Ratiometric G-quadruplex/hemin DNAzymes with low-dosage associative substrates.

BACKGROUND: G-quadruplex (G4)/hemin DNAzymes with conversion of substrates into colorimetric readouts are well recognized as convenient biocatalysis tools in sensor development. However, the previously developed colorimetric G4/hemin DNAzymes are diffusive substrate-based DNAzymes (DSBDs). The current colorimetric DSBDs have several drawbacks including high dosage (∼mM) of diffusive substrates (DSs), colorimetric product toxicity, and single colorimetric readout without tolerance to fluctuation of experimental factors and background. In addition, the usage of high-dosage DSs can smear the G4 foldings and their discard is more harmful to environment. Therefore, exploring alternative DNAzymes with potential to overcome these drawbacks of DSBDs is urgently needed. RESULTS: We herein developed associative substrate-based DNAzymes (ASBDs). Cyanine dyes were selected as associative substrates (ASs) due to their binding competency with G4/hemin DNAzymes. With respect to DSBDs, ASBDs needed only low dosage (∼10 µM) of ASs to be able to cause a rapid and visible substrate conversion. In addition, since cyanine dyes are NIR dyes with high extinction coefficients and their conversion products have absorption bands at shorter wavelength. Therefore, a colorimetric ratio response can be developed to follow activities of G4/hemin DNAzymes with competency to tolerate fluctuation of experimental factors and background. In particular, herein developed ASBDs can endure somewhat concentration fluctuation of H2O2. ASBDs are able to cowork with other enzymes (for example, glucose oxidase) to realize cascade sensing. SIGNIFICANCE: The developed ASBDs can operate at low dosage of substrates with a colorimetric ratio response and can overcome the drawbacks met in DSBDs. We expect that, by designing ASs with fruitful color panel in the future, our work will inspire more interesting in developing environment-benign and low-carbon G4/hemin DNAzymes and desired colorful high-performance sensors.

Polarity-Specific and Pyrimidine-over-Purine Adaptive Triplex DNA Recognition by a Near-Infrared Fluorogenic Molecular Rotor.

Triplex DNA structures have displayed a wide range of applications including nanosensing, molecule switching, and drug delivering. Therefore, it is of great importance to effectively recognize triplex DNA structures by a simple and highly selective manner. Herein, we found that a near-infrared fluorogenic probe of NIAD-4 with a molecular rotor (MR) merit can selectively recognize triplex DNA structures over G-quadruplex, i-motif, and duplex structures (Tri-over-QID selectivity), which is competent over the widely used MR probe of thioflavin T (ThT). Furthermore, NIAD-4 exhibits as well a high selectivity toward the 'pyrimidine-type' triplex structures (Y:R-Y type) with respect to the 'purine-type' triplex structures (R:R-Y type) (a Y-over-R selectivity). Interestingly, NIAD-4 recognizes the Y:R-Y triplex structures by a polarity-dependent manner. The 3' end triplet is the preferential binding field of NIAD-4 with respect to the 5' end one (a 3'-over-5' selectivity) as the 3' end triplet is more stable than the 5' end one in the Hoogsteen hydrogen bond. It is expected that the adaptive stacking interaction between NIAD-4 and the 3' end triplet favors the Tri-over-QID, Y-over-R, and 3'-over-5' selectivities since this MR probe has three rotating shafts matching well with the triplet in topology. Such a high selectivity of NIAD-4 opens a new route in designing sensors with DNA structures switching between triplex, i-motif, and G-quadruplex structures.

Remodeling of the ryanodine receptor isoform 1 channel regulates the sweet and umami taste perception of Rattus norvegicus.

Sweet and umami tastes are elicited by sweet and umami receptors on the tongue and palate epithelium, respectively. However, the molecular machinery allowing the taste reaction remains incompletely understood. Through a phosphoproteomic approach, we identified the key proteins that trigger taste mechanisms based on phosphorylation cascades. Ryanodine receptor isoform 1 (RYR1) was further verified by sensory and behavioral assays. We propose a model of RYR1-mediated sweet/umami signaling in which the RYR1 channel, which mediates Ca2+ release from the endoplasmic reticulum, is closed by dephosphorylation in bud tissue after sweet/umami treatment. The alteration in Ca2+ content in the cytosol induces transient membrane depolarization and generates a cell current for taste signal transduction. We demonstrate that RYR1 is a new channel involved in the regulation of sweet/umami signal transduction and propose a "metabolic clock" notion based on sweet/umami sensing. Our study provides a valuable foundation for a system-level understanding of the taste perception mechanism.

Selectively Identifying Exposed-over-Unexposed C-C+ Pairs in Human Telomeric i-Motif Structures with Length-Dependent Polymorphism.

