Series or parallel toehold-mediated strand displacement and its application in circular RNA detection and logic gates.

Toehold-mediated strand displacement (TMSD) is widely employed in constructing a wide range of chemical reaction networks. In TMSD, single-stranded DNA or RNA can fold back upon itself to form a local short double-strand structure often hindering bimolecular hybridization. Here, based on series and parallel circuits, we introduce two mechanisms: series toehold-mediated strand displacement (STMSD) and parallel toehold-mediated strand displacement (PTMSD). These mechanisms can be highly effective when the target area is blocked by a secondary structure. In addition, these systems allow regulating the reaction rates spanning three to five orders of magnitude by adjusting the length of the two toeholds with the added advantage of multifunctional regulation and selectivity. To demonstrate the impressive function of this approach, a logic operation system based on STMSD was constructed to simulate the signal processing of a half-adder. We believe that the introduction of series and parallel toeholds will provide design flexibility contributing to the development of molecular computers, molecular robotics, and DNA-based biosensors.

Short divalent ethacrynic amides as pro-inhibitors of glutathione S-transferase isozyme Mu and potent sensitisers of cisplatin-resistant ovarian cancers.

The linking of ethacrynic acid with ethylenediamine and 1,4-butanediamine gave EDEA and BDEA, respectively, as membrane-permeable divalent pro-inhibitors of glutathione S-transferase (GST). Their divalent glutathione conjugates showed subnanomolar inhibition and divalence-binding to GSTmu (GSTM) (PDB: 5HWL) at ∼0.35 min-1. In cisplatin-resistant SK-OV-3, COC1, SGC7901 and A549 cells, GSTM activities probed by 15 nM BDEA or EDEA revealed 5-fold and 1.0-fold increases in cisplatin-resistant SK-OV-3 and COC1 cells, respectively, in comparison with the susceptible parental cells. Being tolerable by HEK293 and LO2 cells, BDEA at 0.2 µM sensitised resistant SK-OV-3 and COC1 cells by ∼3- and ∼5-folds, respectively, released cytochrome c and increased apoptosis; EDEA at 1.0 µM sensitised resistant SK-OV-3 and A549 cells by ∼5- and ∼7-fold, respectively. EDEA at 1.7 µg/g sensitised resistant SK-OV-3 cells to cisplatin at 3.3 µg/g in nude mouse xenograft model. BDEA and EDEA are promising leads for probing cellular GSTM and sensitising cisplatin-resistant ovarian cancers.

Tuning the specificity of DNA probes using bulge-loops for low-abundance SNV detection.

Tuning the free energy difference between a molecular probe and the target has been regarded as a feasible way to realize selective mutant recognition. But due to limited extent of variation on the probing sequences, it remains a challenge to moderately leverage the thermodynamic kinetics simply by changing the base composition of probes. Herein we propose the modulation of discrimination capability for single nucleotide variations (SNVs) detection by insertion of bulge-loop into duplex DNA probes. Based on controllable tuning of free energy change (ΔG) before and after strand exchange with either mutated or wild-type DNAs, much higher specificity than conventional linear probes is obtained. As-proposed bulge-loop probes allows excellent discrimination of SNVs in high guanine and cytosine (GC) rich regions, and reaches a detection limit of 0.02% abundance with down to 2 femtomolar target gene. The probes also demonstrate excellent consistence with droplet digital PCR (ddPCR) in identifying low abundant L858R mutant in lung tissue samples that are not resolved by either a commercial PCR kit or Sanger sequencing. Our work not only provides insight into the rational design of strand exchange probes for point-of-care diagnosis but also advance the construction of customizable cascade reactions in dynamic DNA nanotechnology more broadly.

Method of damage location determination based on a neural network using a single fiber Bragg grating sensor.

In this paper, a structural damage identification algorithm based on a single fiber Bragg grating (FBG) sensor is proposed. The signal detected by the FBG can be analyzed by the wavelet packet decomposition and back propagation neural network to obtain the damage location information. A high-speed FBG demodulation system based on a tunable Fabry-Perot filter and unbalanced Mach-Zehnder (M-Z) interferometer is designed to respond to a signal with a frequency range from 0 to 4 kHz, which will increase the sensing accuracy. This algorithm is verified by the aluminum plate model, which can simulate the generation of damage in reality. The experimental results show that a single FBG sensor is enough to realize accurate damage location identification according to this algorithm, and identification accuracy can reach 90.0%.

