue of o-Gemini without a biotin unit displayed a higher signal as well as faster internalization into the cells. We suppose that less hydrophilic carboxyrhodamine compared to sulforhodamine can more readily penetrate through the cell plasma membrane and, together with its higher affinity to o-Coral, provide the observed improvement in the imaging experiments. The promiscuity of the o-Coral RNA aptamer to the fluorogenic dimer allowed us to tune a fluorogen chemical structure and thus drastically improve the fluorescence response of the probe to o-Coral-tagged RNAs.
Geersens E., Vuilleumier S., Ryckelynck M.
Growth-Associated Droplet Shrinkage for Bacterial Quantification, Growth Monitoring, and Separation by Ultrahigh-Throughput Microfluidics Journal Article
In: ACS Omega, vol. 7, no. 14, pp. 12039-12047, 2022, ISBN: 35449964, (2470-1343 (Electronic) 2470-1343 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Labex, RYCKELYNCK, Unité ARN
@article{nokey,
title = {Growth-Associated Droplet Shrinkage for Bacterial Quantification, Growth Monitoring, and Separation by Ultrahigh-Throughput Microfluidics},
author = {E. Geersens and S. Vuilleumier and M. Ryckelynck},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=35449964},
doi = {10.1021/acsomega.2c00248},
isbn = {35449964},
year = {2022},
date = {2022-01-01},
journal = {ACS Omega},
volume = {7},
number = {14},
pages = {12039-12047},
abstract = {Microbiology still relies on en masse cultivation for selection, isolation, and characterization of microorganisms of interest. This constrains the diversity of microbial types and metabolisms that can be investigated in the laboratory also because of intercellular competition during cultivation. Cell individualization by droplet-based microfluidics prior to experimental analysis provides an attractive alternative to access a larger fraction of the microbial biosphere, miniaturizing the required equipment and minimizing reagent use for increased and more efficient analytical throughput. Here, we show that cultivation of a model two-strain bacterial community in droplets significantly reduces representation bias in the grown culture compared to batch cultivation. Further, and based on the droplet shrinkage observed upon cell proliferation, we provide proof-of-concept for a simple strategy that allows absolute quantification of microbial cells in a sample as well as selective recovery of microorganisms of interest for subsequent experimental characterization.},
note = {2470-1343 (Electronic)
2470-1343 (Linking)
Journal Article},
keywords = {Labex, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Microbiology still relies on en masse cultivation for selection, isolation, and characterization of microorganisms of interest. This constrains the diversity of microbial types and metabolisms that can be investigated in the laboratory also because of intercellular competition during cultivation. Cell individualization by droplet-based microfluidics prior to experimental analysis provides an attractive alternative to access a larger fraction of the microbial biosphere, miniaturizing the required equipment and minimizing reagent use for increased and more efficient analytical throughput. Here, we show that cultivation of a model two-strain bacterial community in droplets significantly reduces representation bias in the grown culture compared to batch cultivation. Further, and based on the droplet shrinkage observed upon cell proliferation, we provide proof-of-concept for a simple strategy that allows absolute quantification of microbial cells in a sample as well as selective recovery of microorganisms of interest for subsequent experimental characterization.
Baudrey Stéphanie, Cubi Roger, Ryckelynck Michael
Droplet-Based Microfluidic Chip Design, Fabrication, and Use for Ultrahigh-Throughput DNA Analysis and Quantification Journal Article
In: Adv Exp Med Biol, vol. 1379, pp. 445–460, 2022, ISSN: 0065-2598.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{pmid35761003,
title = {Droplet-Based Microfluidic Chip Design, Fabrication, and Use for Ultrahigh-Throughput DNA Analysis and Quantification},
author = {Stéphanie Baudrey and Roger Cubi and Michael Ryckelynck},
doi = {10.1007/978-3-031-04039-9_18},
issn = {0065-2598},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Adv Exp Med Biol},
volume = {1379},
pages = {445--460},
abstract = {DNA is widely used as a biomarker of contamination, infection, or disease, which has stimulated the development of a wide palette of detection and quantification methods. Even though several analytical approaches based on isothermal amplification have been proposed, DNA is still mainly detected and quantified by quantitative PCR (qPCR). However, for some analyses (e.g., in cancer research) qPCR may suffer from limitations arising from competitions between highly similar template DNAs, the presence of inhibitors, or suboptimal primer design. Nevertheless, digitalizing the analysis (i.e., individualizing DNA molecules into compartments prior to amplifying them in situ) allows to address most of these issues. By its capacity to generate and manipulate millions of highly similar picoliter volume water-in-oil droplets, microfluidics offers both the required miniaturization and parallelization capacity, and led to the introduction of digital droplet PCR (ddPCR). This chapter aims at introducing the reader to the basic principles behind ddPCR while also providing the key guidelines to fabricate, set up, and use his/her own ddPCR platform. We further provide procedures to detect and quantify DNA either purified in solution or directly from individualized cells. This approach not only gives access to DNA absolute concentration with unrivaled sensitivity, but it may also be the starting point of more complex in vitro analytical pipelines discussed at the end of the chapter.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
DNA is widely used as a biomarker of contamination, infection, or disease, which has stimulated the development of a wide palette of detection and quantification methods. Even though several analytical approaches based on isothermal amplification have been proposed, DNA is still mainly detected and quantified by quantitative PCR (qPCR). However, for some analyses (e.g., in cancer research) qPCR may suffer from limitations arising from competitions between highly similar template DNAs, the presence of inhibitors, or suboptimal primer design. Nevertheless, digitalizing the analysis (i.e., individualizing DNA molecules into compartments prior to amplifying them in situ) allows to address most of these issues. By its capacity to generate and manipulate millions of highly similar picoliter volume water-in-oil droplets, microfluidics offers both the required miniaturization and parallelization capacity, and led to the introduction of digital droplet PCR (ddPCR). This chapter aims at introducing the reader to the basic principles behind ddPCR while also providing the key guidelines to fabricate, set up, and use his/her own ddPCR platform. We further provide procedures to detect and quantify DNA either purified in solution or directly from individualized cells. This approach not only gives access to DNA absolute concentration with unrivaled sensitivity, but it may also be the starting point of more complex in vitro analytical pipelines discussed at the end of the chapter.
2021
Husser C, Dentz N, Ryckelynck M
Structure-Switching RNAs: From Gene Expression Regulation to Small Molecule Detection Journal Article
In: Small Structures, vol. 2, no. 4, pp. 2000132, 2021.
