RNA Selection and Evolution<i>In Vitro:</i>Powerful Techniques for the Analysis and Identification of new Molecular Tools

Romero-López, Cristina; Díaz-González, R.; Berzal‐Herranz, Alfredo

doi:10.1080/13102818.2007.10817461

Cited by 6 publications

(3 citation statements)

References 124 publications

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“…However, these finding fell into oblivion until 1990, when the great potential of in vitro selection and evolution techniques was reported by three independent groups [ 2 , 3 , 4 , 5 ]. Since then, numerous authors have used these technologies to study the chemical and catalytic properties of nucleic acids (for further information see [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. They have also helped uphold the RNA world hypothesis, and it seems likely that they may soon have applications in biomedicine.…”

Section: Introductionmentioning

confidence: 99%

In Vitro and Ex Vivo Selection Procedures for Identifying Potentially Therapeutic DNA and RNA Molecules

et al. 2010

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It was only relatively recently discovered that nucleic acids participate in a variety of biological functions, besides the storage and transmission of genetic information. Quite apart from the nucleotide sequence, it is now clear that the structure of a nucleic acid plays an essential role in its functionality, enabling catalysis and specific binding reactions. In vitro selection and evolution strategies have been extremely useful in the analysis of functional RNA and DNA molecules, helping to expand our knowledge of their functional repertoire and to identify and optimize DNA and RNA molecules with potential therapeutic and diagnostic applications. The great progress made in this field has prompted the development of ex vivo methods for selecting functional nucleic acids in the cellular environment. This review summarizes the most important and most recent applications of in vitro and ex vivo selection strategies aimed at exploring the therapeutic potential of nucleic acids.

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Section: Introductionmentioning

confidence: 99%

In Vitro and Ex Vivo Selection Procedures for Identifying Potentially Therapeutic DNA and RNA Molecules

et al. 2010

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“…In addition, systematic evolution of ligands by exponential enrichment (SELEX) technology has been applied to in vitro selection of artificial combinative RNA sequences, known as RNA-aptamers, from random sequence libraries , . RNA-aptamers targeting not only natural proteins and peptides but also organic small molecules and nonnatural drugs have been discovered by the SELEX technology − . Since many of the RNA-aptamers induce conformational changes in response to target binding, they can be utilized as biosensor moieties, if the conformational changes will be detected by an obvious output signal − .…”

Section: Introductionmentioning

confidence: 99%

Detection of Bioactive Small Molecules by Fluorescent Resonance Energy Transfer (FRET) in RNA−Protein Conjugates

et al. 2009

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Bioactive small molecules such as metabolites and drugs play important roles in regulating biological functions. A technique for visualizing such small molecules is very useful to understand their molecular mechanisms. In this study, an RNA-protein conjugate, which consists of an RRE-RNA sensor protein (EYFP-Rev-ECFP) and an altered RRE-RNA, was constructed to detect bioactive small molecules by fluorescent resonance energy transfer (FRET). We designed a theophylline-aptamer-inserted RRE-RNA (Theo-RRE) to detect theophylline as a model target molecule. Theo-RRE formed an RNA-protein conjugate with EYFP-Rev-ECFP in the presence of theophylline. As a result, theophylline was specifically detected down to 10 microM by the FRET increase in distinction from theophylline analogue, caffeine, in cell lysates.

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“…In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities to study the origin of life or develop new molecular tools (Romero-Lopez et al 2007;Ellington et al 2009). Among them, Systematic Evolution of Ligand by Exponential enrichment (SELEX) (Ellington and Szostak 1990;Tuerk and Gold 1990) is the most widely used strategy and has proved to be an extremely effective method for the selection of highly specific RNA (and DNA) aptamers binding a wide range of ligands with high affinity (Stoltenburg et al 2007).…”

Section: Introductionmentioning

confidence: 99%

Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions

Ryckelynck

Baudrey

Rick

et al. 2015

RNA

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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, k ss 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.

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RNA Selection and EvolutionIn Vitro:Powerful Techniques for the Analysis and Identification of new Molecular Tools

Cited by 6 publications

References 124 publications

In Vitro and Ex Vivo Selection Procedures for Identifying Potentially Therapeutic DNA and RNA Molecules

In Vitro and Ex Vivo Selection Procedures for Identifying Potentially Therapeutic DNA and RNA Molecules

Detection of Bioactive Small Molecules by Fluorescent Resonance Energy Transfer (FRET) in RNA−Protein Conjugates

Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions

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