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• W2944604860 abstract "RNA is a key molecule for living organism in all kingdom of life. A major task performed by RNA is the regulation of gene expression. One of the strategies adopted by RNA to control and adapt gene expression to the environment of the cell, is the use of riboswitches that can activate or inhibit gene expression in response to changes in the presence of a specific molecule. Riboswitches are composed of two domains, one where binding of the target happens is called the aptamer and the other domain is called expression platform. Based on this principle, scientists could engineer synthetic riboswitches using in vitro selected aptamers by a technique called SELEX. So far, several synthetic riboswitches have been developed but they are still a few compared to natural riboswitches. One of the causes is not the lack of aptamers that recognize a target molecule, but that most of these aptamers are not suitable for riboswitch engineering. In fact, the majority of in vitro selected aptamers are not able to regulate gene expression. It requires the aptamers undergo a conformational change during ligand binding. In classical SELEX, however, only binding and not conformational changes are selected. Recently a new technique was developed for DNA, called Capture-SELEX allowing the selection of structure switching aptamers against small molecules in solution.The first part of this work included the development and the adaptation of Capture-SELEX for RNA. A complete adaptation of the protocol was needed to perform this SELEX with RNA. Some improvements were designed compared to the original protocol to increase efficiency that lead to a first successful enrichment towards neomycin. Improvements was mostly done aiming a better elution of specific binders but also the decrease of non-specific recovered sequence during the selection. A heat elution step was introduced to remove weak binders, shorten the time of specific elution, select only aptamers with a fast kon and optimize the design of the library. Thanks to these modification, selection against different types of molecules could be successfully achieved and their characterisation revealed the development of aptamers with high binding affinity and specificity.As the main goal of this project was the development of synthetic riboswitches, the paromomycin enriched SELEX library was used to screen for in vivo active sequences. After the screening of about 1200 different candidates, several hits were found and one candidate was selected for further engineering. he secondary structure of this candidate was analysed by inline probing and a ligand binding was determined by ITC measurement of KD=21 nM. High specificity towards its target could be shown both in vitro and in vivo. The candidate was then used for partial randomisation to search for improved mutants. By combining several of these mutations, a paromomycin specific riboswitch with 8.5-fold regulation could be engineered. Further engineering was performed based on the structural analysis to exchange a part of the riboswitch with the neomycin riboswitch for the assembly of an in vivo NOR Boolean logic gate resulting in 30-fold regulation." @default.
• W2944604860 created "2019-05-16" @default.
• W2944604860 creator A5000546131 @default.
• W2944604860 date "2018-09-15" @default.
• W2944604860 modified "2023-09-27" @default.
• W2944604860 title "Development of RNA aptamers and synthetic riboswitch using Capture-SELEX" @default.
• W2944604860 hasPublicationYear "2018" @default.
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