Hydrogen sulfide (H₂S) is an endogenously produced gaseous signaling molecule with multiple biological functions. In order to visualize the endogenous in situ production of H₂S in living cells in real time, here we developed multi-fluorinated azido coumarins as fluorescent probes for the rapid and selective detection of biological H₂S. Kinetic studies indicated that an increase in fluorine substitution leads to an increased rate of H₂S-mediated reduction reaction, which is also supported by our theoretical calculations. To our delight, tetra-fluorinated coumarin 1 could react with H₂S fast (t_1/2≈1 min) and selectively, which could be further used for continuous enzymatic assays and for visualization of intracellular H₂S. Bioimaging results obtained with 1 revealed that d-Cys could induce a higher level of endogenous H₂S production than l-Cys in a time-dependent manner in living cell.

RNA interference (RNAi) is a cellular process for gene silencing. Because of poor serum stability, transferring dsRNA directly into the target cells is a challenge. We report a facile and universal strategy to construct short hairpin RNA (shRNA) transcription nanoparticles with multiple shRNA transcription templates by PCR with flexible branched primers (branch-PCR). Compared with conventional linear shRNA transcription templates, these shRNA transcription nanoparticles show excellent stability against digestion by exonuclease III. Importantly, we found that our highly stable shRNA transcription nanoparticles can also be transcribed and thus induce efficient and long-lasting RNAi with picomolar activity in living mammalian cells. These chemically well-defined branch-PCR-generated stable shRNA transcription nanoparticles might facilitate RNAi delivery with a long-lasting RNAi effects.

Hydrogen sulfide (H₂S) is an endogenously produced gaseous signaling molecule with multiple biological functions. To visualize the endogenous in situ production of H₂S in real time, new coumarin- and boron-dipyrromethene-based fluorescent turn-on probes were developed for fast sensing of H₂S in aqueous buffer and in living cells. Introduction of a fluoro group in the ortho position of the aromatic azide can lead to a greater than twofold increase in the rate of reaction with H₂S. On the basis of o-fluorinated aromatic azides, fluorescent probes with high sensitivity and selectivity toward H₂S over other biologically relevant species were designed and synthesized. The probes can be used to in situ to visualize exogenous H₂S and D-cysteine-dependent endogenously produced H₂S in living cells, which makes them promising tools for potential applications in H₂S biology.

Chlorsulfuron is the first commercialized sulfonylurea herbicide, which targets acetohydroxyacid synthase (AHAS). Mutations in AHAS have caused serious herbicide resistance to chlorsulfuron. Quantitative description of the herbicide resistance in molecular level will benefit the understanding of the resistance mechanism and aid the design of resistance-evading herbicide. We have recently established a MB-QSAR (Mutation-dependent Biomacromolecular Quantitative Structure-Activity Relationship) method to conduct the 3D-QSAR study in biomacromolecules. Herein, based on the herbicide resistance data measured for a series of AHAS mutants against chlorsulfuron, we constructed MB-QSAR models to quantitatively predict the herbicide resistance and interpret the structure resistance relationships for AHAS mutants against chlorsulfuron. Quite well correlations between the experimental and the predicted pK_i values were achieved for MB-QSAR/CoMFA (q²=0.705, r²=0.918, r²_pred=0.635) and MB-QSAR/CoMSIA (q²=0.558, r²=0.940, r²_pred=0.527) models, and interpretation of the MB-QSAR models gave chemical intuitive information to guide the resistance-evading herbicide design.

Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched-chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co-workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C-terminal, N-terminal, and C- and N-terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be Δ_N14–Δ_C89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (K_d) of Δ_N14–Δ_C89 when binding CSUs of different species was found to be 9.3–66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (K_d) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide Δ_N14–Δ_C89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs.