Abstract | Links | BibTeX | Tags: biosensing, Gene Expression Regulation, riboswitches, RNA aptamers, RYCKELYNCK, Synthetic Biology, Unité ARN
@article{C2021c,
title = {Structure-Switching RNAs: From Gene Expression Regulation to Small Molecule Detection},
author = {C Husser and N Dentz and M Ryckelynck},
url = {https://doi.org/10.1002/sstr.202000132},
doi = {10.1002/sstr.202000132},
year = {2021},
date = {2021-01-01},
journal = {Small Structures},
volume = {2},
number = {4},
pages = {2000132},
abstract = {RNA is instrumental to cell life in many aspects, especially gene expression regulation. Among the various known regulatory RNAs, riboswitches are particularly interesting cis‐acting molecules as they do not need cellular factor to achieve their function and are therefore highly portable from one organism to the other. These molecules usually found in the 5′ untranslated region of bacterial messenger RNAs are able to specifically sense a target ligand via an aptamer domain prior to transmitting this recognition event to an expression platform that turns on, or off, the expression of downstream genes. In addition to their obvious scientific interest, these modular molecules can also serve for the development of synthetic RNA devices with applications ranging from the control of transgene expression in gene therapy to the specific biosensing of small molecules. The engineering of such nanomachines is greatly facilitated by the proper understanding of their structure as well as the introduction of new technologies. Herein, a general overview of the current knowledge on natural riboswitches prior to explaining the main strategies used to develop new synthetic structure‐switching molecules (riboswitches or biosensors) controlled by small molecules is given.},
keywords = {biosensing, Gene Expression Regulation, riboswitches, RNA aptamers, RYCKELYNCK, Synthetic Biology, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
RNA is instrumental to cell life in many aspects, especially gene expression regulation. Among the various known regulatory RNAs, riboswitches are particularly interesting cis‐acting molecules as they do not need cellular factor to achieve their function and are therefore highly portable from one organism to the other. These molecules usually found in the 5′ untranslated region of bacterial messenger RNAs are able to specifically sense a target ligand via an aptamer domain prior to transmitting this recognition event to an expression platform that turns on, or off, the expression of downstream genes. In addition to their obvious scientific interest, these modular molecules can also serve for the development of synthetic RNA devices with applications ranging from the control of transgene expression in gene therapy to the specific biosensing of small molecules. The engineering of such nanomachines is greatly facilitated by the proper understanding of their structure as well as the introduction of new technologies. Herein, a general overview of the current knowledge on natural riboswitches prior to explaining the main strategies used to develop new synthetic structure‐switching molecules (riboswitches or biosensors) controlled by small molecules is given.
Bouhedda F, Cubi R, Baudrey S, Ryckelynck M
microIVC-Seq: A Method for Ultrahigh-Throughput Development and Functional Characterization of Small RNAs Book Chapter
In: Rederstorff, M (Ed.): Small Non-Coding RNAs, vol. 2300, pp. 203-237, Springer Protocols, Humana Press, New York, NY, Small Non-Coding RNAs, 2021, ISBN: 978-1-0716-1385-6/ISSN, (1940-6029 (Electronic) 1064-3745 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Artificial RNAs, directed evolution, Droplet microfluidics, Functional screening, In vitro selection, RNA, RYCKELYNCK, RYCKELYNCK Artificial RNAs, Unité ARN
@inbook{Bouhedda2021,
title = {microIVC-Seq: A Method for Ultrahigh-Throughput Development and Functional Characterization of Small RNAs},
author = {F Bouhedda and R Cubi and S Baudrey and M Ryckelynck},
editor = {M Rederstorff},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33792882},
doi = {10.1007/978-1-0716-1386-3_17},
isbn = {978-1-0716-1385-6/ISSN},
year = {2021},
date = {2021-01-01},
booktitle = {Small Non-Coding RNAs},
volume = {2300},
pages = {203-237},
publisher = {Springer Protocols, Humana Press},
address = {New York, NY},
edition = {Small Non-Coding RNAs},
series = {Methods in Molecular Biology},
abstract = {For a long time, artificial RNAs have been developed by in vitro selection methodologies like Systematic Evolution of Ligands by EXponential enrichment (SELEX). Yet, even though this technology is extremely powerful to isolate specific and high-affinity binders, it is less suited for the isolation of RNAs optimized for more complex functions such as fluorescence emission or multiple-turnover catalysis. Whereas such RNAs should ideally be developed by screening approaches, conventional microtiter plate assays become rapidly cost-prohibitive. However, the advent of droplet-based microfluidics recently enabled us to devise microfluidic-assisted In Vitro Compartmentalization (muIVC), a strongly miniaturized and highly parallelized screening technology allowing to functionally screen millions of mutants in a single day while using a very low amount of reagent. Used in combination with high-throughput sequencing, the resulting muIVC-seq pipeline described in this chapter now allows rapid and semiautomated screening to be performed at low cost and in an ultrahigh-throughput regime.},
note = {1940-6029 (Electronic)
1064-3745 (Linking)
Journal Article},
keywords = {Artificial RNAs, directed evolution, Droplet microfluidics, Functional screening, In vitro selection, RNA, RYCKELYNCK, RYCKELYNCK Artificial RNAs, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
For a long time, artificial RNAs have been developed by in vitro selection methodologies like Systematic Evolution of Ligands by EXponential enrichment (SELEX). Yet, even though this technology is extremely powerful to isolate specific and high-affinity binders, it is less suited for the isolation of RNAs optimized for more complex functions such as fluorescence emission or multiple-turnover catalysis. Whereas such RNAs should ideally be developed by screening approaches, conventional microtiter plate assays become rapidly cost-prohibitive. However, the advent of droplet-based microfluidics recently enabled us to devise microfluidic-assisted In Vitro Compartmentalization (muIVC), a strongly miniaturized and highly parallelized screening technology allowing to functionally screen millions of mutants in a single day while using a very low amount of reagent. Used in combination with high-throughput sequencing, the resulting muIVC-seq pipeline described in this chapter now allows rapid and semiautomated screening to be performed at low cost and in an ultrahigh-throughput regime.
