Draft:DogTag/DogCatcher

Quick info box:

• Number of Amino Acids

○ DogTag: 23 ○ DogCatcher: 104

• Residues from RrgA

○ DogTag: 838-860 ○ DogCatcher: 734-838

• Reactive Residues

○ DogTag: Asn854 ○ DogCatcher: Lys742 and Glu803

Two systems were developed for DogTag and DogTag/DogCatcher. Both are derived from splitting the C-terminal domain (domain 4) of RrgA.,^[1] a pilus-associated adhesin protein from Streptococcus pneumoniae Streptococcus Pneumoniae. To develop the 3-part system, RrgA was split into three components, each containing a single reactive residue. These included the peptides SnoopTag, later mutated and termed SnoopTagJr, with a reactive Lysine (residues 734-745) and DogTag with a reactive Asparagine (residues 838-860), and the protein RrgA ligase, later mutated and termed SnoopLigase, with a catalytic Glutamate (residues 743-846).^[2] To develop the 2-part system, RrgA was split into two components. These included the peptide R2Tag with a reactive asparagine (residues 838-846) and the protein R2Catcher with both a reactive lysine and catalytic glutamate (residues 734-838). DogTag was created from R2Tag by introducing the mutations G842T, N847D, and D848G. DogCatcher was created by predicting mutations using Rosetta prediction in R2Catcher to form R2CatcherB and then creating a library of mutations in R2CatcherB followed by phage display Phage Dislpay.^[3]

DogTag is a small, 23-amino acid (DIPATYEFTDGKHYITNEPIPPK) peptide formed from residues 838-860 of RrgA adhesin. In the 2-part system, it contains mutations G842T, N847D, and D848G. The peptide forms a β-hairpin structure and includes a reactive asparagine (Asn854) that forms an isopeptide bond with Lys742.^[2][3]

DogCatcher is a 15 kDa, 104-amino acid protein formed from residues 734-838 of RrgA adhesin with mutations at D737E, N744D, N746T, N780D, K792T, F802I, A808P, A820S, Q822R, and N825D. The protein includes a reactive lysine (Lys742) and a catalytic glutamate (Glu803).^[3]

Researchers have previously developed and optimized a protein-protein ligation pair called SpyTag and SpyCatcher SpyCatcher for use at the protein/peptide termini, but its use in internal protein regions has been unsuccessful due to the rigidity and size of the tag, as loop regions need to retain their flexibility to not disrupt the protein structure. Internal loop tagging can be beneficial, as terminal tagging can affect structure/function depending on the protein of interest. Additionally, some proteins, such as transmembrane proteinsTransmembrane Protein, do not have exposed termini, rendering terminal ligation impossible for mechanistic studies. As such, researchers developed DogTag, which can be effectively inserted directly into a nonfunctional loop of a protein, allowing for protein-peptide covalent binding via a spontaneous isopeptide bond Isopeptide Bond formation of Asn 854 residue on the inserted DogTag and the lysine 742 residue on the complementing DogCatcher upon mixing. Via a combination of rational design and phage evolution, the DogTag-DogCatcher reaction yields 98% conversion at nanomolar concentrations^[3].

The researchers inserted the DogTag into loop regions of several natural proteins that have struggled to be efficiently tagged on their termini, including a GFP variant, a transmembrane ion channel protein, and a functional enzyme. Upon optimizing the placement of the DogTag within the loop region of the protein, researchers achieved effective insertion with sustained high affinity for the DogCatcher protein, with minimal inhibition of native protein function^[3].

In addition to a single peptide-protein ligation between DogTag and DogCatcher, other chemistry has been developed to incorporate a single ligation between two tagged proteins. In this mechanism, the same base structure, RrgA domain 4, was split into 3 components (as described above).^[4] In these studies, the 12 residue SnoopTagJr containing the reactive lysine residue can be conjugated to the terminus of one protein of interest while the 23 residue DogTag can be conjugated to either a terminal region or internal loop region of an additional protein of interest. The SnoopLigase protein catalyzes the ligation of the two tags to generate the same Lys 742-Asn 854 isopeptide bond, thereby facilitating the conjugation of two tagged proteins or peptides.^{[citation needed]}

The initial proof of concept for the three-component ligation involved a nanoparticle with numerous DogTags conjugated to its surface and a library of antigens engineered to express a terminal SnoopTagJr. In the presence of SnoopLigase, the researchers produced a multivalent nanoparticle with a library of covalently attached antigens for potential use as a therapeutic for rapidly-mutating pathogens.^[4]

Dog Tag/DogCatcher–based modification systems represent a novel and highly versatile platform for site-specific bioconjugation in both in vitro and in vivo therapeutic applications. This technology enables rapid, covalent, and highly specific attachment of functional molecules to biological carriers, allowing for enhanced targeting, stability, and modular design of next-generation therapeutics. Dog Tag/DogCatcher systems have been explored across multiple domains, including gene therapy, cell engineering, oncolytic therapies, and vaccine development, demonstrating broad translational potential. For gene therapy Gene Therapy use, Dog Tag/DogCatcher has been applied to modify adeno-associated viruses Adeno-Associated Virus to improve targeting specificity and therapeutic precision.^[5][6] By conjugating targeting ligands or receptor-binding proteins to the viral capsid, these engineered AAVs increase transduction efficiency in the desired cells, reduce off-target effects, and have the potential to deliver lower doses in therapeutics.

For cell engineering Cell Engineering use, Dog Tag/DogCatcher has been added to the exterior of nucleocapsids^[6][7] that deliver RNA. The DogTag/DogCatcher binds to the desired cells via specific receptors on the cell surfaces. DogTag/DogCatcher modified nucleocapsids have shown increased RNA uptake and no excess release of nucleocapsids or RNA from cells. Desired cells were identified by the display proteins generated after incorporation of the nucleocapsid and RNA.

For oncolytic^[8] use, DogTag/DogCatcher has been used to develop modular prodrugs that allow selective loading of therapeutics, reduced off-target effects, and specified activation. While similar to SpyTag/Spy Catcher, DogTag/DogCatcher had faster reaction kinetics.

For vaccine use Vaccine, Dog Tag/DogCatcher is being investigated as a platform for next-generation vaccines. Its ability to covalently display antigens on viral or nanoparticle surfaces enables precise antigen presentation and improved immune activation. Current investigations into the synthesis of a new HPV vaccine^[5] have shown increased immunogenicity in mice, as evidenced by neutralizing and cross-neutralizing antibody titers. DogTag/DogCatcher has been used in an adenovirus vector COVID-19 vaccine^[6] to protect the spike proteins, reduce neutralization, improve delivery efficacy, and enhance antigen presentation to strengthen immunoresponses.