Covalent protein–oligonucleotide (POC) conjugates (POC) are intensely studied for a broad range of applications in biology, biotechnology, bioimaging, and DNA nanotechnology. They are additionally interesting for diagnostic assays and biosensors. [1], [2], [3] Various methods have been attempted to bind oligonucleotides to proteins covalently and to achieve a homogeneous protein tethering, characterised by a single oligonucleotide attached to a specific position on the protein. [4] The most commonly used protocols involve the direct attachment of an oligonucleotide to the reactive functional groups on a protein, usually to free cysteines, [5] amino-groups or carboxy groups. [4], [6]

The direct covalent attachment of the maleimide modified oligonucleotides (prepared via Sulfo-SMCC cross-linkers or obtained through commercial suppliers7) to a free cysteine residue is widely applied. The maleimide−thiol addition reaction is performed without a catalyst in aqueous buffers and the pH range of 6.5−7.5.. Still, aside from limited reaction yield, it suffers from other drawbacks. In proteins, most thiols are often inaccessible within the folded protein and form disulfide bonds essential for protein stability. Reducing reagents prior to the conjugation can cleave disulphide bonds and inactivate the proteins. Furthermore, maleimide-functionalized oligonucleotides sometimes undergo autohydrolysis in aqueous solutions, showing photo-induced dimerisation at higher concentrations. [8] These disadvantages are significant for the horseradish peroxidase protein (HRP), since it contains disulphide bonds essential for enzymatic activity. [9] Less-reactive carboxylic acids present in glutamic acid, aspartic acid, and at the C-terminal end can also be conjugated, as well as serine, tyrosine, and histidine, which, similarly to other methods, give low yields and protein mixtures usually with altered activities. To overcome such difficulties, mild, efficient, and general methods promoting site-selective modifications of proteins are increasingly important.

Alternatively, labelling amino groups that emerge predominantly on the outside surfaces of protein tertiary structures is one of the oldest and most versatile techniques for protein conjugation due to their nucleophilicity and accessibility to conjugation reagents introduced into the aqueous media. [10] Surface lysine residues are among the most available functional groups present in most proteins. However, the chemistry is rarely site-specific due to the abundance of secondary amines (lysine, N-terminal amino group), [11] resulting in heterogeneous mixtures of conjugates containing several oligonucleotides per protein. Notably, the N-terminal residue represents a unique reactive site for chemical modifications as this position is usually well-exposed (80.3%) [12] to the solvent and is commonly accessible for chemical modifications. Several chemical techniques can be used to modify N-terminal amino acids directly or convert them into unique functional groups for further ligations. [13] Azidation of the N-terminal α-amino group is one potentially useful approach to achieve the site-selective modification of proteins. Azides can be introduced in proteins in various ways: via genetic engineering, [14], [15], [16], [17] chemical or enzymatic modifications. [18], [19], [20], [21] For instance, Van Dongen and co-workers reported the introduction of azides non-selectively at all amino residues of HRP by using an aqueous diazotransfer reaction. [22] This approach involved Cu(II) as a catalyst, which is difficult to remove after the reaction, and thus includes some adverse effects for potential downstream reactions [23] related to the cytotoxicity of the copper cation and the risk of Cu-mediated protein inactivation. [24] The same group further developed a novel methodology on non-glycosylated proteins (not HRP) omitting Cu-catalyst, and by fine-tuning the pH, they achieved the highly selective conversion to azide only at the N-terminal amine, while the lysine side chain amines remained intact. [18] This high selectivity was based on the difference in the pKa values of the N-terminal α-amine and ε-amine of the lysine residues (8.5 and 10.5 for α- and ε-amines, respectively). Since the diazotransfer reagent (hydrochloric salt of imidazole-1-sulfonyl azide, ISA·HCl) requires a deprotonated amine as a reactive nucleophile, by pH adjustment, the reactivity of the α-amino group versus ε-amines can be selectively controlled, allowing the site-selective modification at the N-terminus (α-amine) at pH 8.5. [13]

Such obtained protein-N-terminal azides are usually further reacted by various “click” chemistry methodologies, among which the cyclooctyne-based strain promoted alkyne-azide cycloaddition (SPAAC) is characterised by exceptionally mild and bioapplicable conditions in aqueous solutions to get almost quantitative yields of stable triazoles without the need for a toxic copper catalyst. 23,25

Thiol-based linking procedures relying on all available HRP-amines (4 lysines, N-terminus amine) and thiol-modified oligonucleotides are already available. [26], [27] However, they are non-selective and are based on the SMCC bifunctional linker methodology, which has several disadvantages. Namely, SMCC-modified proteins can be unstable and self-reactive since they often contain both amine and thiol groups that cause a significant amount of homo-crosslinking. The conjugation linkage is exchangeable when other thiol groups are present.

Another methodology based on a proprietary, protected linker [28] also does not ensure specific binding at only one position of HRP since it reacts non-selectively with all available protein-amines. [29]

A further approach is based on the conjugation of a 5'-modified oligonucleotide directly linked to several available HRP carboxylates via an EDC coupling method, whereby careful control of reagents can yield dominantly 1:1 stoichiometry of HRP:DNA conjugates. [30] However, it remains unclear which carboxylate is primarily targeted or, more likely, several different positions are involved, thus resulting in a heterogeneous HRP modification.

In the present study, we developed a copper-free method for the regio-selective DNA-tagging of HRP. It is based on a pH-controlled diazotransfer reaction allowing the highly selective introduction of an azide only to the N-terminus of the HRP (Scheme 1). Then, the N-terminally azide-modified HRP could be “clicked” to DBCO-modified oligonucleotides using copper-free click chemistry (SPAAC). To complement the experimental research and to provide insight into the molecular level, molecular dynamics (MD) simulations of the novel HRP-DNA conjugate were conducted, as well as of the HRP alone. The simulations focused on possible structural shielding of the enzyme’s active site entrance by the oligonucleotide and on possible influences that the DNA might have on the structural and dynamical properties of the protein.