Cubi R, Bouhedda F, Collot M, Klymchenko A S, Ryckelynck M
In: Rna, vol. 27, no. 7, pp. 841-853, 2021, ISBN: 33952671, (1469-9001 (Electronic) 1355-8382 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: Bioinformatics, droplet-based microfluidics, high-throughput screening, light-up RNA aptamer, RNA engineering, RYCKELYNCK, Unité ARN
@article{,
title = {microIVC-Useq: a microfluidic-assisted high-throughput functionnal screening in tandem with next generation sequencing and artificial neural network to rapidly characterize RNA molecules},
author = {R Cubi and F Bouhedda and M Collot and A S Klymchenko and M Ryckelynck},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=33952671},
doi = {10.1261/rna.077586.120},
isbn = {33952671},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Rna},
volume = {27},
number = {7},
pages = {841-853},
abstract = {The function of an RNA is intimately linked to its three-dimensional structure. X-ray crystallography or NMR allow the fine structural characterization of small RNA (e.g., aptamers) with a precision down to atomic resolution. Yet, these technics are time consuming, laborious and do not inform on mutational robustness and the extent to which a sequence can be modified without altering RNA function, an important set of information to assist RNA engineering. On another hand, thought powerful, in silico predictions still lack the required accuracy. These limitations can be overcome by using high-throughput microfluidic-assisted functional screening technologies, as they allow exploring large mutant libraries in a rapid and cost-effective manner. Among them, we recently introduced the microfluidic-assisted In Vitro Compartmentalization (microIVC), an efficient screening strategy in which reactions are performed in picoliter droplets at rates of several thousand per second. We later improved microIVC efficiency by using in tandem with high throughput sequencing, thought a laborious bioinformatic step was still required at the end of the process. In the present work, we strongly increased the automation level of the pipeline by implementing an artificial neural network enabling unsupervised bioinformatic analysis. We demonstrate the efficiency of this "microIVC-Useq" technology by rapidly identifying a set of sequences readily accepted by a key domain of the light-up RNA aptamer SRB-2. This work not only shed some new light on the way this aptamer can be engineered, but it also allowed us to easily identify new variants with an up-to 10-fold improved performance.},
note = {1469-9001 (Electronic)
1355-8382 (Linking)
Journal Article},
keywords = {Bioinformatics, droplet-based microfluidics, high-throughput screening, light-up RNA aptamer, RNA engineering, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The function of an RNA is intimately linked to its three-dimensional structure. X-ray crystallography or NMR allow the fine structural characterization of small RNA (e.g., aptamers) with a precision down to atomic resolution. Yet, these technics are time consuming, laborious and do not inform on mutational robustness and the extent to which a sequence can be modified without altering RNA function, an important set of information to assist RNA engineering. On another hand, thought powerful, in silico predictions still lack the required accuracy. These limitations can be overcome by using high-throughput microfluidic-assisted functional screening technologies, as they allow exploring large mutant libraries in a rapid and cost-effective manner. Among them, we recently introduced the microfluidic-assisted In Vitro Compartmentalization (microIVC), an efficient screening strategy in which reactions are performed in picoliter droplets at rates of several thousand per second. We later improved microIVC efficiency by using in tandem with high throughput sequencing, thought a laborious bioinformatic step was still required at the end of the process. In the present work, we strongly increased the automation level of the pipeline by implementing an artificial neural network enabling unsupervised bioinformatic analysis. We demonstrate the efficiency of this "microIVC-Useq" technology by rapidly identifying a set of sequences readily accepted by a key domain of the light-up RNA aptamer SRB-2. This work not only shed some new light on the way this aptamer can be engineered, but it also allowed us to easily identify new variants with an up-to 10-fold improved performance.
Singh G., Pereira D., Baudrey S., Hoffmann E., Ryckelynck M., Asnacios A., Chaboute M. E.
Real-time tracking of root hair nucleus morphodynamics using a microfluidic approach Journal Article
In: Plant J, vol. 108, iss. 2, pp. 303-313, 2021, ISBN: 34562320, (1365-313X (Electronic) 0960-7412 (Linking) Journal Article).
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{nokey,
title = {Real-time tracking of root hair nucleus morphodynamics using a microfluidic approach},
author = {G. Singh and D. Pereira and S. Baudrey and E. Hoffmann and M. Ryckelynck and A. Asnacios and M. E. Chaboute},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=34562320},
doi = {10.1111/tpj.15511},
isbn = {34562320},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {Plant J},
volume = {108},
issue = {2},
pages = {303-313},
abstract = {Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RH shows a fast apical growth, constituting a unique single cell model system to analyse cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, but high-resolution imaging is still lacking to study accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) which enables us to capture high resolution confocal imaging of Arabidopsis RH development with real-time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, in order to illustrate the possibilities offered by the CMD, we have compared the real-time variations in the circularity, area, and aspect ratio of nuclei moving in growing and mature RH. Interestingly, we observed higher aspect ratios in the nuclei of mature RH, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development.},
note = {1365-313X (Electronic)
0960-7412 (Linking)
Journal Article},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RH shows a fast apical growth, constituting a unique single cell model system to analyse cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, but high-resolution imaging is still lacking to study accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) which enables us to capture high resolution confocal imaging of Arabidopsis RH development with real-time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, in order to illustrate the possibilities offered by the CMD, we have compared the real-time variations in the circularity, area, and aspect ratio of nuclei moving in growing and mature RH. Interestingly, we observed higher aspect ratios in the nuclei of mature RH, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development.
Bouhedda F., Ryckelynck M.
Compartmentalization-based technologies for in vitro selection and evolution of ribozymes and light-up RNA aptamers Book Chapter
In: & B. Masquida S. Müller, W. Winkler (Ed.): Ribozymes, vol. 28, pp. 721-738, John Wiley & Sons, Ltd, 2021.
Abstract | Links | BibTeX | Tags: catalytic RNAs, Droplet microfluidics, fluorescence, in vitro evolution, In vitro selection, light-up RNA aptamers, ribozymes, RYCKELYNCK, screening, Unité ARN
@inbook{nokey,
title = {Compartmentalization-based technologies for in vitro selection and evolution of ribozymes and light-up RNA aptamers},
author = {F. Bouhedda and M. Ryckelynck},
editor = {W. Winkler & B. Masquida S. Müller},
url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/9783527814527.ch28},
doi = {10.1002/9783527814527.ch28},
year = {2021},
date = {2021-01-01},
booktitle = {Ribozymes},
volume = {28},
pages = {721-738},
publisher = {John Wiley & Sons, Ltd},
abstract = {Summary Catalytic RNAs, also known as ribozymes, are naturally found in every living cell where they can occupy functions as important as peptide bond formation catalysis or intron splicing just as two examples. Besides, ribozymes are thought to be very ancient molecules that might have been the key actors of the so-called RNA world, but they also hold great promise for plenty of modern applications. These features have stimulated the development of in vitro evolution methodologies aiming at characterizing existing but also isolate new artificial ribozymes. Whereas bulk approaches in which all the RNA sequences of library are assayed in a single reaction mixture may be efficient to select fast, single-turn-over and/or self-modifying catalysts, this format is less adapted to the isolation of multiple turnover trans-acting molecules. Instead, a compartmentalization approach in which each variant is isolated and assayed into an individual compartment is better suited. In this chapter, we review the different strategies available to perform such compartmentalization and that range from hand-made water-in-oil emulsion to more advanced microfluidic-assisted ultrahigh-throughput screening. We finally extend the applications scope of these technologies to other RNAs (i.e., light-up RNA aptamers) for which a functional screening may also reveal more efficient than more conventional bulk in vitro selections.},
keywords = {catalytic RNAs, Droplet microfluidics, fluorescence, in vitro evolution, In vitro selection, light-up RNA aptamers, ribozymes, RYCKELYNCK, screening, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
Summary Catalytic RNAs, also known as ribozymes, are naturally found in every living cell where they can occupy functions as important as peptide bond formation catalysis or intron splicing just as two examples. Besides, ribozymes are thought to be very ancient molecules that might have been the key actors of the so-called RNA world, but they also hold great promise for plenty of modern applications. These features have stimulated the development of in vitro evolution methodologies aiming at characterizing existing but also isolate new artificial ribozymes. Whereas bulk approaches in which all the RNA sequences of library are assayed in a single reaction mixture may be efficient to select fast, single-turn-over and/or self-modifying catalysts, this format is less adapted to the isolation of multiple turnover trans-acting molecules. Instead, a compartmentalization approach in which each variant is isolated and assayed into an individual compartment is better suited. In this chapter, we review the different strategies available to perform such compartmentalization and that range from hand-made water-in-oil emulsion to more advanced microfluidic-assisted ultrahigh-throughput screening. We finally extend the applications scope of these technologies to other RNAs (i.e., light-up RNA aptamers) for which a functional screening may also reveal more efficient than more conventional bulk in vitro selections.
