Protein labeling and crosslinking by covalent aptamers

Abstract

In this chapter, a new approach to the selective modification of native proteins is discussed, using electrophilic covalent aptamers. These biochemical tools are generated through the site-specific incorporation of a label-transferring or crosslinking electrophile into a DNA aptamer. Covalent aptamers provide the ability to transfer a variety of functional handles to a protein of interest or to irreversibly crosslink to the target. Methods for the aptamer-mediated labeling and crosslinking of thrombin are described. Thrombin labeling is fast and selective, in both simple buffer and in human plasma and outcompetes nuclease-mediated degradation. This approach provides facile, sensitive detection of labeled protein by western blot, SDS-PAGE, and mass spectrometry.

Keywords: Aptamer; Electrophile; Nucleic acid; Protein bioconjugation; Protein labeling.

Fig. 1

Incorporation of 5-(triisopropylsilyl)ethynyl-5’-DMTr-2′-deoxyuridine (TIPS-EdU) into the aptamer sequence through solid-phase synthesis (SPS) and subsequent reaction with a cleavable N-acylsulfonamide electrophile generates the covalent aptamer. Incubation of the covalent aptamer with its protein target leads to selective, covalent amino acid side chain modification and transfer of a label (e.g., biotin).

Fig. 2

Electrophile position-dependent biotinylation of thrombin. (A) Individual TBAs modified with the biotin-transferring electrophile at different positions were incubated with thrombin for 1 h. Thrombin biotinylation was observed by western blot. Electrophile installed at position 3 of TBA biotinylates thrombin most efficiently. (B) Data represent averages of two experiments and error bars are standard deviation from at least two independent experiments. This figure was adapted with permission from Tivon, Y., Falcone, G., & Deiters, A. (2021). Protein labeling and crosslinking by covalent aptamers. Angewandte Chemie (International Ed. in English), 60(29), 15899–15904. doi:10.1002/anie.202101174.

Fig. 3

Crystal structure of TBA (blue) complexed to thrombin (purple) with biotinylated lysines highlighted in green. The aptamer position T3 is in proximity to the labeled lysines K36, K81, and K109-110 as determined by mass spec sequencing. One of the biotinylated lysines, K149, is missing in the crystal structure. PDB 4DII. This figure was adapted with permission from Tivon, Y., Falcone, G., & Deiters, A. (2021). Protein labeling and crosslinking by covalent aptamers. Angewandte Chemie (International Ed. in English), 60(29), 15899–15904. doi:10.1002/anie.202101174.

Fig. 4

Overview of aptamer-mediated crosslinking to target protein. (A) Structure of the crosslinking warhead. (B) Overview of the crosslinking reaction. (C) Position-dependent crosslinking of modified TBA with thrombin. (D) Fibrinogen clotting assay. Crosslinking TBA prolongs clotting time compared to unmodified TBA. Error bars are standard deviations from averages of two independent experiments. This figure was adapted with permission from Tivon, Y., Falcone, G., & Deiters, A. (2021). Protein labeling and crosslinking by covalent aptamers. Angewandte Chemie (International Ed. in English), 60(29), 15899–15904. doi:10.1002/anie.202101174.