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Identification and characterization of nucleobase-modified aptamers by click-SELEX

Nature Protocols volume 13, pages 1153–1180 (2018)Cite this article

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Abstract

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.

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Figure 1: Overview of the click-SELEX process.
Figure 2: Azide-modified competitor oligonucleotides.
Figure 3: HPLC-chromatogram of nucleoside digest of unmodified and benzyl-modified FT2-pool.
Figure 4: PAGE gel of 32P-labeled starting library and background binding determination in the presence of different competitors.
Figure 5: Background binding determination of differently modified starting libraries on THC-modified and unmodified beads.
Figure 6: Impact of clicked competitor on sequence frequency and kD determination of p5.
Figure 7: Binding assays of libraries of SELEX cycles as well as clones from click-SELEX for THC.
Figure 8: Modulation of binding of clickmers C12 and p5 by changing the clicked-in moiety.

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Acknowledgements

This work was made possible through funding by BMWi-ZIM (grant no. KF3058901SK2) and the Deutsche Forschungsgemeinschaft (grant nos. MA3442/4-1 and MA3442/4-2). We thank J.L. Schultze, K. Händler and M. Beyer (Life and Medical Sciences Institute and German Center for Neurodegenerative Diseases, Bonn) for their support regarding next-generation sequencing and M. Blank and C. Gröber (AptaIT, Munich, Germany) for their support regarding the NGS analysis. We also thank Protzek Gesellschaft für biomedizinische Technik mbH (Lörrach, Germany) for support and helpful discussions.

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Author notes

  1. Fabian Tolle & Gerhard Markus Brändle

    Present address: Present addresses: Department of Biosystems Science and Engineering, ETH Zurich, Zurich, Germany (F.T.); CHT Germany GmbH, Oyten, Germany (G.M.B.).,

  2. Franziska Pfeiffer and Fabian Tolle: These authors contributed equally to this work.

Authors and Affiliations

  1. Life and Medical Sciences Institute, University of Bonn, Bonn, Germany

    Franziska Pfeiffer, Fabian Tolle, Malte Rosenthal, Gerhard Markus Brändle, Jörg Ewers & Günter Mayer

  2. Center of Aptamer Research and Development, University of Bonn, Bonn, Germany

    Franziska Pfeiffer, Fabian Tolle, Malte Rosenthal, Gerhard Markus Brändle, Jörg Ewers & Günter Mayer

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Contributions

F.P., F.T. and G.M. conceived and designed the protocol and wrote the manuscript. F.P., F.T. and M.R. performed the experiments. G.B. and J.E. synthesized the azides used in this study.

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Correspondence to Günter Mayer.

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Pfeiffer, F., Tolle, F., Rosenthal, M. et al. Identification and characterization of nucleobase-modified aptamers by click-SELEX. Nat Protoc 13, 1153–1180 (2018). https://doi.org/10.1038/nprot.2018.023

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