2020
Hayoun K, Geersens E, Laczny C C, Halder R, Sánchez C Lázaro, Manna A, Bringel F, Ryckelynck M, Muller P Wilmesand E E L, Alpha-Bazin B, Armengaud J, Vuilleumier S
Dichloromethane Degradation Pathway from Unsequenced Hyphomicrobium sp. MC8b Rapidly Explored by Pan-Proteomics Journal Article
In: Microorganisms, vol. 8, no. 12, pp. E1876, 2020.
Abstract | Links | BibTeX | Tags: dcm genes, dehalogenation, dichloromethane, differential proteomics, genome sequencing, Hyphomicrobium, Nanopore, pan-proteomics, RYCKELYNCK, Unité ARN
@article{Hayoun2020,
title = {Dichloromethane Degradation Pathway from Unsequenced Hyphomicrobium sp. MC8b Rapidly Explored by Pan-Proteomics },
author = {K Hayoun and E Geersens and C C Laczny and R Halder and C Lázaro Sánchez and A Manna and F Bringel and M Ryckelynck and P Wilmesand E E L Muller and B Alpha-Bazin and J Armengaud and S Vuilleumier
},
url = {https://www.mdpi.com/2076-2607/8/12/1876},
doi = { 10.3390/microorganisms8121876 },
year = {2020},
date = {2020-11-27},
journal = {Microorganisms},
volume = {8},
number = {12},
pages = {E1876},
abstract = {Several bacteria are able to degrade the major industrial solvent dichloromethane (DCM) by using the conserved dehalogenase DcmA, the only system for DCM degradation characterised at the sequence level so far. Using differential proteomics, we rapidly identified key determinants of DCM degradation for Hyphomicrobium sp. MC8b, an unsequenced facultative methylotrophic DCM-degrading strain. For this, we designed a pan-proteomics database comprising the annotated genome sequences of 13 distinct Hyphomicrobium strains. Compared to growth with methanol, growth with DCM induces drastic changes in the proteome of strain MC8b. Dichloromethane dehalogenase DcmA was detected by differential pan-proteomics, but only with poor sequence coverage, suggesting atypical characteristics of the DCM dehalogenation system in this strain. More peptides were assigned to DcmA by error-tolerant search, warranting subsequent sequencing of the genome of strain MC8b, which revealed a highly divergent set of dcm genes in this strain. This suggests that the dcm enzymatic system is less strongly conserved than previously believed, and that substantial molecular evolution of dcm genes has occurred beyond their horizontal transfer in the bacterial domain. Our study showed the power of pan-proteomics for quick characterization of new strains belonging to branches of the Tree of Life that are densely genome-sequenced. },
keywords = {dcm genes, dehalogenation, dichloromethane, differential proteomics, genome sequencing, Hyphomicrobium, Nanopore, pan-proteomics, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Several bacteria are able to degrade the major industrial solvent dichloromethane (DCM) by using the conserved dehalogenase DcmA, the only system for DCM degradation characterised at the sequence level so far. Using differential proteomics, we rapidly identified key determinants of DCM degradation for Hyphomicrobium sp. MC8b, an unsequenced facultative methylotrophic DCM-degrading strain. For this, we designed a pan-proteomics database comprising the annotated genome sequences of 13 distinct Hyphomicrobium strains. Compared to growth with methanol, growth with DCM induces drastic changes in the proteome of strain MC8b. Dichloromethane dehalogenase DcmA was detected by differential pan-proteomics, but only with poor sequence coverage, suggesting atypical characteristics of the DCM dehalogenation system in this strain. More peptides were assigned to DcmA by error-tolerant search, warranting subsequent sequencing of the genome of strain MC8b, which revealed a highly divergent set of dcm genes in this strain. This suggests that the dcm enzymatic system is less strongly conserved than previously believed, and that substantial molecular evolution of dcm genes has occurred beyond their horizontal transfer in the bacterial domain. Our study showed the power of pan-proteomics for quick characterization of new strains belonging to branches of the Tree of Life that are densely genome-sequenced.
Ryckelynck M
Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More Book Chapter
In: vol. 2166, pp. 73-102, Methods in Molecular Biology, 2020.
Abstract | Links | BibTeX | Tags: aptamer, biosensing, Engineering, fluorogen, Functional screening, Live-cell imaging, RNA, RYCKELYNCK, SELEX, Unité ARN
@inbook{Ryckelynck2020,
title = {Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More},
author = {M Ryckelynck},
url = {https://link.springer.com/protocol/10.1007%2F978-1-0716-0712-1_5},
doi = {10.1007/978-1-0716-0712-1_5 },
year = {2020},
date = {2020-07-25},
journal = {RNA Tagging},
volume = {2166},
pages = {73-102},
publisher = {Methods in Molecular Biology},
abstract = {The central role of RNA in living systems made it highly desirable to have noninvasive and sensitive technologies allowing for imaging the synthesis and the location of these molecules in living cells. This need motivated the development of small pro-fluorescent molecules called “fluorogens” that become fluorescent upon binding to genetically encodable RNAs called “light-up aptamers.” Yet, the development of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs. Then, the vast application potential of these molecules for live-cell RNA imaging and other applications is presented and discussed.},
keywords = {aptamer, biosensing, Engineering, fluorogen, Functional screening, Live-cell imaging, RNA, RYCKELYNCK, SELEX, Unité ARN},
pubstate = {published},
tppubtype = {inbook}
}
The central role of RNA in living systems made it highly desirable to have noninvasive and sensitive technologies allowing for imaging the synthesis and the location of these molecules in living cells. This need motivated the development of small pro-fluorescent molecules called “fluorogens” that become fluorescent upon binding to genetically encodable RNAs called “light-up aptamers.” Yet, the development of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs. Then, the vast application potential of these molecules for live-cell RNA imaging and other applications is presented and discussed.