The extra unpaired base(s) or bulged structures of nucleic acids are capable either of forming complexes with nucleic-acid-binding proteins or of acting as binding sites for small molecules. We are interested in developing bulge-specific agents as potential drugs or chemical tools in biological research. Antofine can selectively bind with DNA and RNA bulged structures (Xi et al., Bioorg. Med. Chem. Lett. 2006, 16, 4300–4304). Furthermore, a series of antofine analogues suitable for selective binding with TMV RNA rather than with TMV coat protein (CP) were found. Biochemical studies indicated that antofine and its analogues disrupt in vitro virus assembly through small-molecule–RNA interactions. A structural model to illustrate these effects has been proposed. It is suggested that antofine analogues bind selectively with RNA bulged structures and therefore disrupt interaction between TMV RNA and TMV CP.

During the formation of RNA-induced silencing complex (RISC), the passenger and guide strand of an siRNA duplex separate from each other to generate an active RISC complex. Accumulating evidence shows that an siRNA passenger strand can also assemble into a RISC complex and mediate RNA interference, thereby causing undesired off-target effects. To reduce this effect, the so-called “universal base” 5-nitroindole nucleotides were incorporated into an siRNA passenger strand. Melting temperature and circular dichroism spectrum measurements showed no significant changes compared to the unmodified duplex, thus indicating the formation of normal A-form conformation. Using a dual luciferase reporter assay, we have further shown that 5-nitroindole modification at position 15 of the siRNA passenger strand drastically decreased the RNAi (RNA interfering) potency of this strand, whereas the potency of the RNA guide strand was not much affected. These results could provide a practical approach for reducing off-target effects mediated by the siRNA passenger strand.

Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is composed of catalytic and regulatory subunits. The enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). However, the crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and RSU is also unknown. Herein, we introduced a global-surface, site-directed labeling scanning method to determine the potential interaction region of the RSU. This approach relies on the insertion of a bulky fluorescent probe at the designated site on the surface of the RSU to cause a dramatic change in holoenzyme activity by perturbing subunit interaction. Then, the key amino acid residues in the potential interaction regions were identified by site-directed mutagenesis. Compared to the wild-type, the single-point mutants R26A and D69A showed 54 and 64 % activity, respectively, whereas the double mutant (R26A+D69A) gave 14 %, thus suggesting that residues Arg26 and Asp69 are the key residues of subunit interaction with cooperative action. Additionally, the results of GST pull-down assays and pH-dependence experiments suggested that polar interaction is the main force for subunits interaction. A plausible protein–protein interaction model of the holoenzyme of Escherichia coli AHAS III is proposed, based on the mutagenesis and protein docking studies. The protocol established here should be useful for the identification of the molecular interactions between proteins.

Sparsomycin is an antibiotic that targets the peptidyl transferase center of the ribosome and has the ability to promote ribosomal translocation in the absence of EF-G and GTP. Here we show that changes in the configurations at the two chiral centers of sparsomycin, especially at the chiral carbon, can greatly affect its capability to promote ribosomal translocation. More importantly, the incorporation of the pseudo-uracil moiety of sparsomycin into linezolid through a covalent linkage conferred on linezolid derivatives the ability to promote translocation, thus indicating the importance of interactions between this pseudo-uracil moiety, rRNA, and tRNA for promoting translocation. In addition, these translocation promoters can also effectively inhibit spontaneous reverse translocation; this suggests that they might promote forward translocation by trapping the ribosome in the post-translocation state and shifting the equilibrium between the pre- and post-translocation ribosome in the forward direction.

In order to design efficient and thermostable azo-type regulators, a series of azo-type compounds were synthesized and characterized. While introducing an inductive electron-withdrawing group to an azobenzene para or meta-position, the obtained compound can be an excellent photoswitch. 3,3′-Azo-di-benzyl alcohol was designed and synthesized as one of thermostable and efficient photoswitches, which can efficiently reversibly photoregulate the nucleic acid structure with its cis-isomer being sufficiently stable at physiological temperature.