Glutathione S­transferase isozyme alpha 1 is predominantly involved in the cisplatin resistance of common types of solid cancer.

The roles of glutathione S­transferase pi 1 (GSTP1), glutathione S­transferase mu 2 (GSTM2) and glutathione S­transferase alpha 1 (GSTA1) in cisplatin (DDP)­resistance of solid cancer cells (A549/DDP, SKOV3/DDP and SGC7901/DDP) were compared following expression downregulation with small interfering RNAs (siRNAs). DDP cytotoxicity was reflected by its half maximal inhibition concentration (IC50) calculated from data using a Cell Counting Kit­8 assay; cell apoptosis was examined using flow cytometry and Hoechst 33342 staining. Higher activities of GST were detected in the cytosol of DDP­resistant cells, compared with those in the parental DDP­susceptible cells. The silencing efficacy of each positive siRNA was supported by western blot analysis. GSTP1 silencing resulted in a 4­fold sensitization of SGC7901/DDP cells to DDP cytotoxicity, but negligible sensitization of SKOV3/DDP and A549/DDP cells. GSTM2 silencing sensitized SKOV3/DDP and A549/DDP cells to DDP cytotoxicity by ~2­fold, but did not sensitize SGC7901/DDP cells. Notably, GSTA1 silencing enhanced DDP cytotoxicity in SGC7901/DDP cells by 6­fold, in A549/DDP cells by 5­fold and in SKOV3/DDP cells by 2­fold. The combined actions of positive siRNAs and DDP increased the percentages of apoptotic cells in the DDP­resistant solid cancer cells compared with the combined actions of DDP and the negative siRNAs. The present findings indicated that GSTA1 is a predominant GST isozyme associated with DDP resistance of SGC7901/DDP, A549/DDP and SKOV3/DDP cells; GSTA1­specific inhibitors may be general sensitizers of SGC7901/DDP, A549/DDP and SKOV3/DDP cells to DDP cytotoxicity through the promotion of cell apoptosis.

High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells.

While detection of microRNA with or without signal amplification is highly informative, nanosensors with high specificity for cell-specific RNA detection are rare. Methods: In this study, a tetrahedral DNA nanostructure (TDN) with a specific function was combined with gold nanoparticles (Au-NP) possessing fluorescence quenching effects and a large surface area to fabricate a fluorescence resonance energy transfer based nanosensor (Au-TDNN). The presence of miR-21 (target) can separate the fluorescent dye-labeled detection probe on Au-TDNNs from Au-NPs, which separates the donor and acceptor, thus inducing an intensive fluorescence signal. High specificity for discerning point mutation targets was achieved by rationally designing the nucleic acid strand displacement reaction to occur spontaneously with ΔG0 ≈ 0 based on thermodynamic parameters; under this condition, slight thermodynamic changes caused by base mismatch exert significant effects on hybridization yield. Results: Chemically synthesized DNA of three single-base-changed analogues of target, let-7d, and miR-200b were tested. A discrimination factor (DF) of 15.4 was produced by the expected detection probe on Au-NPs for proximal single-base mismatch. As the control group, the DF produced by an ordinary detection probe on Au-NPs only reached 2.4. The feasibility of the proposed strategy was also confirmed using hepatocyte cancer cells (HepG2). Conclusion: This improved nanosensor opens a new avenue for the specific and easy detection of microRNA in live cells.

Fluorometric titration assay of affinity of tight-binding nonfluorescent inhibitor of glutathione S-transferase.

