Photoactive Probe Research Articles

Rapid Assembly of Ultrafine Palladium Nanoparticle-Decorated HOF-101 Triggered by Guest Enzyme Encapsulation.

Rapid enzyme immobilization is essential for enzyme catalysis and sensing applications, yet constructing effective immobilization systems is challenging due to the need to balance enzyme activity with the properties of the surrounding framework. Herein, taking glucose oxidase (GOx) as a model, a rapid and straightforward approach was presented for synthesizing palladium nanoparticles (PdNPs)-decorated GOx encapsulated in HOF-101 nanocomposite materials (designated as PdNPs/GOx@HOF-101) through an in situ photoreduction and enzyme-triggering HOF-101 encapsulation. The enzyme's surface residues trigger the nucleation of HOF-101 around it through the hydrogen-bonded bio interface, completing the self-assembly of HOF-101 in 0.5 h. Furthermore, the biocomposites loaded with ultrafine PdNPs show satisfactory photoelectrochemical (PEC) properties. As a proof-of-concept, a PEC biosensor was constructed by utilizing PdNPs/GOx@HOF-101 as a photoactive probe, which can quickly and sensitively detect glucose and simultaneously remain stable within the circumstance of 30-60 °C and pH 4-8. These attributes pave the way for diverse applications, including improved enzyme immobilization techniques, advanced biosensors, and more efficient biocatalytic processes.

Responsive photoactive probe based photoelectrochemical sensor for sulfur dioxide

AbstractThis study introduces a novel photoelectrochemical (PEC) sensor for the highly selective detection of sulfur dioxide (SO2) using an organic photoactive molecule probe (OPM). OPM is synthesized through a one‐step coupling reaction, featuring a typical photosensitizer D‐π‐A structure. By covalently bonding OPM with a TiO2 substrate, a PEC sensor is constructed, exhibiting a significant photocurrent response under visible light excitation. The specific addition reaction between SO2 and OPM disrupts its conjugated structure, reducing the photocurrent response and achieving highly selective detection of SO2. The sensor demonstrates excellent performance in real water samples, emphasizing its practical applications.

Upconverting Nanoparticle-Based Photoactive Probes for Highly Efficient Labeling and Isolation of Target Proteins.

Photoaffinity labeling (PAL) has blossomed into a powerful and versatile tool for capture and identification of biomolecular targets. However, low labeling efficiency for specific targets such as lectins, the tedious process for protein purification, inevitable cellular photodamage, and less tissue penetration of UV light are significant challenges. Herein, we reported a near-infrared (NIR) light-driven photoaffinity labeling approach using upconverting nanoparticle (UCNP)-based photoactive probes, which were constructed by assembling photoactive groups and ligands onto NaYF4:Yb,Tm nanoparticles. The novel probes were easily prepared and functionalized, and the labeled proteins can be isolated and purified through simple centrifugation and washing. The advantages of this approach were demonstrated by labeling and isolation of peanut agglutinin (PNA), asialoglycoprotein receptor (ASGPR), and human carbonic anhydrase II (hCAII) from mixed proteins or cell lysates with good selectivity and efficiency, especially for PNA and ASGPR, two lectins that showed low binding affinity to their ligands. More importantly, successful labeling of PNA through pork tissues and ASGPR in mice strongly proved the good tissue penetrating capacity of NIR light and the application potential of UCNP-based photoactive probes for protein labeling in vivo. Biosafety of this approach was experimentally validated by enzyme, cell, and animal work, and we demonstrated that NIR light caused minimal photodamage to enzyme activity compared to UV light, and the UCNP-based photoactive probe presents good biosafety both in vitro and in vivo. We believe that this novel PAL approach will provide a promising tool for study of ligand-protein interactions and identification of biomolecular targets.

Electrochemical Polymerization Induced Chirality Fixation of Crystalline Pillararene-Based Polymer and Its Application in Interfacial Chiral Sensing.

A new strategy has been developed for the direct chirality fixation, which is induced by electrochemical polymerization, of macrocyclic hosts pillar[5]arene. Taking advantage of electrochemical polymerization, thiophene-modified pillar[5]arene monomers (Th-P[5]A) have been regularly arranged under the action of an electric field to form chiral nanofiber-like crystalline pillar[5]arene-based polymers (poly-Th-P[5]A), showing a significant circular dichroism (CD) signal. With the active photochemical properties, poly-Th-P[5]A is first used as a photoelectrochemical (PEC) chiral sensor for the identification and determination of l- and d-ascorbic acid (l-AA, d-AA) without adding any extra photoactive probes. Importantly, the chiral recognition between poly-Th-P[5]A and l-AA also triggers a polarity conversion for the photocurrent of the polymer, and it greatly results in a broad chiral detection range for l-AA, crossing 6 orders of magnitude. This work provides a promotional strategy for building a PEC chiral recognition platform based on pillararenes.

