Tolle, Fabian: Click-SELEX : A versatile approach towards nucleobase-modified aptamers. - Bonn, 2016. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-44920
@phdthesis{handle:20.500.11811/6896,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-44920,
author = {{Fabian Tolle}},
title = {Click-SELEX : A versatile approach towards nucleobase-modified aptamers},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2016,
month = nov,

note = {Aptamers are short nucleic acids that can be selected to bind specifically and with high affinity to a variety of targets, including small organic molecules and proteins. However, for a multitude of targets, traditional SELEX methods still fail to yield suitable ligands. One possible cause is the limited chemical diversity found in the mere four different building blocks from which nucleic acids are constituted. Therefore, the addition of naturally not represented functional groups, e.g. the ones found in the side chains of proteinogenic amino acids, can increase the repertoire of possible interactions of nucleic acids and thereby expand the addressable target spectrum, enabling the selection of novel high-affinity binders.
Click-SELEX is a versatile method to facilitate the modular introduction of chemical entities during the SELEX process. In this approach chemical groups are introduced into the DNA library before the selection step (in situ) via click chemistry and are subsequently removed during the amplification step, thereby avoiding enzymatic incompatibility problems associated with larger nucleobase modifications. Hence, this system allows for an easy implementation of a multitude of different chemical functionalities adapted to the imposed requirements.
The development of all associated methods has been validated by the selection of a highly specific aptamer (”clickmer”) interacting with the Green Fluorescent Protein (GFP). A 3-(2- azidoethyl)-indole functionalized DNA library was enriched for C3-GFP immobilized via a His-tag on magnetic beads. Traditional and next-generation sequencing of the enriched library revealed two aptamer families. C12, the most abundant monoclonal sequence, was shown to interact with high affinity with C3-GFP immobilized on beads and in solution. C12’s binding affinity towards C3-GFP was demonstrated to be critically dependent on the correct functionalization state of three positions, as omission or substitution of the indole functionalization at these nucleotides led to a complete loss of binding affinity. The C12 clickmer also displayed a high degree of specificity, being capable of differentiating between two members of the GFP family.
Having pioneered the modular introduction of nucleobase functionalization the methods developed in this proof of concept study can now be applied to a variety of other functionalizations, selection strategies, and targets. The vast applicability of the click-SELEX method will rapidly advance the application of in vitro selection approaches beyond what was previously feasible, allowing, for example, the generation of aptamers to yet non-targetable molecules.},

url = {https://hdl.handle.net/20.500.11811/6896}
}

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