To determine inhibition constant (K(i)) of tight-binding inhibitor, the putative method estimated an apparent K(i) from the response of initial rates to total concentrations of the inhibitor considering its depletion during binding for conversion into the true K(i), but was impractical with glutathione S-transferase of sophisticated kinetics. A fluorometric titration assay of dissociation constant (K(d)) was thus proposed. Schistosoma japonicum glutathione S-transferase (SjGST) action on a nonfluorescent divalent pro-inhibitor and glutathione yielded a divalent product in active site to act as a tight-binding inhibitor, whose binding quenched fluorescence of SjGST at 340 nm under the excitation at 280 nm. K(d) was estimated from the response of fluorescence of SjGST at 340 nm to total concentrations of the divalent product considering its depletion during binding. By fluorometric titration assay, K(d) of two tested nonfluorescent divalent products varied from subnanomolar to nanomolar, but both were resistant to change of SjGST levels and consistent with their apparent K(i) estimated via the putative method. Hence, fluorometric titration assay of K(d) of nonfluorescent tight-binding inhibitors/ligands was effective to GST and may be universally applicable to common enzymes/proteins; affinities of tight-binding inhibitors of GST can be approximated by their apparent K(i) estimated via the putative method.

Fluorometric titration approach for calibration of quantity of binding site of purified monoclonal antibody recognizing epitope/hapten nonfluorescent at 340 nm.

A fluorometric titration approach was proposed for the calibration of the quantity of monoclonal antibody (mcAb) via the quench of fluorescence of tryptophan residues. It applied to purified mcAbs recognizing tryptophan-deficient epitopes, haptens nonfluorescent at 340 nm under the excitation at 280 nm, or fluorescent haptens bearing excitation valleys nearby 280 nm and excitation peaks nearby 340 nm to serve as Förster-resonance-energy-transfer (FRET) acceptors of tryptophan. Titration probes were epitopes/haptens themselves or conjugates of nonfluorescent haptens or tryptophan-deficient epitopes with FRET acceptors of tryptophan. Under the excitation at 280 nm, titration curves were recorded as fluorescence specific for the FRET acceptors or for mcAbs at 340 nm. To quantify the binding site of a mcAb, a universal model considering both static and dynamic quench by either type of probes was proposed for fitting to the titration curve. This was easy for fitting to fluorescence specific for the FRET acceptors but encountered nonconvergence for fitting to fluorescence of mcAbs at 340 nm. As a solution, (a) the maximum of the absolute values of first-order derivatives of a titration curve as fluorescence at 340 nm was estimated from the best-fit model for a probe level of zero, and (b) molar quantity of the binding site of the mcAb was estimated via consecutive fitting to the same titration curve by utilizing such a maximum as an approximate of the slope for linear response of fluorescence at 340 nm to quantities of the mcAb. This fluorometric titration approach was proved effective with one mcAb for six-histidine and another for penicillin G.

Comparison of Förster-resonance-energy-transfer acceptors for tryptophan and tyrosine residues in native proteins as donors.

Homogenous bioaffinity analysis with tryptophan/tyrosine residues in native proteins as FÖrster-resonance-energy-transfer (FRET) donors is feasible when suitable fluorophors can act as FRET acceptors in ligands (FRET probes) and FRET efficiency in complexes of proteins and FRET probes is high enough. In complexes of proteins and FRET probes, suitable acceptors should have excitation peaks around 335 nm and high rotation freedom, are preferred to have sufficient quantum yields and excitation valleys around 280 nm. In protein binding sites mimicked with mixtures of neutral phosphate buffer and organic solvents, quantum yields of candidate acceptors are altered inconsistently but their excitation peaks show tiny changes. Fluorophores as acceptors in such FRET probes are buried inside glutathione-S-transferase and have low rotation freedom, but are localized on streptavidin surface and display high rotation freedom; FRET efficiency in complexes of streptavidin and its FRET probes is much stronger than that in complexes of glutathione-S-transferase and its FRET probes. Specially, the quantum yield is about 0.70 for free 1-naphthylamine probe in neutral phosphate buffer, about 0.50 for 1-naphthylamine probe bound by streptavidin, and about 0.15 for that bound by glutathione-S-transferase. The quantum yield is about 0.06 for free dansylamide probe, about 0.11 for dansylamide probe bound by streptavidin and about 0.27 for that bound by glutathione-S-transferase. Therefore, 1-naphthylamine and dansylamide are effective acceptors when they localize on surfaces of complexes of proteins and FRET probes.