Unreacted Probe Research Articles

A bio-friendly biotin-coupled and azide-functionalized naphthalimide for real-time endogenous hydrogen sulfide analysis in living cells

Hydrogen sulfide (H2S) is involved in various biological processes. Thereby, abnormal levels of H2S are reported to be related to various human diseases including cancer. Currently, many fluorescent probes are pioneered to detect H2S by taking advantages of naphthalimides’ unique internal charge transfer (ICT) property. However, most probes often require a high content of organic solvents or surfactants, and are limited to the analysis of exogenous H2S treated externally in live cell studies, and have difficulties in analyzing endogenous H2S, thus limiting their practical use. In this study, we developed a bio-friendly biotin-coupled and azide-functionalized naphthalimide (1) as a fluorescent probe enabling real-time analysis of H2S in living system. Probe was able to provide a fluorescence at 545 nm via H2S-mediated azide reduction selectively without interference by biologically abundant constituents and pH effects. In a biological study using A549 cells, probe readily penetrated living cells without cytotoxicity, and unreacted probes showed almost no fluorescence, enabling real-time detection of H2S in living cells without requiring separate washing process. More importantly, under stimulation with various H2S inducers and inhibitors, probe was able to provide an effective fluorescence response against fluctuations in endogenous H2S, a key requirement for H2S studies. Probe 1 can be applied as a useful chemical tool and enables the analysis of H2S and the study of H2S-related cell functions in a variety of environments.

Molecular Design of Bioorthogonal Probes and Imaging Reagents Derived from Photofunctional Transition Metal Complexes.

For more than 15 years, bioorthogonal chemistry has received increasing attention due to its successful applications in the detection and imaging of biomolecules in their native biological environments. The method typically proceeds with the incorporation of a biological substrate appended with a bioorthogonal functional group (chemical reporter), followed by the introduction of the substrate to biological systems. Biomolecules containing the substrate are then recognized by an exogenously delivered bioorthogonal probe. Despite the fact that many useful chemical reporters and bioorthogonal reactions have been developed, most of the bioorthogonal probes reported thus far are fluorescent dyes. A limitation is that stringent washing is required due to the interference caused by the background fluorescence of unreacted probes. Thus, fluorogenic probes with turn-on emission properties upon bioorthogonal labeling have been designed as an alternative strategy. These probes are highly appealing because excellent images can be obtained without the need for washing steps. Nearly all fluorogenic bioorthogonal probes designed are essentially organic dyes, their emission is limited to fluorescence, and the utilization of the probes is confined to bioimaging applications. Recently, there has been a growing interest in the bioimaging and therapeutic applications of luminescent inorganic and organometallic transition metal complexes due to their intriguing photophysical and photochemical properties, high membrane permeability, controllable cellular uptake, intracellular localization, and cytotoxicity. We anticipate that photofunctional transition metal complexes can be exploited as valuable bioorthogonal probes due to these appealing advantages. In this Account, we introduce the molecular design, photophysical and photochemical properties, and biological applications of various bioorthogonal probes and imaging reagents based on photofunctional transition metal complexes. The presence of a cationic metal center significantly enhances the bioorthogonal reactivity of the probes, yet their stability in aqueous solutions can be maintained. Interestingly, some of these metal complexes are strategically modified to display phosphorogenic properties, that is, phosphorescence turn-on upon bioorthogonal labeling reactions. Importantly, these probes not only exhibit favorable photophysical properties after bioorthogonal labeling, but also efficient photoinduced singlet oxygen (1O2) generation. This interesting bioorthogonal reaction-triggered photosensitization capability allows the modulation of 1O2 generation efficiency and contributes to the development of controllable photocytotoxic agents. The exploration of transition metal complex-based probes not only significantly widens the scope of bioorthogonal labeling but also further highlights the unique advantages of these complexes in the design of theranostic reagents. The development of these innovative reagents is expected to contribute to the basic understanding of biological processes in living systems and provide exciting opportunities for new diagnostic and therapeutic applications.

Modified beacon probe assisted dual signal amplification for visual detection of microRNA

In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity.

Photoluminescence Lifetime Imaging of Synthesized Proteins in Living Cells Using an Iridium-Alkyne Probe.

