Can a Six‐Letter Alphabet Increase the Likelihood of Photochemical Assault to the Genetic Code?

In 2014, two unnatural nucleosides, d5SICS and dNaM, were shown to selectively base pair and replicate with high fidelity in a modified strain of E. coli, thus effectively expanding its genetic alphabet from four to six letters. More recently, a significant reduction in cell proliferation was reported in cells cultured with d5SICS, and putatively with dNaM, upon exposure to brief periods of near‐visible radiation. The photosensitizing properties of the lowest‐energy excited triplet state of both d5SICS and dNaM were implicated in their cytotoxicity. Importantly, however, the excited‐state mechanisms by which near‐visible excitation populates the triplet states of d5SICS and dNaM are currently unknown. In this study, steady‐state and time‐resolved spectroscopies are combined with quantum‐chemical calculations in order to reveal the excited‐state relaxation mechanisms leading to efficient population of the triplet states in these unnatural nucleosides in solution. It is shown that excitation of d5SICS or dNaM with near‐visible light leads overwhelmingly to ultrafast population of their triplet states on the femtosecond time scale. The results presented in this work lend strong support to the proposal that photoexcitation of these unnatural nucleosides can accelerate oxidatively generated damage to DNA and other biomolecules within the cellular environment.     

A Convenient Laboratory Preparation of N,N-Dimethyl-dichloro-methylene-iminium Chloride (Phosgene-iminium chloride)

A method of preparation of the title compound 1a avoiding the use of gaseous chlorine is described. Tetramethylthiuram disulfide 4 is oxidized with sulfuryl chloride to dime thyl thiocarbamoyl chloride 5, which is converted to 1a by means of phosphorus pentachloride.     

Click‐Tag and Amine‐Tag: Chemical Tag Approaches for Efficient Protein Labeling In Vitro and on Live Cells using the Naturally Split Npu DnaE Intein

Protein labeling with synthetic moieties remains in many cases a technically challenging or unresolved task. Two new and simple concepts are presented. In both approaches, a very short tag of only a few amino acids is prepared with the desired chemical modification and, in a second step, it is transferred to the protein of interest by protein trans‐splicing. For the amine‐tag, a recombinant intein fragment free of lysine residues was generated such that the amine group of the N terminus could be selectively modified with regular amine‐reactive reagents. Thus, standard bioconjugation procedures without any chemical synthesis could be applied without modification of lysines in the protein of interest. For the click‐tag, protein trans‐splicing was combined with unnatural amino acid mutagenesis and subsequent bioorthogonal side chain modification, as demonstrated for click chemistry using p‐azidophenylalanine. By the two‐step strategy, exposure of the protein of interest to the copper catalyst was avoided.     

Construction of a Live‐Attenuated HIV‐1 Vaccine through Genetic Code Expansion

A safe and effective vaccine against human immunodeficiency virus type 1 (HIV‐1) is urgently needed to combat the worldwide AIDS pandemic, but still remains elusive. The fact that uncontrolled replication of an attenuated vaccine can lead to regaining of its virulence creates safety concerns precluding many vaccines from clinical application. We introduce a novel approach to control HIV‐1 replication, which entails the manipulation of essential HIV‐1 protein biosynthesis through unnatural amino acid (UAA*)‐mediated suppression of genome‐encoded blank codon. We successfully demonstrate that HIV‐1 replication can be precisely turned on and off in vitro.     

Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen‐binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single‐domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o‐Nitrobenzyl‐protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti‐GFP photobody exhibited an at least 10 000‐fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody–photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.     

利用光交联探针研究pH值调控的蛋白-蛋白相互作用

pH值是几乎影响到所有蛋白质分子表面电荷分布和相关结构变化的关键因素,许多蛋白质分子之间的相互作用也受到pH值的调控.近年来,基于非天然氨基酸的光交联探针被广泛应用于捕捉活细胞内的蛋白-蛋白相互作用.然而,由于环境pH值的改变往往导致蛋白质分子结构、带电性质的显著变化,因此现有的非天然氨基酸光交联探针难以实现在极端pH值条件下对相互作用的蛋白质分子的捕获和研究.本文将介绍本课题组新近发展的基于烷基双吖丙啶活性基团的非天然氨基酸光交联探针-DIZPK,通过这一探针,我们成功捕获到大肠杆菌中一种重要的酸性分子伴侣HdeA在膜间质内酸性胁迫过程中的作用对象.在捕获到的HdeA底物中,我们发现了两个膜间质中重要的分子伴侣蛋白：DegP和SurA.通过实验我们证明了在酸性胁迫条件下,DegP和SurA能够被HdeA保护不形成聚集体,并进而在随后的回复中性过程中能够协助HdeA对其他底物进行重折叠.这种不依赖于ATP的分子伴侣间协作模式可能起到了帮助肠道型细菌抵抗酸性胁迫的功能.基于上述实验结果,我们提出了一个＂分子伴侣协同作用＂的模型,用以阐释细菌利用抗酸性分子伴侣提高其在酸胁迫下逃逸的机理.推而广之,在原核和真核细胞中定点引入高可适性的非天然氨基酸光交联探针可广泛适用于在活体内探测众多的由pH值调控的蛋白-蛋白相互作用.     

Geometric quantities of lower doubly excited bound states of helium

Expectation values of single electron and interelectronic geometric quantities such as $\langle r\rangle$, $\langle r_{12}\rangle$, $\langle r_<\rangle$, $\langle r_>\rangle$, $\langle \cos\theta_{12}\rangle$ and $\langle \theta_{12}\rangle$ are calculated for doubly excited $2{\rm p}n{\rm p}\,{}^1P^{\,\rm e}\,(3\leq n\leq5),\, 2{\rm p}n{\rm p}\,{}^3\!P^{\,\rm e}\,(2\leq n\leq5)$ and $2{\rm p}n{\rm d}\,{}^{1,3}D^{\,\rm o}\,(3\leq n\leq5)$ states of helium using Hylleraas-$B$-spline basis set. The energy levels converge to at least 10 significant digits in our calculations. The extrapolated values of geometric quantities except for $\langle \theta_{12}\rangle$ reach 10 significant digits as well; $\langle \theta_{12}\rangle$ reaches at least 7 significant digits using a multipole expansion approach. Our results provide a precise reference for future research.     

The Expanded Genetic Alphabet

All biological information, since the last common ancestor of all life on Earth, has been encoded by a genetic alphabet consisting of only four nucleotides that form two base pairs. Long‐standing efforts to develop two synthetic nucleotides that form a third, unnatural base pair (UBP) have recently yielded three promising candidates, one based on alternative hydrogen bonding, and two based on hydrophobic and packing forces. All three of these UBPs are replicated and transcribed with remarkable efficiency and fidelity, and the latter two thus demonstrate that hydrogen bonding is not unique in its ability to underlie the storage and retrieval of genetic information. This Review highlights these recent developments as well as the applications enabled by the UBPs, including the expansion of the evolution process to include new functionality and the creation of semi‐synthetic life that stores increased information.