OaAEP1-mediated PNA-protein conjugation enables erasable imaging of membrane proteins

Abstract

We report the use of a protein ligase to covalently ligate a protein to a peptide nucleic acid (PNA). The rapid ligation demands only an N-terminal GL dipeptide in target...

Synthesis of Protein-Oligonucleotide Conjugates

“…Similarly, the asparaginyl endopeptidase OaAEP1 can transfer a molecule bearing an N-terminal GL tag to a protein containing a NGL consensus sequence through the liberation of the C-terminal GL dipeptide. [ 9 ] Enzymes can also be used to create a reactive intermediate such as in the case of the formylglycine generating enzyme (FGE) which recognizes a CXPXR consensus sequence and converts the requisite cysteine residue into a formylglycine, allowing for the site-specific placement of a reactive aldehyde within the protein sequence. [ 40 ] This residue can then react with an incoming nucleophile such as an alkoxyamine to facilitate conjugation.…”

“…In addition to these readout applications protein-oligonucleotide conjugates can also be utilized for structural purposes. These predominantly involve the site-specific localization of target proteins to structures including DNA origami, [ 2 , 6 , 9 , 11 , 23 , 24 , 37 , 46 , 49 ] arrays, [ 35 ] nanoparticles [ 25 ] or even the surfaces of phages [ 41 ] to impart new activity. The oligonucleotide tags can also be used to bring labelled protein units together to form new entities with precise three-dimensional arrangements, [ 7 , 39 ] such as the characteristic Y-shape to be able to mimic antibody binding [ 26 , 40 ] or to recapitulate enzyme activity [ 22 , 28 , 33 , 45 ].…”

“…For these reasons nucleic acids are often used for specific recognition of partner strands enabling the formation of defined nano objects, or for the physical transfer of information through conformational change by acting as molecular circuitry. It is important to note that such systems can rely equally on native DNA or on designer nucleic acids (such as peptide nucleic acids (PNA) [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ] or threoninol nucleic acids [ 11 ]) to fulfill this role. In contrast, proteins have a much wider array of potentially accessible structures as a result of their larger number of component building blocks, as well as the broader range of interactions these can undergo to influence global structure [ 1 ].…”

Tertiary amide bond formation by an engineered asparaginyl ligase

“…[C247A]OaAEP1 is a versatile ligase that has been used for a broad range of protein engineering and biotechnological applications. [7][8][9][10][11][12][13] This enzyme acts on substrates bearing a tripeptide Asn-Gly-Leu (P1YP1 0 -P2 0 ) recognition motif, with cleavage of the Asn-Gly peptide bond generating a thioesterlinked acyl-enzyme intermediate that is highly susceptible to nucleophilic attack. Conventional nucleophiles include peptides or proteins with an N-terminal Gly-Leu sequence, with the resulting transpeptidation reaction providing direct access to N-or C-terminally labelled proteins, 9,[13][14][15][16] head-to-tail cyclisation of peptides or proteins, 14,17 or dened protein-protein fusions.…”

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization

“…[41] More recently Lu et al used a protein ligase to connect PNA to a cell surface protein. [42] In this article, we exploited the orthogonality and biostability of XNAs combined with SNAP-tag engineering to develop a robust and versatile methodology for mediating artificial cellcell interactions. To do so, we genetically engineered E. coli to express the protein SNAP-tag at the cell surface and subsequently conjugated it to both complementary unmodified and modified oligonucleotides at the surface of two distinct cell populations forming a bacterial tissue.…”