Temporal Control of Efficient In Vivo Bioconjugation Using a Genetically Encoded Tetrazine-Mediated Inverse-Electron-Demand Diels–Alder Reaction

Abstract

An inverse-electron-demand Diels–Alder (IEDDA) reaction using genetically encoded tetrazine variants enables rapid bioconjugation for diverse applications in vitro and in cellulo. However, in vivo bioconjugation using genetically encoded tetrazine variants is challenging, because the IEDDA coupling reaction competes with rapid elimination of reaction partners in vivo. Here, we tested the hypothesis that a genetically encoded phenylalanine analogue containing a hydrogen-substituted tetrazine (frTet) would increase the IEDDA reaction rate, thereby allowing for successful bioconjugation in vivo. We found that the in vitro IEDDA reaction rate of superfolder green fluorescent protein (sfGFP) containing frTet (sfGFP-frTet) was 12-fold greater than that of sfGFP containing methyl-substituted tetrazine (sfGFP-Tet_v2.0). Additionally, sfGFP variants encapsulated with chitosan-modified, pluronic-based nanocarriers were delivered into nude mice or tumor-bearing mice for in vivo imaging. The in vivo-delivered sfGFP-frTet exhibited almost complete fluorescence recovery upon addition of trans-cyclooctene via the IEDDA reaction within 2 h, whereas sfGFP-Tet_v2.0 did not show substantial fluorescence recovery. These results demonstrated that the genetically encoded frTet allows an almost complete IEDDA reaction in vivo upon addition of trans-cyclooctene, enabling temporal control of in vivo bioconjugation in a very high yield.

Thermostable and Long-Circulating Albumin-Conjugated Arthrobacter globiformis Urate Oxidase

“…In order to prepare 14 AgUox-frTet variants, an amber codon was introduced to each of the14 sites of AgUox-WT by PCR-mediated mutagenesis. Then, C321delAexp E. coli cells [ 34 ] were co-transformed into pDule C11RS plasmid [ 35 ] encoding the engineered MjtRNA Tyr /MjTyrRS specific for ftTet as well as the vectors with each AgUox variant. The transformants were cultured to express each AgUox-frTet variant as described in ‘Materials and Methods’ ( Section 2.3 ).…”

Chemical Modification of Cysteine with 3-Arylpropriolonitrile Improves the In Vivo Stability of Albumin-Conjugated Urate Oxidase Therapeutic Protein

“…First, we prepared AgUox containing frTet (Figure 1) by adapting expression and purification methods previously described except that AgUox was used instead of Aspergillus flavus Uox [36]. Among the non-natural amino acids containing the tetrazine group for the IEDDA reaction, frTet, whose reactivity has been well defined [32,36,40], was successfully incorporated into the protein.…”

“…To compare the in vivo stabilities of thiol-maleimide and thiol-APN reaction products, we first site-specifically incorporated a phenylalanine containing a fast-reacting tetrazine (frTet) into urate oxidase (Uox) using the optimized Methanococcus jasnacci tyrosyl-tRNA/synthetase (MjtRNA Tyr /MjTyrRS) pair [32]. The recombinant Uox and poly-ethylene glycol-conjugated Uox are therapeutic proteins to treat tumor lysis syndrome and gout, respectively [33][34][35].…”

Effect of C-terminus Conjugation via Different Conjugation Chemistries on In Vivo Activity of Albumin-Conjugated Recombinant GLP-1

“…To append the sfGFP-GLP1_8G37C functional group for reaction with HSA, TET-PEG4-MAL was reacted with sfGFP-GLP1_8G37C to generate sfGFP-GLP1_8G37C-TET. We chose this linker in order to use the IEDDA, which is known for its high reactivity compared to that of the formerly used bioorthogonal SPAAC [31]. The reactivity of methyltetrazine was evaluated by conjugating the linker to a fluorescent dye.…”