3rd R J Trachman, Cojocaru R, Wu D, Piszczek G, Ryckelynck M, Unrau P J, Ferré-D'Amaré A R
Structure-Guided Engineering of the Homodimeric Mango-IV Fluorescence Turn-on Aptamer Yields an RNA FRET Pair Journal Article
In: Structure, vol. 28, no. 7, pp. 776-785.e773, 2020, ISBN: 32386573.
Abstract | Links | BibTeX | Tags: RNA aptamer RNA fluorescence RNA structure fluorescent dye structure-guided engineering, RYCKELYNCK, Unité ARN
@article{,
title = {Structure-Guided Engineering of the Homodimeric Mango-IV Fluorescence Turn-on Aptamer Yields an RNA FRET Pair},
author = {3rd R J Trachman and R Cojocaru and D Wu and G Piszczek and M Ryckelynck and P J Unrau and A R Ferré-D'Amaré},
url = {https://www.ncbi.nlm.nih.gov/pubmed/32386573?dopt=Abstract},
doi = {10.1016/j.str.2020.04.007},
isbn = {32386573},
year = {2020},
date = {2020-01-01},
journal = {Structure},
volume = {28},
number = {7},
pages = {776-785.e773},
abstract = {Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking transcripts. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV. Of these, Mango-IV was best at imaging RNAs in both fixed and live mammalian cells. To understand how Mango-IV achieves activity in cells, we determined its crystal structure complexed with TO1-Biotin. The structure reveals a domain-swapped homodimer with two independent G-quadruplex fluorophore binding pockets. Structure-based analyses indicate that the Mango-IV core has relaxed fluorophore specificity, and a tendency to reorganize binding pocket residues. These molecular properties may endow it with robustness in the cellular milieu. Based on the domain-swapped structure, heterodimers between Mango-IV and the fluorescent aptamer iSpinach, joined by Watson-Crick base pairing, were constructed. These exhibited FRET between their respective aptamer-activated fluorophores, advancing fluorescent aptamer technology toward multi-color, RNA-based imaging of RNA coexpression and colocalization.},
keywords = {RNA aptamer RNA fluorescence RNA structure fluorescent dye structure-guided engineering, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking transcripts. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV. Of these, Mango-IV was best at imaging RNAs in both fixed and live mammalian cells. To understand how Mango-IV achieves activity in cells, we determined its crystal structure complexed with TO1-Biotin. The structure reveals a domain-swapped homodimer with two independent G-quadruplex fluorophore binding pockets. Structure-based analyses indicate that the Mango-IV core has relaxed fluorophore specificity, and a tendency to reorganize binding pocket residues. These molecular properties may endow it with robustness in the cellular milieu. Based on the domain-swapped structure, heterodimers between Mango-IV and the fluorescent aptamer iSpinach, joined by Watson-Crick base pairing, were constructed. These exhibited FRET between their respective aptamer-activated fluorophores, advancing fluorescent aptamer technology toward multi-color, RNA-based imaging of RNA coexpression and colocalization.
Pernod K, Schaeffer L, Chicher J, Hok E, Rick C, Geslain R, Eriani G, Westhof E, Ryckelynck M, Martin F
In: Nucleic Acids Res, vol. 48, no. 11, pp. 6170-6183, 2020, ISBN: 32266934.
Abstract | Links | BibTeX | Tags: ERIANI, PPSE, RYCKELYNCK, Unité ARN, WESTHOF
@article{,
title = {The Nature of the Purine at Position 34 in tRNAs of 4-codon Boxes Is Correlated With Nucleotides at Positions 32 and 38 to Maintain Decoding Fidelity},
author = {K Pernod and L Schaeffer and J Chicher and E Hok and C Rick and R Geslain and G Eriani and E Westhof and M Ryckelynck and F Martin},
url = {https://www.ncbi.nlm.nih.gov/pubmed/32266934?dopt=Abstract},
doi = {10.1093/nar/gkaa221},
isbn = {32266934},
year = {2020},
date = {2020-01-01},
journal = {Nucleic Acids Res},
volume = {48},
number = {11},
pages = {6170-6183},
abstract = {Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.},
keywords = {ERIANI, PPSE, RYCKELYNCK, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.
Bouhedda F, Fam K T, Collot M, Autour A, Marzi S, Klymchenko A, Ryckelynck M
A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells Journal Article
In: Nat Chem Biol, vol. 16, no. 1, pp. 69-76, 2020, ISBN: 31636432.
Abstract | Links | BibTeX | Tags: ROMBY, RYCKELYNCK, Unité ARN
@article{,
title = {A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells},
author = {F Bouhedda and K T Fam and M Collot and A Autour and S Marzi and A Klymchenko and M Ryckelynck},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31636432},
doi = {https://doi.org/10.1038/s41589-019-0381-8},
isbn = {31636432},
year = {2020},
date = {2020-01-01},
journal = {Nat Chem Biol},
volume = {16},
number = {1},
pages = {69-76},
abstract = {Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.},
keywords = {ROMBY, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.
2019
3rd R J Trachman, Autour A, Jeng S C Y, Abdolahzadeh A, Andreoni A, Cojocaru R, Garipov R, Dolgosheina E V, Knutson J R, Ryckelynck M, Unrau P J, Ferré-D'Amaré A R
Structure and functional reselection of the Mango-III fluorogenic RNA aptamer Journal Article
In: Nat Chem Biol, vol. 15, no. 5, pp. 472-479, 2019, ISBN: 30992561.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{,
title = {Structure and functional reselection of the Mango-III fluorogenic RNA aptamer},
author = {3rd R J Trachman and A Autour and S C Y Jeng and A Abdolahzadeh and A Andreoni and R Cojocaru and R Garipov and E V Dolgosheina and J R Knutson and M Ryckelynck and P J Unrau and A R Ferré-D'Amaré},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30992561?dopt=Abstract},
doi = {10.1038/s41589-019-0267-9},
isbn = {30992561},
year = {2019},
date = {2019-01-01},
journal = {Nat Chem Biol},
volume = {15},
number = {5},
pages = {472-479},
abstract = {Several turn-on RNA aptamers that activate small-molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to the nucleobase planes of those motifs. The improved iMango-III and Mango-III(A10U) fluoresce ~50% brighter than enhanced green fluorescent protein, making them suitable tags for live cell RNA visualization.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Several turn-on RNA aptamers that activate small-molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to the nucleobase planes of those motifs. The improved iMango-III and Mango-III(A10U) fluoresce ~50% brighter than enhanced green fluorescent protein, making them suitable tags for live cell RNA visualization.
