Fast detection of cellular thiols in aqueous medium was achieved using a newly developed fluorescence probe (see picture). Based on this probe, a high-throughput fluorescence assay for glutathione reductase was developed.

In an effort to seek novel agents targeting prostatespecific membrane antigen (PSMA), 16 ligands (L1−L16) with structural modifications in S1′ binding pocket were synthesized and evaluated for PSMA inhibition. (S)-3-(Carboxyformamido)-2-(3-(carboxymethyl)ureido)propanoic acids proved to be potent PSMA ligands with K i values ranging from 0.08 nM to 8.98 nM, which are in the range of or are higher in potency compared to previously published urea-based ligands. Computational docking was performed to study the binding mode of the two most potent ligands discovered. FITC-conjugated L14 could selectively stain PSMA + LNCaP cells over PSMA − PC3 cells. IRDye800CW conjugated L16 can effectively image tumors in a murine xenograft model of prostate cancer.

No abstract

8-17 and 10-23 are the two most comprehensively studied RNA-cleaving DNAzymes to date and have the ability to carry out sequence-specific cleavage of both all-RNA or chimeric RNA/DNA substrates. Mutagenesis studies of 8-17 and 10-23 DNAzymes using alternative natural nucleotides to substitute a given nucleotide in the DNAzyme sequence have found that both DNAzymes are able to tolerate a variety of alterations at many sequence locations. Chemical modification studies employing nucleotides containing nonnatural nucleobases have led to findings that some specific entities of selected nucleobases are irreplaceable by other functional groups. In this work, we set out to carry out a mutagenesis study on both 8-17 and 10-23 by substituting individual nucleotides in their catalytic cores with a baseless (abasic) nucleotide or a baseless/sugarless nucleotide containing only acyclic C3 spacer. We observed that the substitution with an abasic nucleotide or C3 spacer at many locations within the catalytic core of both 8-17 and 10-23 was still able to support a significant level of catalytic activity of each DNAzyme, suggesting that both DNAzymes have considerable structural plasticity to maintain their catalytic functions. We also observed that almost all nucleobases in the catalytic core of each DNAzyme appeared to make either an absolutely essential contribution to the function of each DNAzyme or exhibit a "chaperone-like" activity that is important for the optimal function of each DNAzyme; in contrast, only one sugar ring in 8-17 and four in 10-23 were inferred to make some contribution to the optimal function of the relevant DNAzyme. Finally, our study also raised a possibility that the 10-23 DNAzyme might be a special structural variant of the larger 8-17 DNAzyme family.

In situ generation of 5-formylcytosine (5fC) in nucleosome core particles (NCPs) reveals that 5fC leads to essential DNA-protein crosslinks (DPCs). Mechanistic studies using chemical models and mutated histones demonstrate that DPCs form reversibly between the formyl function of 5fC and primary amines on histones. These results suggest that DPC formation from 5fC in chromatin occurs in addition to its role in DNA demethylation.

5-Methylene pyrrolones (5MPs) are highly thiol-specific and tracelessly removable bioconjugation tools. 5MPs are readily prepared from primary amines in one step. 5MPs exhibit significantly improved stability under physiologically relevant conditions and cysteine specificity compared to commonly used analogues, maleimides. Michael addition of thiol to 5MPs occurs rapidly, cleanly and does not generate a stereocenter. The conjugates efficiently release thiols via retro-Michael reaction in alkaline buffer (pH 9.5) or via thiol exchange at pH 7.5. This unique property makes 5MPs valuable for the controlled release of conjugated cargo and temporary thiol protection. The utilization of 5MPs for protein immobilization and pull-down of active complexes is illustrated using E. coli. acetohydroxyacid synthase isozyme I.

Human protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein, converts protoporphyrinogen IX to protoporphyrin IX in the heme biosynthetic pathway. Mutations in the hPPO gene cause the inherited human disease variegate porphyria (VP). In this study, we report the crystal structure of hPPO in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of 1.9 Å. The structural and biochemical analyses revealed the molecular details of FAD and acifluorfen binding to hPPO as well as the interactions of the substrate with hPPO. Structural analysis and gel chromatography indicated that hPPO is a monomer rather than a homodimer in vitro. The founder-effect mutation R59W in VP patients is most likely caused by a severe electrostatic hindrance in the hydrophilic binding pocket involving the bulky, hydrophobic indolyl ring of the tryptophan. Forty-seven VP-causing mutations were purified by chromatography and kinetically characterized in vitro. The effect of each mutation was demonstrated in the high-resolution crystal structure.

A highly sensitive H2S-specific near-infrared fluorescence-enhanced probe was developed for real-time imaging of endogenous H2S in colorectal cancer cells (HCT116 and HT29) in mice.