Abstract
While proteins are highly biochemically competent, DNA offers the ability to program, both reactions and the assembly of nanostructures, with a control that is unprecedented by any other molecule. Their joining: DNA–protein conjugates - offer the ability to combine the programmability of DNA with the competence of proteins to form novel tools enabling exquisite molecular control and the highest biological activity in one structure. However, in order for tools like these to become viable for biological applications, their production must be scalable, and an entirely enzymatic process is one way to achieve this. Here, we present a step in this direction: enzymatic production of DNA–protein conjugates using a new self-labeling tag derived from a truncated VirD2 protein of Agrobacterium tumefaciens. Using our previously reported MOSIC method for enzymatic ssDNA oligo production, we outline a pipeline for protein–DNA conjugates without the need for any synthetic chemistry in a one-pot reaction. Further, we validate HER2 staining using a completely enzymatically produced probe, enable the decoration of cell membranes and control of genetic expression. Establishing a method where protein–DNA conjugates can be made entirely using biological or enzymatic processing, opens a path to harvest these structures directly from bacteria and ultimately in-vivo assembly.
Highlights
Achieving a successful DNA–protein conjugate, despite the availability of several different approaches [1,2,3,4,5,6,7], remains a pitfall-prone process that is highly protein dependent and requires optimizations for each new conjugate
The purified self-tagging domain mVirD2 was eluted in TKM buffer, which provides the amount of magnesium (1 mM) required for the conjugation reaction to take place [16]
We have shown that the minimal version of VirD2 retains the self-tagging properties of the full-length protein
Summary
Achieving a successful DNA–protein conjugate, despite the availability of several different approaches [1,2,3,4,5,6,7], remains a pitfall-prone process that is highly protein dependent and requires optimizations for each new conjugate. Among the plethora of naturally occurring proteins some have evolved a very peculiar function: the ability to establish a covalent bond at a specific sequence location of an unmodified nucleic acid molecule with the use of divalent ions as sole cofactor [10]. Such proteins are involved in genetic material transfer from a pathogen to its host [11], in DNA digestion [12], remodelling [13,14] and replication [15]. The capability of these proteins to form strong bonds is essential to avoid a loss of their DNA cargo while subjected to mechanical stresses
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