ENZYME REACTION IN AQUEGUB-ORGANIC BYSTEM —KINETIC RESOLUTION OP RACEMIC OXIRANECARBOXYLIC ESTERS WITH LIPASE

The chiral oxiranecarboxylic acids or esters 2a-e were obtained with lipase (CCL) in aqueous-organic system.The ee and the absolute configurations of the products were determined.

One-step detection of T4 polynucleotide kinase activity based on single particle-confined enzyme reaction and digital particle counting

T4 polynucleotide kinase(T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle,the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope(TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.

Comparison of Measurements for Enhanced Diffusion Problem in Chemical Reaction Systems

The problem of molecular diffusion in the soup of chemical reactions attracts mounting interest across fields ranging from chemistry to biophysics to material science.Chemical reactions involve bond breakup and formation,whose time scale is typically on the orders of fs to ps,while molecular diffusion occurs at time scales ofμs to ms.The two processes are often considered orthogonal,given the vastly different scales.The serial results show that the enzyme's diffusion is enhanced in a substrate-dependent manner,which was further extended to small molecule reaction systems,challenging this classical paradigm.However,the results from different groups using different techniques do not quantitatively agree,and a general mechanism is yet to be understood.We summarize experimental studies on diffusion problems and seek to reconcile the interpretation with understanding the limits of measurement tools and the chemical nature of reaction systems.Understanding molecular diffusion in chemical reactions will provide fresh thoughts in designing chemical systems such as molecular machines that harvest work at the nanoscale in a controllable manner.

In Vivo Activation of Pro-Protein Therapeutics via Chemically Engineered Enzyme Cascade Reaction

Selective and temporal control over protein activity is of great importance for the advancement of the protein of interest into precise molecular medicine.Simply installing synthetic ligands to proteins for activity regulation,however,is often obscured by either nonspecificity or insufficient efficiency.This study reports a chemical approach in which enzymatic cascade reactions were designed for selective activation of pro-protein both in vitro and in vivo.Specifically,the system consisted of aromatic boronic-acid-modified nanoparticles,reactive oxygen species(ROS)-responsive pro-protein(RNase A-NBC),a small molecule drug,β-Lapachone(β-Lap),and strategically screened synthetic lipids,required for the assembly of the nanocomplexes.Once target-delivered into tumor cells,the reduction ofβ-Lap produces massive H2O2 in response to NAD(P)H quinone oxidoreductase 1(NQO1),a tumor-specific enzyme,which triggers further induction by selective chemical modification of ROS-responsive cytosolic protein ribonuclease A(RNase A)-NBC,thus,switching from“inert”pro-protein to active therapeutics,that ultimately prohibit tumor cell growth.Moreover,the designed enzymatic cascade activation of the pro-protein was effective in vivo,displaying superior therapeutic efficacy to either the pro-protein alone or theβ-Lap via tumor-targeted delivery and the consequent suppression of the tumor growth.As both RNase A andβ-Lap have been evaluated clinically as antitumor therapeutics,our chemical multi-step cascade methodology is,therefore,a promising strategy for selective modulation of pro-protein chemistry in the living system for fundamental investigations,favorable toward potential anticancer applications.

Point mutations of muscle mitochondrial DNA from patients with mitochondrial encephalomyopathies

Objective To study the relation between point mutations at nt3243 and nt8344 of muscle mitochondrial DNA from patients with mitochondrial encephalomyopathies and phenotypes. Methods DNA was extracted from muscle specimens from 5 patients with mitochondrial encephalomyopathies and amplified by PCR method, using corresponding oligonucleotide primers. DNA fragments were digested with restriction enzymes BglⅠ and ApaⅠ, then the digested DNA fragments were analyzed with an electrophoresis method.Results The point mutation at nt3243 of mtDNA was found in 2 patients, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) and another with myoclonic epilepsy with ragged red fibers (MERRF). The point mutation at nt8344 was found in 2 patients with MERRF, including the one with point mutation at nt3243.Conclusion The point mutation of DNA at nt3243 correlated with MELAS and nt8344 correlated with MERRF. In addition, the detection of point mutations at both nt3243 and nt8344 in a patient with MERRF shows the association of mutation with diversity in clinical manifestations of mitochondrial encephalomyopathies.