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12. Temporal Control of Efficient In VivoBioconjugation Using a Genetically Encoded Tetrazine-Mediated Inverse-Electron-Demand Diels–Alder Reaction

Author

Yang, Byungseop, Kwon, Kiyoon, Jana, Subhashis, Kim, Seoungkyun, Avila-Crump, Savanna, Tae, Giyoong, Mehl, Ryan A., and Kwon, Inchan

Abstract

An inverse-electron-demand Diels–Alder (IEDDA) reaction using genetically encoded tetrazine variants enables rapid bioconjugation for diverse applications in vitroand in cellulo. However, in vivobioconjugation 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 vitroIEDDA 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 vivoimaging. 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 vivoupon addition of trans-cyclooctene, enabling temporal control of in vivobioconjugation in a very high yield.

Published
2020
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