Studying the "Rigid-Flexible" Properties of Polymeric Micelle Core-Forming Segments with a Hydrophobic Phthalocyanine Probe Using NMR and UV Spectroscopy.

The aim of the performed studies was to thoroughly examine the internal structure of self-assembled nanocarriers (i.e., polymeric micelles—PMs) by means of a hydrophobic phthalocyanine probe in order to identify the crucial features that are required to enhance the photoactive probe stability and reactivity. PMs of hydrophilic poly(ethylene glycol) and hydrophobic poly(ε-caprolactone) (PCL) or poly(d,l-lactide) (PDLLA) were fabricated and loaded with tetra tert-butyl zinc(II) phthalocyanine (ZnPc-t-but4), a multifunctional spectroscopic probe with a profound ability to generate singlet oxygen upon irradiation. The presence of subdomains, comprising “rigid” and “flexible” regions, in the studied block copolymers’ micelles as well as their interactions with the probe molecules, were assessed by various high-resolution NMR measurements [e.g., through-space magnetic interactions by the 1D NOE effect, pulsed field gradient spin-echo, and spin–lattice relaxation time (T1) techniques]. The studies of the impact of the core-type microenvironment on the ZnPc-t-but4 photochemical performance also included photobleaching and reactive oxygen species measurements. ZnPc-t-but4 molecules were found to exhibit spatial proximity effects with both (PCL and PDLLA) hydrophobic polymer chains and interact with both subdomains, which are characterized by different rigidities. It was deduced that the interfaces between particular subdomains constitute an optimal host space for probe molecules, especially in the context of photochemical stability, photoactivity (i.e., for significant enhancement of singlet oxygen generation rates), and aggregation prevention. The present contribution proves that the combination of an appropriate probe, high-resolution NMR techniques, and UV–vis spectroscopy enables one to gain complex information about the subtle structure of PMs essential for their application as nanocarriers for photoactive compounds, for example, in photodynamic therapy, nanotheranostics, combination therapy, or photocatalysis, where the micelles constitute the optimal microenvironment for the desired photoreactions.

Immunoediting role for major vault protein in apoptotic signaling induced by bacterial N-acyl homoserine lactones

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

Analysis and Identification of Tumorigenic Targets of MicroRNA in Cancer Cells by Photoreactive Chemical Probes.

Photoactive RNA probes have unique advantages in the identification of microRNA (miR) targets due to their ability for efficient conjugation to the target sequences by covalent crosslinking, providing stable miR-mRNA complexes for further analysis. Here, we report a highly efficient and straightforward method for miR target identification that is based on photo-reactive chemical probes and RNA-seq technology (denotes PCP-Seq). UV reactive probes were prepared by incorporating psoralen in the specific position of the seed sequence of miR. Cancer cells that were transfected with the miR probes were treated with UV, following the isolation of poly(A) RNA and sequencing of the transcriptome. Quantitative analysis of RNA-seq reads and subsequent validation by qPCR, dual luciferase assay as well as western blotting confirmed that PCP-Seq could highly efficiently identify multiple targets of different miRs in the lung cancer cell line, such as targets PTTG1 and PTGR1 of miR-29a and ILF2 of miR-34a. Collectively, our data showed that PCP-Seq is a robust strategy for miR targets identification, and unique in the identification of the targets that escape degradation by miRISC and maintain normal cellular level, although their translation is repressed.

Interrogating Amyloid Aggregates using Fluorescent Probes.

Amyloids are a broad class of proteins and peptides that can misfold and assemble into long unbranched fibrils with a cross-β conformation. These misfolding and aggregation events are associated with the onset of a variety of human diseases, among them, Alzheimer's disease, Parkinson's disease, and Huntington disease. Our understanding of amyloids has been greatly supported by fluorescent molecular probes, such as thioflavin-T, which shows an increase in fluorescence emission upon binding to fibrillar aggregates. Since the first application of thioflavin-T in amyloid studies nearly 30 years ago, many probes have emerged exhibiting a variety of responses to amyloids, such as intensity changes, shifts in fluorescence maxima, and variations in lifetimes, among many others. These probes have shed light on a variety of topics including the kinetics of amyloid aggregation, the effectiveness of amyloid aggregation inhibitors, the elucidation of binding sites in amyloid structures, and the staining of amyloids aggregates in vitro, ex vivo, and in vivo. In this Review, we discuss the design, properties, and application of photoactive probes used to study amyloid aggregation, as well as the challenges faced by current probes and techniques, and the novel approaches that are emerging to address these challenges.

A label-free photoelectrochemical aptasensor for facile and ultrasensitive mercury ion assay based on a solution-phase photoactive probe and exonuclease III-assisted amplification.