Designing probes for real-time imaging of dynamic processes in living cells is a continuous challenge. Herein, a novel near-infrared (NIR) photoluminescence probe having a long lifetime was exploited for photoluminescence lifetime imaging (PLIM) using an iridium-alkyne complex. This probe offers the benefits of deep-red to NIR emission, a long Stokes shift, excellent cell penetration, low cytotoxicity, and good resistance to photobleaching. This example is the first PLIM probe applicable to the click reaction of copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with remarkable lifetime shifts of 414 ns, before and after click reaction. The approach fully eliminates the background interference and distinguishes the reacted probes from the unreacted probes, thus enabling the wash-free imaging of the newly synthesized proteins within single living cells. Based on the unique properties of the iridium complexes, it is anticipated to have applications for imaging other processes within living cells.

Photoluminescence Lifetime Imaging of Synthesized Proteins in Living Cells Using an Iridium–Alkyne Probe

AbstractDesigning probes for real‐time imaging of dynamic processes in living cells is a continuous challenge. Herein, a novel near‐infrared (NIR) photoluminescence probe having a long lifetime was exploited for photoluminescence lifetime imaging (PLIM) using an iridium‐alkyne complex. This probe offers the benefits of deep‐red to NIR emission, a long Stokes shift, excellent cell penetration, low cytotoxicity, and good resistance to photobleaching. This example is the first PLIM probe applicable to the click reaction of copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) with remarkable lifetime shifts of 414 ns, before and after click reaction. The approach fully eliminates the background interference and distinguishes the reacted probes from the unreacted probes, thus enabling the wash‐free imaging of the newly synthesized proteins within single living cells. Based on the unique properties of the iridium complexes, it is anticipated to have applications for imaging other processes within living cells.

Fluorescence Turn-On Folding Sensor To Monitor Proteome Stress in Live Cells.

Proteome misfolding and/or aggregation, caused by a thermal perturbation or a related stress, transiently challenges the cellular protein homeostasis (proteostasis) network capacity of cells by consuming chaperone/chaperonin pathway and degradation pathway capacity. Developing protein client-based probes to quantify the cellular proteostasis network capacity in real time is highly desirable. Herein we introduce a small-molecule-regulated fluorescent protein folding sensor based on a thermo-labile mutant of the de novo designed retroaldolase (RA) enzyme. Since RA enzyme activity is not present in any cell, the protein folding sensor is bioorthogonal. The fluorogenic small molecule was designed to become fluorescent when it binds to and covalently reacts with folded and functional RA. Thus, in the first experimental paradigm, cellular proteostasis network capacity and its dynamics are reflected by RA–small molecule conjugate fluorescence, which correlates with the amount of folded and functional RA present, provided that pharmacologic chaperoning is minimized. In the second experimental scenario, the RA–fluorogenic probe conjugate is pre-formed in a cell by simply adding the fluorogenic probe to the cell culture media. Unreacted probe is then washed away before a proteome misfolding stress is applied in a pulse-chase-type experiment. Insufficient proteostasis network capacity is reflected by aggregate formation of the fluorescent RA–fluorogenic probe conjugate. Removal of the stress results in apparent RA–fluorogenic probe conjugate re-folding, mediated in part by the heat-shock response transcriptional program augmenting cytosolic proteostasis network capacity, and in part by time-dependent RA–fluorogenic probe conjugate degradation by cellular proteolysis.

Fluorogenic protein labelling: a review of photophysical quench mechanisms and principles of fluorogen design

Fluorescent labelling of specific proteins in complex biological systems remains an important challenge in chemical biology. One promising approach comprises the use of small molecules designed to react specifically with a targeted protein of interest and to increase in fluorescent intensity following this reaction. This kind of fluorogenic reaction generally derives from fluorescence quenching in the unreacted probe that is abrogated over the course of the reaction. Herein, we review the mechanistic principles of three major photophysical quenching mechanisms involving Förster resonance energy transfer (FRET), through-bond energy transfer (TBET), and photoinduced electron transfer (PeT). We then present design principles for novel fluorogenic probes based on an understanding of these quench mechanisms, with emphasis on the emerging utility of density functional theory (DFT) calculations in the design process.

Design strategies for bioorthogonal smart probes.

Bioorthogonal chemistry has enabled the selective labeling and detection of biomolecules in living systems. Bioorthogonal smart probes, which become fluorescent or deliver imaging or therapeutic agents upon reaction, allow for the visualization of biomolecules or targeted delivery even in the presence of excess unreacted probe. This review discusses the strategies used in the development of bioorthogonal smart probes and highlights the potential of these probes to further our understanding of biology.