Ryckelynck M
Development and engineering of artificial RNAs Journal Article
In: Methods, vol. 161, pp. 1-2, 2019, ISBN: 31195094.
Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{,
title = {Development and engineering of artificial RNAs},
author = {M Ryckelynck},
url = {https://www.ncbi.nlm.nih.gov/pubmed/31195094?dopt=Abstract},
doi = {10.1016/j.ymeth.2019.06.009},
isbn = {31195094},
year = {2019},
date = {2019-01-01},
journal = {Methods},
volume = {161},
pages = {1-2},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Autour A, Bouhedda F, Cubi R, Ryckelynck M
Optimization of fluorogenic RNA-based biosensors using droplet-based microfluidic ultrahigh-throughput screening Journal Article
In: Methods, vol. 161, pp. 46-53, 2019, ISBN: 30902664.
Abstract | Links | BibTeX | Tags: Aptasensors Fluorogenic biosensors High-throughput screening Light-up aptamer Next generation sequencing RNA, RYCKELYNCK, Unité ARN
@article{,
title = {Optimization of fluorogenic RNA-based biosensors using droplet-based microfluidic ultrahigh-throughput screening},
author = {A Autour and F Bouhedda and R Cubi and M Ryckelynck},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30902664?dopt=Abstract},
doi = {10.1016/j.ymeth.2019.03.015},
isbn = {30902664},
year = {2019},
date = {2019-01-01},
journal = {Methods},
volume = {161},
pages = {46-53},
abstract = {Biosensors are biological molecules able to detect and report the presence of a target molecule by the emission of a signal. Nucleic acids are particularly appealing for the design of such molecule since their great structural plasticity makes them able to specifically interact with a wide range of ligands and their structure can rearrange upon recognition to trigger a reporting event. A biosensor is typically made of three main domains: a sensing domain that is connected to a reporting domain via a communication module in charge of transmitting the sensing event through the molecule. The communication module is therefore an instrumental element of the sensor. This module is usually empirically developed through a trial-and-error strategy with the testing of only a few combinations judged relevant by the experimenter. In this work, we introduce a novel method combining the use of droplet-based microfluidics and next generation sequencing. This method allows to functionally characterize up to a million of different sequences in a single set of experiments and, by doing so, to exhaustively test every possible sequence permutations of the communication module. Here, we demonstrate the efficiency of the approach by isolating a set of optimized RNA biosensors able to sense theophylline and to convert this recognition into fluorescence emission.},
keywords = {Aptasensors Fluorogenic biosensors High-throughput screening Light-up aptamer Next generation sequencing RNA, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Biosensors are biological molecules able to detect and report the presence of a target molecule by the emission of a signal. Nucleic acids are particularly appealing for the design of such molecule since their great structural plasticity makes them able to specifically interact with a wide range of ligands and their structure can rearrange upon recognition to trigger a reporting event. A biosensor is typically made of three main domains: a sensing domain that is connected to a reporting domain via a communication module in charge of transmitting the sensing event through the molecule. The communication module is therefore an instrumental element of the sensor. This module is usually empirically developed through a trial-and-error strategy with the testing of only a few combinations judged relevant by the experimenter. In this work, we introduce a novel method combining the use of droplet-based microfluidics and next generation sequencing. This method allows to functionally characterize up to a million of different sequences in a single set of experiments and, by doing so, to exhaustively test every possible sequence permutations of the communication module. Here, we demonstrate the efficiency of the approach by isolating a set of optimized RNA biosensors able to sense theophylline and to convert this recognition into fluorescence emission.
2018
3rd R J Trachman, Abdolahzadeh A, Andreoni A, Cojocaru R, Knutson J R, Ryckelynck M, Unrau P J, Ferré-D'Amaré A R
In: Biochemistry, vol. 57, no. 26, pp. 3544-3548, 2018, ISBN: 29768001.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{,
title = {Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness},
author = {3rd R J Trachman and A Abdolahzadeh and A Andreoni and R Cojocaru and J R Knutson and M Ryckelynck and P J Unrau and A R Ferré-D'Amaré},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29768001?dopt=Abstract},
doi = {10.1021/acs.biochem.8b00399},
isbn = {29768001},
year = {2018},
date = {2018-01-01},
journal = {Biochemistry},
volume = {57},
number = {26},
pages = {3544-3548},
abstract = {Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ~1 nM affinity and enhances its fluorescence by >1,500-fold was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates over 5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases 18% while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analog studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ~1 nM affinity and enhances its fluorescence by >1,500-fold was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates over 5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases 18% while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analog studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.
Bouhedda F, Autour A, Ryckelynck M
Light-Up RNA Aptamers and Their Cognate Fluorogens: From Their Development to Their Applications Journal Article
In: Int J Mol Sci, vol. 19, no. 1, pp. 44, 2018, ISBN: 29295531.
Abstract | Links | BibTeX | Tags: RNA biosensing fluorescence fluorogen fluorogenic dye gene expression monitoring in vitro evolution light-up aptamer live-cell imaging, RYCKELYNCK, Unité ARN
@article{,
title = {Light-Up RNA Aptamers and Their Cognate Fluorogens: From Their Development to Their Applications},
author = {F Bouhedda and A Autour and M Ryckelynck},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29295531?dopt=Abstract},
doi = {10.3390/ijms19010044},
isbn = {29295531},
year = {2018},
date = {2018-01-01},
journal = {Int J Mol Sci},
volume = {19},
number = {1},
pages = {44},
abstract = {An RNA-based fluorogenic module consists of a light-up RNA aptamer able to specifically interact with a fluorogen to form a fluorescent complex. Over the past decade, significant efforts have been devoted to the development of such modules, which now cover the whole visible spectrum, as well as to their engineering to serve in a wide range of applications. In this review, we summarize the different strategies used to develop each partner (the fluorogen and the light-up RNA aptamer) prior to giving an overview of their applications that range from live-cell RNA imaging to the set-up of high-throughput drug screening pipelines. We then conclude with a critical discussion on the current limitations of these modules and how combining in vitro selection with screening approaches may help develop even better molecules.},
keywords = {RNA biosensing fluorescence fluorogen fluorogenic dye gene expression monitoring in vitro evolution light-up aptamer live-cell imaging, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
An RNA-based fluorogenic module consists of a light-up RNA aptamer able to specifically interact with a fluorogen to form a fluorescent complex. Over the past decade, significant efforts have been devoted to the development of such modules, which now cover the whole visible spectrum, as well as to their engineering to serve in a wide range of applications. In this review, we summarize the different strategies used to develop each partner (the fluorogen and the light-up RNA aptamer) prior to giving an overview of their applications that range from live-cell RNA imaging to the set-up of high-throughput drug screening pipelines. We then conclude with a critical discussion on the current limitations of these modules and how combining in vitro selection with screening approaches may help develop even better molecules.
