Aptamers are single-stranded oligonucleotides that are in vitro-selected to recognize their target molecule with high affinity and specificity. As they consist of the four canonical nucleobases, their chemical diversity is limited, which in turn limits the addressable target spectrum. Introducing chemical modifications into nucleic acid libraries increases the interaction capabilities of the DNA and thereby the target spectrum. Here, we describe a protocol to select nucleobase-modified aptamers by using click chemistry (CuAAC) to introduce the preferred chemical modification. The use of click chemistry to modify the DNA library enables the introduction of a wide range of possible functionalities, which can be customized to the requirements of the target molecule and the desired application. This protocol yields modified DNA aptamers with extended interaction properties that are not accessible with the canonical set of nucleotides. After synthesis of the starting library containing a commercially available, alkyne-modified uridine (5-ethynyl-deoxyuridine (EdU)) instead of thymidine, the library is functionalized with the modification of choice by CuAAC. The thus-modified DNA is incubated with the target molecule and the best binding sequences are recovered. The chemical modification is removed during the amplification process. Therefore, this protocol is compatible with conventional amplification procedures and avoids enzymatic incompatibility problems associated with more extensive nucleobase modifications. After single-strand generation, the modification is reintroduced into the enriched library, which can then be subjected to the subsequent selection cycle. The duration of each selection cycle as outlined in the protocol is ∼1 d.
Biomedical sciences require effective tools to manipulate,d etect, and study biological phenomena. Oligo-(deoxy)nucleotide ligands represent such tools,but the current strategies to generate them are restricted. Their limited availability is insufficient to address the broad range of targets related to biomedical research. Exemplified by targeting the hydrophobic molecule (À)-D 9 -tetrahydrocannabinol (THC), we report areceptor-guided design (RGD) strategy to generate chemically modified oligodeoxynucleotide libraries for the tailored selection of clickmers.
Angewandte ChemieCommunications
The post-synthetic functionalization of nucleic acids via click chemistry (CuAAC) has seen tremendous implementation, extending the applicability of nucleobase-modified nucleic acids in fields like fluorescent labeling, nanotechnology, and in vitro selection. However, the production of large quantities of high-density functionalized material via solid phase synthesis has been hampered by oxidative by-product formation associated with the alkaline workup conditions. Herein, we describe a rapid and cost-effective protocol for the high fidelity large-scale production of nucleobase-modified nucleic acids, exemplified with a recently described nucleobase-modified aptamer.
Die Biomedizin bençtigt effektive Werkzeuge um biologische Phänomene zu manipulieren, detektieren und studieren. Oligo(deoxy)nukleotid-basierte Liganden stellen solcheWerkzeuge dar,aber die momentanen Strategien fürihre Selektion sind eingeschränkt. Ihre eingeschränkte Verfügbarkeit genügt nicht, das breite Spektrum an Zielmolekülen abzudecken, das fürdie Biomedizin von Bedeutung ist. Anhand des hydrophoben Moleküls (À)-D 9 -Tetrahydrocannabinol (THC) stellen wir eine rezeptorgeleitete Designstrategie (RGD) vor,d ie die Generierung modular anpassbarer,c hemischm odifizierter Oligodeoxynukleotidbibliothekenz ur Selektion von Clickmeren ermçglicht.
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