Site-Specific and Trigger-Activated Modification of Proteins by Means of Catalytic Hemin/G-quadruplex DNAzyme Nanostructures

Abstract

Catalytic nanostructures have the potency to mimic enzymatic features. In this paper, we show that the complex between hemin and G-quadruplex DNA efficiently catalyzes the modification of proteins with N -methyl luminol derivatives. Final conversions are reached within 15–30 min, and LC-MS analysis of tryptic digests of the proteins shows that the reaction proceeds with chemoselectivity for electron-rich aromatic residues (Tyr ≫ Trp), and the site-specificity of the modification depends on the sequence and secondary structure folding of the G-quadruplex nanostructure. Furthermore, the modification can be applied on proteins with different biomedical functions, and the nanostructure can be designed to contain a regulatory element in order to regulate protein modification by an external stimulus.

Aptamer‐Assisted Bioconjugation of Tyrosine Derivatives with hemin/G‐quadruplex (hGQ) DNAzyme Nucleoapzyme Nanostructures

“…Only the hGQ DNAzyme based on the human telomere (HT) repeat sequence did not display enhanced activity. In accordance with previous reports by us [3] and others, [16,17] the anti-parallel conformation of the hGQ DNAzyme is the least active, followed by the hybrid/mixed and intermolecular parallel structures, and with the parallel conformation being the most active. Differences in activity among DNAzymes with the same conformation type can be attributed to subtle differences in the interaction between the DNA G-tetrads and hemin, caused by the other nucleobases in the DNA sequence.…”

“…Specifically, whereas hemin or the PW17 DNAzyme hardly modified this amino acid residue, the nucleoapzymes gave significantly higher conversions (14-16 %). Although this result supports our earlier finding that Trp can also be modified by hGQ DNAzyme-catalyzed NML conjugation, 3 we have not yet been able to identify the site of modification with atomic precision. Still, conversions on tyrosine almost reach 40 %, which indicates that the aptamer is more efficient in assisting transformations on this residue.…”

“…Recently, we [3] and others [4] have shown that the horseradish peroxidase-mimicking hemin/G-quadruplex (hGQ) DNAzyme catalyst [5] has the ability to efficiently modify tyrosine (Tyr) residues in a variety of proteins with N-methyl luminol derivatives. [6] Applications of this hGQ DNAzyme as proteinmodifying catalyst in a switchable nanostructure showed that the modification could also be performed in more advanced settings.…”

“…[6] Applications of this hGQ DNAzyme as proteinmodifying catalyst in a switchable nanostructure showed that the modification could also be performed in more advanced settings. [3] Although the hGQ nanostructure itself provided a basic level of substrate selectivity, its conjugation to structural elements that specifically recognize a substrate could, in principle, enable a higher level of selectivity.…”

Site-selective and inducible acylation of thrombin using aptamer-catalyst conjugates

“…Although conditions have been established to derivatize specific residues in some proteins, [6][7][8] tedious optimization is often required when applied to different proteins. Alternatively, catalytic modification of reactive residues 9,10 has resulted in various degrees of selective protein modification, especially when guided by protein-binding ligands. 11,12 Theoretically, this principle can also be applied using protein-binding aptamers, [13][14][15][16] but this has not yet been shown.…”