Template-Directed Fluorogenic Oligonucleotide Ligation Using “Click” Chemistry: Detection of Single Nucleotide Polymorphism in the Human p53 Tumor Suppressor Gene

A novel nonfluorescent alkyne-modified coumarin phosphoramidite was synthesized and successfully incorporated into oligonucleotides, which were then used in highly efficient DNA interstrand cross-linking and ligation reactions via "click" chemistry. The template-directed fluorogenic ligation "click" chemistry reaction was used for single nucleotide polymorphism analysis, where the target DNA catalyzes the ligation of two nonfluorescent probes to generate a fluorescent product. The upstream oligonucleotide probe is a nonfluorescent alkyne-modified coumarin and the downstream probe is an azide-modified oligonucleotide. When bound to a fully complementary template, the oligonucleotides ligated to produce a fluorescent product with a fluorophore at the ligation point. Wild-type and mutant p53 alleles were used to demonstrate that template-directed fluorogenic oligonucleotide ligation is sequence-specific and is capable of single nucleotide discrimination under mild conditions, even without the removal of unreacted probes.

Universal Drag Tag for Direct Quantitative Analysis of Multiple MicroRNAs

Using microRNA (miRNA) as molecular markers of diseases requires a method for accurate measurement of multiple miRNAs in biological samples. Direct quantitative analysis of multiple miRNAs (DQAMmiR) has been recently developed based on a classical hybridization approach. In DQAMmiR, miRNAs are hybridized with excess fluorescently labeled complementary DNA probes. Capillary electrophoresis (CE) is used to separate the unreacted probes from the hybrids and the hybrids from each other. The challenging separation is achieved by using two types of mobility modifiers. Single-strand DNA binding protein (SSB) is added to the running buffer to bind and shift the single-stranded unreacted probes from the double-stranded hybrids. Different drag tags are built into the probes to introduce significant differential mobility between their respective hybrids. For the method to be practical it requires a universal extendable drag tag. Polymers are a logical choice for making extendable drag tags. Our recent theoretical work suggested that short peptides could provide a sufficient mobility shift to facilitate DQAMmiR. Here, we experimentally confirm this prediction in the analysis of five miRNAs: mir10b, mir21, mir125b, mir145, and mir155. We conjugated four fluorescently labeled DNA molecules with peptides of 5, 10, 15, or 20 neutral amino acids in length; the fifth probe was peptide-free. The peptide tags showed no interference with SSB binding to the probes and facilitated separation of the five hybrids. The mobilities of the five hybrids were used to refine the previously suggested theory. The analysis was performed in both a pure buffer and in cell lysate. Our analysis of the experimental data suggests that using DNA-peptide probes can readily facilitate simultaneous analysis of more than 10 miRNAs.

DNAzyme based gap-LCR detection of single-nucleotide polymorphism

Fast and accurate detection of single-nucleotide polymorphism (SNP) is thought more and more important for understanding of human physiology and elucidating the molecular based diseases. A great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. However most of those methods developed to date incorporate complicated probe labeling and depend on advanced equipment. The DNAzyme based Gap-LCR detection method averts any chemical modification on probes and circumvents those problems by incorporating a short functional DNA sequence into one of LCR primers. Two kinds of exonuclease are utilized in our strategy to digest all the unreacted probes and release the DNAzymes embedded in the LCR product. The DNAzyme applied in our method is a versatile tool to report the result of SNP detection in colorimetric or fluorometric ways for different detection purposes.

Theoretical estimation of drag tag lengths for direct quantitative analysis of multiple miRNAs (DQAMmiR)

To better understand the regulatory roles of miRNA in biological functions and to use miRNA as molecular markers of diseases, we need to accurately measure amounts of multiple miRNAs in biological samples. Direct quantitative analysis of multiple miRNAs (DQAMmiR) has been recently developed by using a classical hybridization approach where miRNAs are hybridized with fluorescently labeled complementary DNA probes taken in excess, and the amounts of the hybrids and the unreacted probes are measured to calculate the amount of miRNAs. Capillary electrophoresis was used as an instrumental platform for analysis. The problem of separating the unreacted probes from the hybrids was solved by adding SSB to the run buffer. A more difficult problem of separating hybrids from each other was solved by attaching different drag tags to the probes. Biotin and a hairpin-forming extension on the probe were used as two drag tags in the proof-of-principle work. Making DQAMmiR a generic approach requires a generic solution for drag tags. Peptides have been suggested as drag tags for long oligonucleotides in DNA sequencing by electrophoresis. Here we theoretically consider short peptides of different lengths as drag tags for DQAMmiR. We find analytical equations that allow us to estimate mobilities of RNA-DNA hybrids with peptide drag tags of different lengths. Our calculations suggest that the mobility shifts required for DQAMmiR can be achieved with the length of peptide chains in the ranges of 5-20 residues for five miRNAs and 2-47 residues for nine miRNAs. Peptides of these lengths can be feasibly synthesized with good yield and purity. The results of this theoretical study will guide the design and production of hybridization probes for DQAMmiR.