The i-motif structure (iM) has attracted much attention, because of its in vivo bioactivity and wide in vitro applications such as DNA-based switches. Herein, the length-dependent folding of cytosine-rich repeats of the human telomeric 5'-(CCCTAA)n-1CCC-3' (iM-n, where n = 2-8) was fully explored. We found that iM-4, iM-5, and iM-8 mainly form the intramolecular monomer iM structures, while a tetramolecular structure populates only for iM-3. However, iM-6 and iM-7 have the potential to fold as well into the dimeric iM structures besides the monomer ones. The natural hypericin (Hyp) was used as the polymorphism-selective probe to recognize the iM structures. Interestingly, only iM-3, iM-6, and iM-7 can efficiently switch on the Hyp fluorescence by specifically binding with the outmost C-C+ base pairs that are exposed directly to solution. However, other iM structures that fold in a way with a coverage of the outmost C-C+ pairs by loop sequences are totally unavailable for the Hyp binding. Theoretical modeling indicates that adaptive π-π and cation-π interactions contribute to the Hyp recognition toward the exposed C-C+ pairs. This specific iM recognition can be boosted by a photocatalytic DNAzyme construct. Our work provides a reliable fluorescence method to selectively explore the polymorphism of iM structures.

Switching G-quadruplex to parallel duplex by molecular rotor clustering.

Switching of G-quadruplex (G4) structures between variant types of folding has been proved to be a versatile tool for regulation of genomic expression and development of nucleic acid-based constructs. Various specific ligands have been developed to target G4s in K+ solution with therapeutic prospects. Although G4 structures have been reported to be converted by sequence modification or a unimolecular ligand binding event in K+-deficient conditions, switching G4s towards non-G4 folding continues to be a great challenge due to the stability of G4 in physiological K+ conditions. Herein, we first observed the G4 switching towards parallel-stranded duplex (psDNA) by multimolecular ligand binding (namely ligand clustering) to overcome the switching barrier in K+. Purine-rich sequences (e.g. those from the KRAS promoter region) can be converted from G4 structures to dimeric psDNAs using molecular rotors (e.g. thioflavin T and thiazole orange) as initiators. The formed psDNAs provided multiple binding sites for molecular rotor clustering to favor subsequent structures with stability higher than the corresponding G4 folding. Our finding provides a clue to designing ligands with the competency of molecular rotor clustering to implement an efficient G4 switching.

Visible light-driven i-motif-based DNAzymes.

DNA foldings provide variant possibilities to develop DNAzymes with remarkable catalytic performance. In spite of fruitful reports on G-quadruplex DNAzymes, four-stranded cytosine-rich i-motifs have not been explored as the potential skeletons of DNAzymes. In this work, we developed a visible light-driven DNAzyme based on human telomeric i-motifs using a natural photosensitizer of hypericin (Hyp) as the cofactor and dissolved oxygen as the oxidant source. The i-motif folding in acidic solution caused the distal thymine overhangs at the 3' and 5' ends to approach each other to provide a favorable binding site for Hyp via an interaction of fully complementary hydrogen bonding. However, the i-motifs without the distal overhangs or with the inappropriate overhang length and the base identity exhibited no binding with Hyp. The binding event converted Hyp from the fully dark state to the emissive state under visible light illumination. Subsequently, the excited Hyp had an opportunity to transfer energy to dissolved oxygen. Resultantly, singlet oxygen (1O2) was generated to initiate the substrate oxidation. The catalytic performance of the DNAzyme can be improved using a long-lived mediator. Our developed i-motif-based DNAzyme can be driven by almost the whole range of visible lights, suggesting broad applications in the photocatalytic fields, for example, as an alternative strategy in developing biodevices.

Selectively recognizing extrahelical conformations of DNA trinucleotide repeats by a hydroxylated porphyrin ligand.

Trinucleotide repeats (TRs) with abnormal lengths and atypical folding are implicated in various neurodegenerative diseases. The least stable cytosine-cytosine (C-C) mismatches in TRs when structuring into homoduplexes/hairpins have more chance in certain sequence contexts to preferentially adopt an extrahelical (E-motif) conformation with respect to those in polarity-inverted intrahelical counterparts. Herein, we designed a trihydroxyphenyl porphyrin ligand (POH3) to meet the challenge towards resolving the E-motif conformation. POH3 exhibited a specific 2:1 binding with DNAs adopting the E-motif cytosine conformation, independent of the TRs length. The trihydroxyl pattern was very crucial to gain the E-motif selectivity over the polarity-inverted counterparts via the complementary hydrogen bonding that occurred in the minor groove. Our work first elucidates the rationale in designing ligands to selectively resolve the E-motif nucleotides within TRs.