Autour A, Jeng S C Y, Cawte A D, Abdolahzadeh A, Galli A, Panchapakesan SSS., Rueda D, Ryckelynck M, Unrau P J
Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells Journal Article
In: Nat Commun, vol. 9, no. 1, pp. 656, 2018, ISBN: 29440634.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{,
title = {Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells},
author = {A Autour and S C Y Jeng and A D Cawte and A Abdolahzadeh and A Galli and SSS. Panchapakesan and D Rueda and M Ryckelynck and P J Unrau},
url = {https://www.ncbi.nlm.nih.gov/pubmed/29440634?dopt=Abstract},
doi = {10.1038/s41467-018-02993-8},
isbn = {29440634},
year = {2018},
date = {2018-01-01},
journal = {Nat Commun},
volume = {9},
number = {1},
pages = {656},
abstract = {Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.
2017
Autour A, Ryckelynck M
Ultrahigh-Throughput Improvement and Discovery of Enzymes Using Droplet-Based Microfluidic Screening Journal Article
In: Micromachines, vol. 8, no. 4, pp. 128, 2017.
Abstract | Links | BibTeX | Tags: directed evolution, droplet-based microfluidics, enzyme improvement, high-throughput screening, RYCKELYNCK, single-cell, Unité ARN
@article{Autour2017,
title = {Ultrahigh-Throughput Improvement and Discovery of Enzymes Using Droplet-Based Microfluidic Screening},
author = {A Autour and M Ryckelynck},
url = {https://doi.org/10.3390/mi8040128},
doi = {10.3390/mi8040128},
year = {2017},
date = {2017-04-18},
journal = {Micromachines},
volume = {8},
number = {4},
pages = {128},
abstract = {Enzymes are extremely valuable tools for industrial, environmental, and biotechnological applications and there is a constant need for improving existing biological catalysts and for discovering new ones. Screening microbe or gene libraries is an efficient way of identifying new enzymes. In this view, droplet-based microfluidics appears to be one of the most powerful approaches as it allows inexpensive screenings in well-controlled conditions and an ultrahigh-throughput regime. This review aims to introduce the main microfluidic devices and concepts to be considered for such screening before presenting and discussing the latest successful applications of the technology for enzyme discovery. },
keywords = {directed evolution, droplet-based microfluidics, enzyme improvement, high-throughput screening, RYCKELYNCK, single-cell, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
Enzymes are extremely valuable tools for industrial, environmental, and biotechnological applications and there is a constant need for improving existing biological catalysts and for discovering new ones. Screening microbe or gene libraries is an efficient way of identifying new enzymes. In this view, droplet-based microfluidics appears to be one of the most powerful approaches as it allows inexpensive screenings in well-controlled conditions and an ultrahigh-throughput regime. This review aims to introduce the main microfluidic devices and concepts to be considered for such screening before presenting and discussing the latest successful applications of the technology for enzyme discovery.
Fernandez-Millan P, Autour A, Ennifar E, Westhof E, Ryckelynck M
Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI Journal Article
In: RNA, vol. 23, no. 12, pp. 1788-1795, 2017, ISBN: 28939697.
Abstract | Links | BibTeX | Tags: DFHBI Spinach crystal structure fluorescence fluorogenic RNA aptamer, ENNIFAR, RYCKELYNCK, Unité ARN, WESTHOF
@article{,
title = {Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI},
author = {P Fernandez-Millan and A Autour and E Ennifar and E Westhof and M Ryckelynck},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28939697?dopt=Abstract},
doi = {10.1261/rna.063008},
isbn = {28939697},
year = {2017},
date = {2017-01-01},
journal = {RNA},
volume = {23},
number = {12},
pages = {1788-1795},
abstract = {Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach comprising five mutations whom the exact role was not deciphered yet. In this work, we co-crystallized a re-engineered version of iSpinach in complex with the DFHBI and solved the x-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach allows shedding some light on the possible function of the different beneficial mutations carried by iSpinach.},
keywords = {DFHBI Spinach crystal structure fluorescence fluorogenic RNA aptamer, ENNIFAR, RYCKELYNCK, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach comprising five mutations whom the exact role was not deciphered yet. In this work, we co-crystallized a re-engineered version of iSpinach in complex with the DFHBI and solved the x-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach allows shedding some light on the possible function of the different beneficial mutations carried by iSpinach.
2016
Autour A, Westhof E, Ryckelynck M
iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications. Journal Article
In: Nucleic Acids Res, vol. 44, no. 6, pp. 2491-2500, 2016, ISBN: 26932363.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN, WESTHOF
@article{,
title = {iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications.},
author = {A Autour and E Westhof and M Ryckelynck},
url = {http://www.ncbi.nlm.nih.gov/pubmed/26932363?dopt=Abstract},
doi = {10.1093/nar/gkw083},
isbn = {26932363},
year = {2016},
date = {2016-01-01},
journal = {Nucleic Acids Res},
volume = {44},
number = {6},
pages = {2491-2500},
abstract = {Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme.},
keywords = {RYCKELYNCK, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme.
Matsumura S, Kun A, Ryckelynck M, Coldren F, Szilagyi A, Jossinet F, Rick C, Nghe P, Szathmary E, Griffiths A D
Transient compartmentalization of RNA replicators prevents extinction due to parasites. Journal Article
In: Science, vol. 354, no. 6317, pp. 1293-1296, 2016, ISBN: 27940874.
Abstract | Links | BibTeX | Tags: RYCKELYNCK, Unité ARN
@article{,
title = {Transient compartmentalization of RNA replicators prevents extinction due to parasites.},
author = {S Matsumura and A Kun and M Ryckelynck and F Coldren and A Szilagyi and F Jossinet and C Rick and P Nghe and E Szathmary and A D Griffiths},
url = {https://www.ncbi.nlm.nih.gov/pubmed/27940874?dopt=Abstract},
doi = {10.1126/science.aag1582},
isbn = {27940874},
year = {2016},
date = {2016-01-01},
journal = {Science},
volume = {354},
number = {6317},
pages = {1293-1296},
abstract = {The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators would take over and would have prevented life from taking off unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus, TC in natural, abiological compartments could have allowed life to take hold.},
keywords = {RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators would take over and would have prevented life from taking off unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus, TC in natural, abiological compartments could have allowed life to take hold.
