A versatile platform for single-molecule enzymology of restriction endonuclease

Enzymes are the major players for many biological processes.Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level.Yet,these single-molecule experiments are technically di±cult and generally require complicated experimental design.Here,we develop a Holliday junction(HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET(sm-FRET).We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events.Thanks to the multiple-arms structure of HJ,the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm.Therefore,it is possible to independently change the substrate arm to study different enzymes with similar functions.Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations.Moreover,this method can be easily extended to study other types of DNA-binding enzymes without too much modi fication of the design.We anticipate it can find broad applications in single-molecule enzymology.

Analysis of Binding Interaction between Captopril and Human Serum Albumin

The interaction between captopril, an inhibitor of angiotensin converting enzyme and human serum albumin, a principal plasma protein in the liver has been investigated in vitro under a simulated physiological condition by UV-vis spectrophotometry and fluorescence spectrometry. The intrinsic fluorescence intensity of human serum albumin was strongly quenched by captopril. The binding constants and the number of binding sites can be calculated from the data obtained from fluorescence quenching experiments. The negative value of ΔG0 reveals that the binding process is a spontaneous process. According to the van’t Hoff equation, the standard enthalpy change (ΔH0) and standard entropy change (ΔS0) for the reaction were calculated to be 35.98 KJ●mol-1 and 221.25 J●mol-1 K. It indicated that the hydrophobic interactions play a main role in the binding of captopril to human serum albumin. In addition, the distance between captopril (acceptor) and tryptophan residues of human serum albumin (donor) was estimated to be 1.05 nm according to the F?rster’s resonance energy transfer theory. The results obtained herein will be of biological significance in pharmacology and clinical medicine.

In vivo precision imaging of vicinal-dithiol-containing proteins by a FRET molecular probe sensitive to protein environment

We here present a Förster resonance energy transfer(FRET)-based and environment-sensitive fluorescent probe VG-1 for vicinal-dithiol-containing proteins(VDPS).VG-1 uniquely contalns two sites sensitive to the protein environment(SPE)thus it shows weak fluorescence in both blue and green channels(a low FRET efficiency)in solution.After specifically binding with VDPs,its fuorescence in the green channel increases,while that in the blue channel disappears,achieving the specific detection of VDPs.The obvioussignal changes in fluorescence may be attributed to that the increased rigidity of the molecular skeletons causes the enhanced FRET eficiency,The probe also achleved the cell super-resolution imaging of VDPs and the confocal imaging of VDPs in zebrafish.

Effect of excitation wavelength(blue vs near UV)and dopant concentrations on afterglow and fast decay of persistent phosphor SrAl_(2)O_(4):Eu^(2+),Dy^(3+)

The persistent phosphor SrAl_(2)O_(4):Eu^(2+),Dy^(3+)is the subject of numerous investigations.One often neglected aspect is that in this phosphor,as well as in Sr_(4)Al_(14)O_(25):Eu^(2+),Dy^(3+),there are two different Sr^(2)+sites which can be occupied by the dopant Eu^(2+)ions,We first introduce a general scheme of possible energy transfers in these persistent phosphor materials including explicitly both europium ions,This scheme is used as a generic starting point to study experimentally specific pathways.We illustrate this application with the study of the effect of excitation wavelength(444 and 382 nm)on the afterglow of differently doped SrAl_(2)O_(4):Eu^(2+),Dy^(3+)samples,as well as on the emission decay curves.With the same excitation intensity under 444 nm excitation,the resulting afterglow intensity is stronger than under near UV excitation.At 382 nm,Eu^(2+)ions on both Sr^(2)+s ites in SrAl_(2)O_(4)are excited,but at room temperature the blue emission is quenched,leading to a loss of photons.The observed effects can further be associated with the ratio of Eu^(2+)ions and trap states which are modulated by the concentrations of Eu^(2+)and Dy^(3+)in SrAl_(2)O_(4),as well as by temperature,Increasing the nominal Dy^(3+)content from 0.1 mol%to 0.5 mol%with respect to Sr results in the doubling of the integrated afterglow intensity and confirms thus that Dy^(3+)ions are indeed involved in the trapping process,The concentration of trap states is much lower than the concentration of Eu^(2+)ions,as even with low excitation densities,a plateau of integrated afterglow intensity(corresponding to the total number of accessible traps)is reached.We postulate that an important fraction of excited Eu^(2+)ions can potentially transfer their energy to trap states.Once that all traps are filled or in a dynamical filling-depletion process under illumination(with thermal and/or optical depletion processes),for the remaining Eu^(2+)a"normal"steady-state emission is observed.The luminescence decay curves at 520 nm measured at 77 K show a mono-exponential decay with a common lifetime of about 1140 ns for all 5 samples under 437 nm excitation,while under 375 nm excitation,a feed process originating from the energy transfer between Eu^(2+)ions is demonstrated.Under 375 nm excitation,the non-exponential decay observed at 440 nm can be quantitatively associated to a Forster energy transfer process with R_(0)=1.58(8)nm.For the overall understanding of the afterglow processes,it appears that one has to consider the individual contributions of all active ions on different lattice sites.

White Electroluminescence with Simultaneous Three-Color Emission from a Four-Armed Star-Shaped Single-Polymer System

A four-armed star-shaped single-polymer system with 4,7-bis（5-（4-（9H-carbazol-9-yl）phenyl）-4-hexylthio- phen-2-yl）benzo[c][1,2,5]thiadiazole （FTBT） as a red emissive core, polyfluorene （PF） as blue emissive arms and 1,3-benzo thiadiazole （BT） as green emissive dopants was designed and synthesized, in which red, green, and blue （RGB） emission balance can be achieved by adjusting the doping concentration of FTBT and BT discreetly. A typ- ical single-emissive-layer device （ITO/PEDOT：PSS/polymer/TPBI/LiF/Al） was studied and discussed, realizing a pure and stable white emission with a luminous efficiency （LE） of 1.59 cd·A^-1 and CIE coordinates of （0.31, 0.34）. The high-color-quality white electroluminescence of the devices could be mainly attributed to the suppressed in- termolecular interactions, and partial energy transfer from the blue PF arms to the red and green dopants.