In typical photoelectrochemical (PEC) biosensing assays, electrodes are generally modified with photoactive probes and/or target recognition probes, which makes the processes complicated, time-consuming, and difficult to achieve excellent reproducibility. Hence, to overcome such shortcomings, we propose here an immobilization-free and label-free PEC aptasensor using solution-phase methylene blue (MB) as the PEC signal probe. Based on the unique T-Hg2+-T base pairs, and the diffusivity difference between free MB molecules and the MB/G-quadruplex composite towards the ITO electrode surface with negative charge, the "signal-off" approach for Hg2+ detection is developed. In the presence of target Hg2+, via the T-Hg2+-T bond formation, the two sticky ends of the hairpin DNA probe form a rigid duplex stem, which triggers the exonuclease III-facilitated target cycling amplification, and the formation of multiple G-quadruplexes. Upon the intercalation of MB in G-quadruplexes, significantly decreased photocurrent is obtained owing to the increased electrostatic repulsion between the MB/G-quadruplex composite and the ITO electrode. Therefore, highly sensitive and ultrasensitive Hg2+ determination is achieved, with a low detection limit of 1.2 pM, well below the maximum allowable Hg2+ level in drinking water defined by the WHO, China's Ministry of Health, and the US EPA. Due to the avoidance of sophisticated electrode modification and recognition probe immobilization processes, as well as an expensive labeling procedure, the PEC aptasensor proposed here demonstrates the advantages of simplicity, good reproducibility, rapidness and low cost.

Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical Biosensing Strategy for Facile and Highly Sensitive MicroRNA Assay.

In conventional photoelectrochemical (PEC) analysis, photoactive materials are usually immobilized on electrode surfaces, and such immobilization procedures are tedious and time-consuming, and it is also difficult to prepare electrodes with good reproducibility. To circumvent such limitations, we propose here a truly immobilization-free diffusivity-mediated PEC bionsensing strategy for microRNA assay, using methylene blue (MB) in solution as the photoactive probe, and nonmodified indium tin oxide (ITO) glass as the working electrode. The hybridization between the target microRNA and the MB-labeled single-stranded DNA probe (MB-DNA) triggers the digestion of MB-DNA by T7 exonuclease (T7 Exo), thus to generate MB-labeled mononucleotide, and then the released target microRNA initiates the subsequent cycling processes and generates a large amount of MB-labeled mononucleotides. Due to the diffusivity difference between MB-DNAs and MB-labeled mononucleotides, significantly increased photocurrent signal is observed for MB-labeled mononucleotides as compared to that of MB-DNAs. Therefore, via this "signal-on" mode and the T7 Exo facilitated signal amplification, a facile and highly sensitive immobilization-free PEC microRNA assay is readily realized, with a detection limit down to 27 aM. Moreover, this strategy exhibits excellent specificity and is successfully applied in detecting microRNA spiked in serum samples. Since all the reactions take place in homogeneous solutions and no electrode modification is needed, this PEC biosensing strategy exhibits the advantages of simplicity, rapidness, and good reproducibility. More significantly, it provides a novel concept to design truly immobilization-free PEC biosensing systems, and shows potential to be applied in bioanalysis and biochemical research.

Novel ubiquitin binding sites of S. cerevisiae polymerase η identified by a genetically encoded photoactive probe (927.4)

DNA translesion synthesis is carried out by specialized DNA polymerases such as Polη. Polη contains a ubiquitin‐binding zinc finger (UBZ) domain required for its effective recruitment to the stalled replication fork in response to DNA damages. Our previous study reported a novel ubiquitin‐binding mode of S. cerevisiae Polη in which the C‐terminal portion of Polη binds ubiquitin with 30‐fold higher affinity than the UBZ domain alone. However, the mechanism of such higher binding affinity is not clear. In this study, a genetically encoded photoactive probe, p‐benzoyl‐L‐phenylalanine (pBpa), was incorporated at various positions on ubiquitin to map the interaction sites between Polη and ubiquitin. Data suggests that besides the Ile44 hydrophobic patch which was known to bind UBZ domain of Polη, several other residues at the ubiquitin C‐terminus and a surface opposite from the hydrophobic patch on ubiquitin also bind to Polη. These new binding sites increase the binding affinity between ubiquitin and Polη. Our finding is important for understanding the role of ubiquitin in the recruitment of Polη during DNA translesion synthesis.Grant Funding Source: Supported by U.S National Science Foundation Grant MCB‐0953764 to Z. Zhuang

Synthesis and Evaluation of a Photoactive Probe with a Multivalent Carbohydrate for Capturing Carbohydrate–Lectin Interactions

