Expanding the Genetic Code for a Dinitrophenyl Hapten

Abstract

Haptens, such as dinitrophenyl (DNP) are small molecules that induce strong immune responses when attached to proteins or peptides and, as such, have been exploited for diverse applications. We engineered a Methanosarcina barkeri pyrrolysyl-tRNA synthetase (mbPylRS) to genetically encode a DNP-containing unnatural amino acid, N(6) -(2-(2,4-dinitrophenyl)acetyl)lysine (DnpK). Although this moiety was unstable in Escherichia coli, we found that its stability was enhanced in mammalian HEK 293T cells and was able to induce selective interactions with anti-DNP antibodies. The capability of genetically introducing DNP into proteins is expected to find broad applications in biosensing, immunology, and therapeutics.

Bioorthogonal Chemical Activation of Kinases in Living Systems

“… 14 In addition, the nitrobenzyl-based photocaging lysine analogue such as o -nitropiperonyl caged lysine (ONPK, 1 , Figure S1 ) employed in the aforementioned study is not fully bioorthogonal and could be decaged in prokaryotic reducing environments ( Figure S2 ), or by enzymes in eukaryotic cells under hypoxic conditions. 15 The development of two-photon excitation caging groups circumvented the concern of UV cytotoxicity and could be used to improve the tissue penetration ability. 16 Still, a small molecule-based chemical activation strategy has its unique advantages such as better biocompatibility, tunability, and minimal invasiveness, particularly for intact living animals.…”

Inverse electron demand Diels–Alder reactions in chemical biology

“…49a). 310,311 Additionally, Chin et al have genetically encoded caged Tyr 312 and Cys 313 amino acid variants into proteins for protein photoactivation. Even though photodecaging could offer high temporal resolution and potential spatial control, the UV light may however cause photo toxicity and perturb cellular processes.…”

“…Moreover, the nitrobenzyl-based photocaging proved to be not fully bioorthogonal and stable in cells. 310,311 Additionally, the poor penetration of ultraviolet radiation also hamper its compatibility in tissue and animal studies.…”

Therapeutic applications of genetic code expansion

“…Site-specific incorporation of p -nitrophenylalanine (pNO 2 Phe) to murine tumor necrosis factor-α (mTNF-α) successfully generated a sustained high-titer antibody response to both parental and antigenically distinct mTNF-α variants without the need for strong adjuvants [ 122 ]. Another DNP-containing ncAA, N 6 ‐(2‐(2,4‐dinitrophenyl)acetyl)lysine (DnpK) was later incorporated into HEK 293T cells, although unstable in Escherichia coli [ 123 ]. Sulfotyrosine (SO 3 Tyr) and 3-nitrotyrosine (3NO 2 Tyr), two PTM-related ncAAs were introduced to specific sites in mTNF-α and EGF, indicating that PTM might play a role in autoimmune disorders [ 124 ].…”