Activity-based fluorescent probes for monitoring sulfatase activity

A small-molecule probe for sulfatase is developed that shows a significant change in fluorescence upon reaction with sulfatase in an activity-based manner. As this probe is free from interference from background fluorescence caused by an unreacted probe, it could be a simple and efficient tool for the study of sulfatase activity.

A FRET-based probe with a chemically deactivatable quencher

A new concept of a chemically deactivatable quencher is proposed for a FRET-based probe that turns-on its fluorescence by either an enzymatic cleavage or a chemical reagent (sodium dithionite). This concept allowed us to quantify the caspase-3 cleavage activity in solution and to reveal unreacted probes in cell experiments.

A fluorescence-based assay for octreotide in kinetic release from depot formulations

Here we report the validation of a derivatization method that makes use of fluorescamine as a selective reactant for the quantitative analysis of peptide and protein drugs in the dissolution profile from depot formulations. Typical current methods require separation of the nano/microparticles and time-consuming chromatographic runs. In this study we report a method which can be conducted without the need for complete physical separation of the particles or removal of the unreacted probe. This method was used here for the analysis of the release profile of octreotide in a depot formulation, with results in excellent agreement with reported chromatographic assays.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

A Highly Sensitive Aptamer-Nanogold Catalytic Resonance Scattering Spectral Assay for Melamine

The aptamer (ssDNA) was used to label nanogold (NG) particle to fabricate an aptamer-nanogold (NGssDNA) probe for melamine. The probe was stabile in pH6.6 Na(2)HPO(4)-NaH(2)PO(4) buffer solutions and in the presence of high concentration of electrolyte. Upon addition of melamine, it interacted with the probe to form big NGssDNA-melamine aggregations that led to the resonance Rayleigh scattering (RRS) intensity at 566nm increased greatly. The increased RRS intensity (ΔI) is linear to melamine concentration in the range of 1.89-81.98μg/L, with a detection limit of 0.98μg/L melamine. The unreacted probe in the aptamer reaction solution exhibited strong catalytic effect on the slow Cu(2)O particle reaction between glucose and Fehling reagent, but the catalytic activity of NG aggregations is very weak. When melamine concentration increased, the unreacted probe decreased, the RRS peak intensity at 614nm decreased. The decreased RS intensity is linear to melamine concentration in the range of 0.63-47.30ng/L melamine, with a detection limit of 0.38ng/L. The aptamer-modified nanogold catalytic RRS assay was applied to determination of melamine in milk, with high sensitivity and selectivity, simplicity and rapidity.

Activity-based Protein Profiling Identifies a Host Enzyme, Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.

Fluorescence quenched quinone methide based activity probes – a cautionary tale

A carbamate linked quenching group coupled with a pro-quinone methide reactive core provides an effective tool for studying enzyme function without problems associated with background fluorescence from unreacted probe. However, the relatively slow fragmentation of the carbamate linkage in such a strategy may cause problems of loss of signal or a decoupling of enzyme activity and labelling.

Evidence for a Second, High Affinity Gβγ Binding Site on Gαi1(GDP) Subunits

It is well known that Galpha(i1)(GDP) binds strongly to Gbetagamma subunits to form the Galpha(i1)(GDP)-Gbetagamma heterotrimer, and that activation to Galpha(i1)(GTP) results in conformational changes that reduces its affinity for Gbetagamma subunits. Previous studies of G protein subunit interactions have used stoichiometric amounts of the proteins. Here, we have found that Galpha(i1)(GDP) can bind a second Gbetagamma subunit with an affinity only 10-fold weaker than the primary site and close to the affinity between activated Galpha(i1) and Gbetagamma subunits. Also, we find that phospholipase Cbeta2, an effector of Gbetagamma, does not compete with the second binding site implying that effectors can be bound to the Galpha(i1)(GDP)-(Gbetagamma)(2) complex. Biophysical measurements and molecular docking studies suggest that this second site is distant from the primary one. A synthetic peptide having a sequence identical to the putative second binding site on Galpha(i1) competes with binding of the second Gbetagamma subunit. Injection of this peptide into cultured cells expressing eYFP-Galpha(i1)(GDP) and eCFP-Gbetagamma reduces the overall association of the subunits suggesting this site is operative in cells. We propose that this second binding site serves to promote and stabilize G protein subunit interactions in the presence of competing cellular proteins.