Lectins are ubiquitous carbohydrate-binding proteins of nonimmune origin that are characterized by their specific recognition of defined monosaccharide or oligosaccharide structures. However, the use of carbohydrates to study lectin has been restricted by the weak binding affinity and noncovalent character of the interaction between carbohydrates and lectin. In this report, we designed and synthesized a multifunctional photoaffinity reagent composed of a trialkyne chain, a masked latent amine group, and a photoreactive 3-trifluoromethyl-3-phenyl-diazirine group in high overall yield. Two well-defined chemistries, Huisgen-Sharpless click chemistry and amide bond coupling, were the key steps for installing the multivalent character and tag in our designed photoaffinity probe. The photolabeling results demonstrated that the designed probe selectively labeled the target lectin, RCA120 ( Ricinus communis Agglutinin), in an E. coli lysate and an asialoglycoprotein receptor (ASGP-R) on intact HepG2 cell membranes. Moreover, the probe also enabled the detection of weak protein-protein interactions between RCA120 and ovalbumin (OVA).

Magnesium optical one-shot sensor based on a coumarin chromoionophore

The characterization of a new irreversible optical absorption-based one-shot sensor for magnesium is described. The magnesium photoactive probe is 7-diethylamino-3-(3,4-ethylendioxybenzoyl)coumarin immobilized in a plasticized polymeric membrane. The magnesium selectivity can be explained in terms of size and charge density of magnesium and charge-separated resonance forms contribution in the excited state of coumarin. The selectivity obtained for magnesium over a variety of naturally occurring species in natural waters meets the requirements for the determination of this ion in water. The one-shot sensor responds between 0.14 and 14 mg L −1 with a sensor-to-sensor reproducibility of 1.3% as log ⁡ a M g 2 + , at the medium level of the range. The performance of the optical one-shot sensor was tested in the analysis of magnesium in different types of natural waters and soft drinks validating results against a reference procedure.

Membrane orientation of laminin binding protein.

Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.

Phototriggered drug release from functionalized oligonucleotides by a molecular beacon strategy.

More than just a light switch: A phototriggered molecule-releasing system controlled through intramolecular triplet quenching by using a molecular beacon strategy with photoactive probe oligodeoxynucleotides (ODNs) has been developed (see picture).

Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-azido-4-methylcoumarin.

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 +/- 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.

Unusual behaviour of 7-diethylamino-3-(3,4-ethylen-dioxybenzoyl)coumarin towards Group IIA cations: A potential photoactive probe for magnesium

The titled compound showed, upon spectrophotometric titrations with Group IIA cations, a spectacular bathochromic shift ( c.a. 50 nm) with Mg 2+ but not with the other cations. This result is understood in terms of internal charge-transfer species expressly favored by Et 2N and ArCO substituents and best matching of ionic diameter and charge density of Mg 2+. This finding may lead to new simple photoactive probes.

Site specific photo-crosslinking of single stranded oligonucleotides by a complementary sequence equipped with an internal photoactive probe

The photo-crosslinking behaviour of oligonucleotide constructs, incorporating photoactive residues 1–5 at a defined position, has been examined in the presence of their DNA (14-mer) and RNA (15-mer) complementary targets using a two-fold excess; it revealed a moderate but promising irreversible binding efficiency, the results of which could be anticipated by molecular mechanics in the case of 4.

Inhibition of rat steroid 5α-reductase (isozyme 1) by suramin

In this study, we show the inhibition of rat steroid 5α-reductase (isozyme 1) by suramin. The enzyme activity decreased in a dose-dependent manner as suramin concentrations increased with the calculated drug dose required for 50% inhibition (at 5 μM testosterone and 200 μM NADPH) being 13 μM. Suramin showed non-competitive inhibition of 5α-reductase with respect to testosterone (K T I = 2.4 μM) and competitive inhibition with respect to NADPH (K NADPH i = 220 nM). Furthermore, suramin and NADP +, but not NAD +, protected 5α-reductase from labeling by 2-azido-NADP +, a photoactive probe which has recently been used to identify the NADPH binding domain of 5α-reductase. These results suggest that suramin inhibits rat steroid 5α-reductase (isozyme 1) at the level of NADPH binding to the enzyme.

Characterization of a Max:DNA complex by cross-linking to photoactive oligonucleotides.

The structure of the Max:DNA complex was investigated by cross-linking Max to a series of photoactive oligonucleotides. A single photoactive aryl azide was introduced into oligonucleotides at defined positions downstream from the specific CACGTG binding site. Purified Max homodimers bound to and were cross-linked to oligonucleotides containing a photoactive group either 2, 5, 8, or 11 bp downstream from the binding site. Further analysis revealed that an amino-terminal chymotryptic peptide of Max was cross-linked to the oligonucleotide containing a photoactive probe 11 bp downstream from the specific binding site. This result is consistent with the recent crystal structure of the Max:DNA complex (Ferré-D'Amaré et al., 1993) and further suggests that amino acid residues near the amino-terminus of Max are in close proximity to a region of DNA that is separated from the core binding site by one turn of the double helix.