2015
Woronoff G, Ryckelynck M, Wessel J, Schicke O, Griffiths A, P. Soumillion
Activity-Fed Translation (AFT) Assay: A New High-Throughput Screening Strategy for Enzymes in Droplets. Journal Article
In: Chembiochem, vol. 16, no. 9, pp. 1343-1349, 2015.
Abstract | Links | BibTeX | Tags: droplet-based microfluidics enzyme catalysis in vitro translation penicillin acylase protein expression, RYCKELYNCK, Unité ARN
@article{,
title = {Activity-Fed Translation (AFT) Assay: A New High-Throughput Screening Strategy for Enzymes in Droplets.},
author = {G Woronoff and M Ryckelynck and J Wessel and O Schicke and A Griffiths and Soumillion P.},
url = {http://www.ncbi.nlm.nih.gov/pubmed/25914325?dopt=Abstract},
doi = {10.1002/cbic.201500087},
year = {2015},
date = {2015-01-01},
journal = {Chembiochem},
volume = {16},
number = {9},
pages = {1343-1349},
abstract = {There is an increasing demand for the development of sensitive enzymatic assays compatible with droplet-based microfluidics. Here we describe an original strategy, activity-fed translation (AFT), based on the coupling of enzymatic activity to in vitro translation of a fluorescent protein. We show that methionine release upon the hydrolysis of phenylacetylmethionine by penicillin acylase enabled in vitro expression of green fluorescent protein. An autocatalytic setup where both proteins are expressed makes the assay highly sensitive, as fluorescence was detected in droplets containing single PAC genes. Adding a PCR step in the droplets prior to the assay increased the sensitivity further. The strategy is potentially applicable for any activity that can be coupled to the production of an amino acid, and as the microdroplet volume is small the use of costly reagents such as in vitro expression mixtures is not limiting for high-throughput screening projects},
keywords = {droplet-based microfluidics enzyme catalysis in vitro translation penicillin acylase protein expression, RYCKELYNCK, Unité ARN},
pubstate = {published},
tppubtype = {article}
}
There is an increasing demand for the development of sensitive enzymatic assays compatible with droplet-based microfluidics. Here we describe an original strategy, activity-fed translation (AFT), based on the coupling of enzymatic activity to in vitro translation of a fluorescent protein. We show that methionine release upon the hydrolysis of phenylacetylmethionine by penicillin acylase enabled in vitro expression of green fluorescent protein. An autocatalytic setup where both proteins are expressed makes the assay highly sensitive, as fluorescence was detected in droplets containing single PAC genes. Adding a PCR step in the droplets prior to the assay increased the sensitivity further. The strategy is potentially applicable for any activity that can be coupled to the production of an amino acid, and as the microdroplet volume is small the use of costly reagents such as in vitro expression mixtures is not limiting for high-throughput screening projects
Ryckelynck M, Baudrey S, Rick C, Marin A, Coldren F, Westhof E, Griffiths A D
Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions. Journal Article
In: RNA, vol. 21, no. 3, pp. 458-469, 2015, ISBN: 25605963.
Abstract | Links | BibTeX | Tags: RNA droplet-based microfluidics high-throughput screening in vitro evolution ribozymes, RYCKELYNCK, Unité ARN, WESTHOF
@article{,
title = {Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions.},
author = {M Ryckelynck and S Baudrey and C Rick and A Marin and F Coldren and E Westhof and A D Griffiths},
url = {http://www.ncbi.nlm.nih.gov/pubmed/25605963?dopt=Abstract},
doi = {10.1261/rna.048033.114},
isbn = {25605963},
year = {2015},
date = {2015-01-01},
journal = {RNA},
volume = {21},
number = {3},
pages = {458-469},
abstract = {In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities. While Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an efficient approach to generate new RNA aptamers, it is less suited for the isolation of efficient ribozymes as it does not select directly for the catalysis. In vitro compartmentalization (IVC) in aqueous droplets in emulsions allows catalytic RNAs to be selected under multiple-turnover conditions but suffers severe limitations that can be overcome using the droplet-based microfluidics workflow described in this paper. Using microfluidics, millions of genes in a library can be individually compartmentalized in highly monodisperse aqueous droplets and serial operations performed on them. This allows the different steps of the evolution process (gene amplification, transcription, and phenotypic assay) to be uncoupled, making the method highly flexible, applicable to the selection and evolution of a variety of RNAs, and easily adaptable for evolution of DNA or proteins. To demonstrate the method, we performed cycles of random mutagenesis and selection to evolve the X-motif, a ribozyme which, like many ribozymes selected using SELEX, has limited multiple-turnover activity. This led to the selection of variants, likely to be the optimal ribozymes that can be generated using point mutagenesis alone, with a turnover number under multiple-turnover conditions, kss cat, ∼28-fold higher than the original X-motif, primarily due to an increase in the rate of product release, the rate-limiting step in the multiple-turnover reaction.},
keywords = {RNA droplet-based microfluidics high-throughput screening in vitro evolution ribozymes, RYCKELYNCK, Unité ARN, WESTHOF},
pubstate = {published},
tppubtype = {article}
}
In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities. While Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an efficient approach to generate new RNA aptamers, it is less suited for the isolation of efficient ribozymes as it does not select directly for the catalysis. In vitro compartmentalization (IVC) in aqueous droplets in emulsions allows catalytic RNAs to be selected under multiple-turnover conditions but suffers severe limitations that can be overcome using the droplet-based microfluidics workflow described in this paper. Using microfluidics, millions of genes in a library can be individually compartmentalized in highly monodisperse aqueous droplets and serial operations performed on them. This allows the different steps of the evolution process (gene amplification, transcription, and phenotypic assay) to be uncoupled, making the method highly flexible, applicable to the selection and evolution of a variety of RNAs, and easily adaptable for evolution of DNA or proteins. To demonstrate the method, we performed cycles of random mutagenesis and selection to evolve the X-motif, a ribozyme which, like many ribozymes selected using SELEX, has limited multiple-turnover activity. This led to the selection of variants, likely to be the optimal ribozymes that can be generated using point mutagenesis alone, with a turnover number under multiple-turnover conditions, kss cat, ∼28-fold higher than the original X-motif, primarily due to an increase in the rate of product release, the rate-limiting step in the multiple-turnover reaction.