A catalytic triplex DNAzyme for porphyrin metalation.

Trihydroxyphenyl porphyrin (POH3) was designed to specifically bind with a triplex DNA resulting in a turn-on fluorescence response. This ensemble can be developed into a catalytic triplex DNAzyme towards porphyrin metalation. The catalytic activity is initiated by the enhanced basicity of POH3 upon binding with the triplex DNA.

A bombykol electrochemical receptor sensor and its kinetics.

This study aimed to explore the interaction between bombykol and BmOR1 and also provide a paradigm for agroforestry pest control. The electrochemical biosensor signal amplification system was used: nanogold with horseradish peroxidase. An electrochemical bilayer nanogold membrane receptor sensor was developed using the following schemes and processes: twice self-assembly of nanogold and succeeding absorption of Bombyx mori olfactory receptor 1 (BmOR1); sex pheromone-binding protein; spectral scanning and transmission electron microscope to characterize nanogold sol; and atomic force microscope, cyclic voltammetry, and AC impedance methods to characterize individual processes of sensor assembly. The amperometric I-T curve was adopted to measure the response current upon interaction with different concentrations of bombykol (diluted in phosphate-buffered saline) and BmOR1. The results demonstrated the receptor-ligand interaction pattern, which was similar to enzymatic reaction kinetics, with the activation constant Ka of up to 8.57 × 10-20 mol/L and signal magnification of about 10,000-fold. In this study, the simulation of intracellular receptor signaling cascade by an electrochemical signal amplification system helped in directly measuring BmOR1-bombykol ligand interaction and exploring the kinetics after the self-assembly of BmOR1 on the biosensor. It provided a novel platform for future studies on receptor-ligand interaction.

Study on Bombykol Receptor Self-Assembly and Universality of G Protein Cellular Signal Amplification System.

The G protein cascade amplification system couples with several receptors to sense/amplify the cellular signal, implying universal application. In order to explore whether GPCRs can trigger G protein signal amplification in tissues/cells from different species, bombykol receptor was isolated and purified from antennas of male Bombyx mori, which subsequently self-assembled on the cell membrane in rat taste buds/rat vomeronasa/catfish tentacles/taste bud tissues of rabbits/pig/cattle in those lacking endogenous bombykol receptor, followed by immobilization between two sheets of nucleopore membranes fixed by sodium alginate-starch gel, forming the sandwich-type sensing membrane, which in turn was immobilized on the glass-carbon electrode. Thus, bombykol receptor sensors were established with different tissues. The response current of bombykol receptor sensor toward bombykol was measured with an electrochemical workstation. Every bombykol receptor sensor could sense bombykol based on enzyme-substrate kinetics. The double reciprocal plot and the activation constant values of bombykol receptor sensors assembled with rat taste buds, rat vomeronasa, catfish tentacles, rabbit taste buds, pig taste buds, and cattle taste buds were calculated. Approximately 2-3 receptors could trigger the G protein cascade amplification system and achieve the maximum signal output. Moreover, the detection lower limit indicated that the bombykol receptor self-assembled on the cell membranes of different tissues that transmitted and amplified the bombykol signal with hypersensitivity. Also, cattle taste bud tissues served as an ideal system for heterogeneous GPCRs self-assembly and signal sensing/amplification. This sensing technique and method had promising potential in studies of biological pest control, sex pheromone detection, and receptor structure and function.

A novel electrochemical immunosensor based on Au nanoparticles and horseradish peroxidase signal amplification for ultrasensitive detection of α-fetoprotein.

An electrochemical double-layer Au nanoparticle membrane immunosensor was developed using an electrochemical biosensing signal amplification system with Au nanoparticles, thionine, chitosan, and horseradish peroxidase, which was fabricated using double self-adsorption of Au nanoparticle sol followed by anti-α-fetoprotein Balb/c mouse monoclonal antibody adsorption. The AuNPs sol was characterized by spectrum scanning and transmission electron microscopy. The immunosensor was characterized by atomic force microscopy, cyclic voltammetry, and alternating-current impedance during each stage of adsorption and assembly. The amperometric I-t curve method was used to measure α-fetoprotein (AFP) diluted in phosphate buffered saline. The result indicated a wide linear range, and the change rate of steady-current before and after immune response had linear correlation within the range 0.1-104 pg/mL AFP. The current change rate equation was △I = 5.82334 lgC + 37.01195 (R2 = 0.9922). The lowest limit of detection was 0.03 pg/mL (S/N = 3), and the reproducibility of the sensor was good. Additionally, the sensor could be stably stored above phosphate buffered saline at 4 °C for more than 24 days. More importantly, the sensor is label-free, reagentless and low fouling, making it capable of assaying AFP in real serum samples without suffering from significant interference or biofouling.