Synthesis of Protein-Oligonucleotide Conjugates

Abstract

Nucleic acids and proteins form two of the key classes of functional biomolecules. Through the ability to access specific protein-oligonucleotide conjugates, a broader range of functional molecules becomes accessible which leverages both the programmability and recognition potential of nucleic acids and the structural, chemical and functional diversity of proteins. Herein, we summarize the available conjugation strategies to access such chimeric molecules and highlight some key case study examples within the field to showcase the power and utility of such technology.

Keywords: bioconjugation; oligonucleotides; proteins.

Figure 1

Comparison of nucleic acids and proteins as molecular building blocks.

Figure 2

Conjugation chemistries utilized in the formation of protein oligonucleotide conjugates including (A) NHS-esters, (B) maleimides, (C) disulfide bond formation, (D) NCL, (E) CuAAC and SPAAC, (F) oxime ligation, (G) tetrazine click, (H) enzyme conjugation and (I) self-labelling proteins.

Figure 3

Templated conjugation relies on the regiospecific binding of a recognition motif to the protein surface, thereby imparting regiospecificity to the conjugation event. R: see red box for examples of interaction; C: see blue box for examples of crosslinking moieties; POI: protein of interest; PPI: protein-protein interaction.

Figure 4

Case Study 1: lysine directed labelling of IgG1 for templated reactivity. (A) Initial iminium ion formation activates the p-phenyl ester towards attack from a second nearby lysine residue, enabling site specific conjugation to the hinge region of IgG1 antibodies (B). This can be exploited for proximity templated chemistry through the use of an orthogonal pair of antibodies against a single target (C).

Figure 5

Case Study 2: engineered Protein G-oligonucleotide conjugates as generic handles for labelling antibodies and Fc-fusion proteins. A bifunctional linker is used to conjugate oligonucleotides to a mutant Protein G containing a single cysteine residue. The specificity of Protein G for the Fc domain of antibodies is then harnessed to facilitate site specific conjugation through a BPA residue (represented as a red circle).

Figure 6

Case Study 3: Coiled-coiled templation for live-cell imaging. (A) Coiled-coil templation allows for site specific conjugation of an oligonucleotide to a membrane protein via native chemical ligation (B).

Figure 7

Case Study 4: hijacking post-translational processing to facilitate conjugation. (A) the HhC domain undergoes self-splicing and is liberated from an N-terminal fusion protein by the addition of an incoming sterol. (B) This can be exploited for the synthesis of nucleic acid sensing enzymatic beacons.

Figure 8

Case Study 5: conjugation facilitates the formation of a precise three-dimensional reaction center for precise drug release. SNAP labelling and ligand templation allow the nLuc luciferase to be brought into close proximity to the photocatalyst to enable BRET-based uncaging of cargo molecules.