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2021
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.
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.
