Split Fragments Research Articles

Fluorometric detection of copper ions using click chemistry and the target-induced conjunction of split DNAzyme fragments

ABSTRACTThis study aimed to develop a fluorescence assay for Cu2+ monitoring with cascade signal amplification using split Mg2+-dependent DNAzyme subunits as the sensing system and fluorescence as the signal reporter. A subunit of DNAzyme can be formed through a Cu2+-catalysed click reaction between azide- and alkyne-modified short sequences immobilized on magnetic beads, eliminating the washing steps of conventional assays. The immobilized subunit recruits the assisting oligonucleotides to form an autocatalytic DNAzyme as the signal amplifier and a caged fluorescent molecule as the signal reporter. This assay is ultrasensitive, enabling the detection of trace levels of Cu2+ as low as 2 nM, which enhances the detection sensitivity by approximately 100-fold. In addition, the whole reaction process does not require a long manipulation time or a complicated procedure, making the system simple and cost-effective. This method is robust and can be applied to the determination of spiked Cu2+ in water samples with satisfactory recovery. The combination of click chemistry with fluorescent DNAzyme for a simple on-site detection assay can be used for the rapid and sensitive screening of Cu2+.

Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art.

G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR–GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein–protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR–GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR–GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays.

Bioassays to study biased signalling of novel synthetic opioids

For hundreds of years, opioids have been used in the management of pain, mediating their analgesic effect via binding to the Mu opioid receptor (MOR). Fentanyl and morphine are examples of MOR agonists whose analgesic effects are associated with unwanted side effects such as tolerance and dependence. While analgesia is mainly realized through the G protein-signalling pathway, the undesirable effects of opioids have been linked to the β-arrestin pathway. Little is known about a potential “bias” of the synthetic opioids – including fentanyl analogs – that have emerged on the illegal drug market. To develop a novel, robust platform to study the activity of synthetic opioids through both the G protein and β-arrestin pathways. We developed bioassays that are based on functional complementation of two split fragments of Nanoluciferase (LgBiT and SmBiT), either fused to the receptor or to the cytosolic proteins: mini G protein and β-arrestin. These assays were performed in 96-well plates using transiently transfected HEK293 T cells, using pure reference standards. Assays were set up in which MOR, fused to LgBiT, was combined with cytosolic proteins, fused to SmBiT. Both the mini G protein and the β-arrestin bioassays demonstrated concentration-dependent responses at MOR, using hydromorphone, DAMGO, fentanyl and carfentanil, with activation as low as 10 −13 M still being detectable for carfentanil. A wide range of fentanyl analogs is currently being tested–the results of which will be discussed. The present study provides an in vitro platform to study functional activation of Mu opioid receptor by NPS by means of their capacity to couple to G protein and recruit β-arrestin. The developed bioassays not only allow insight into the signalling of synthetic opioids, which may help to better understand why certain opioids are associated with higher toxicity but may also be used to screen biofluids for opioid activity.

Hybridization-activated spherical DNAzyme for cascading two-photon fluorescence emission: Applied for intracellular miRNA measurement by two-photon microscopy

Two-photon excitation (TPE) has excellent properties, such as lower tissue self-absorption and autofluorescence, reduced photodamage and photobleaching, and higher spatial resolution. Meanwhile, DNAzyme has significant signal amplification ability, and the gold nanoparticle (AuNP)-based spherical nucleic acid (SNA) has the properties of resistance to enzymatic degradation, enhanced nucleic acid binding and excellent cellular uptake. Taking advantage of their properties, in this work, we constructed a novel SNA-based TPE fluorescent DNAzyme probe for the selective and sensitive detection of target miRNA in living cells. Briefly, AuNPs were first functionalized with the substrate sequences hybridized to two split fragments of the 8–17 DNAzyme-contained sequence. Then, numerous two-photon absorption (TPA)-dye molecules of Ethyl-4-[3,6-Bis (1-methyl-4-vinylpyridium iodine)-9H-carbazol-9-yl)] (EBMVC) were inserted into the substrate/split-DNAzyme duplexes on the surface of the AuNP to form the TPE SNA nanoprobe in the “off” fluorescence emission state. When the target miRNA is bound, the conformation of the split DNAzymes will change resulting in the activation of their catalytic ability, thus leading to cleavage of the substrate sequences and the release of the TPA-dye molecules-inserted DNAzyme motif from the surface of the AuNP. Concurrently, the released target miRNA would autonomously hybridize with another split DNAzyme motif and activate further cleavage reactions. This would induce the gradual dissociation of the TPA-dye molecules-inserted DNAzyme motifs from the surface of the AuNP through the repeated target miRNA hybridization-activated cleavage reaction and thereby trigger the cascade TPE fluorescence emission, ultimately achieving the detection of the miRNA through amplification of the fluorescence signals. The assay was successfully validated with clean buffer conditions as well as with cells.

Homo-molecular Fluorescence Complementation for Direct Visualization of Receptor Oligomerization in Living Cells.

Cell surface receptor oligomerization is an attractive target process for drug screening. However, simple but reliable (and thus high-throughput) visualization methods for receptor oligomerization are still lacking. Herein, we report on a new single-construct homo-molecular fluorescence complementation (Homo-FC) probe, which shows strong fluorescence signals by oligomerization of fused receptors in living cells with unexpectedly low background signals. Importantly, this high signal-to-noise ratio was not affected by expression level variations of fused receptors. The Homo-FC probe was developed by optimized flopped fusion of split fragments of superfolder green fluorescence protein and subsequent surface charge engineering. Homo-FC reliably visualized the oligomerization of diverse natural receptors such as GPCR, EGFR, and even cytosolic DAI.

Homo‐molecular Fluorescence Complementation for Direct Visualization of Receptor Oligomerization in Living Cells

AbstractCell surface receptor oligomerization is an attractive target process for drug screening. However, simple but reliable (and thus high‐throughput) visualization methods for receptor oligomerization are still lacking. Herein, we report on a new single‐construct homo‐molecular fluorescence complementation (Homo‐FC) probe, which shows strong fluorescence signals by oligomerization of fused receptors in living cells with unexpectedly low background signals. Importantly, this high signal‐to‐noise ratio was not affected by expression level variations of fused receptors. The Homo‐FC probe was developed by optimized flopped fusion of split fragments of superfolder green fluorescence protein and subsequent surface charge engineering. Homo‐FC reliably visualized the oligomerization of diverse natural receptors such as GPCR, EGFR, and even cytosolic DAI.

A label-free and enzyme-free aptasensor for visual Cd2+detection based on split DNAzyme fragments

A label-free and enzyme-free three-way G-quadruplex junction sensing system was developed for Cd2+detection with a naked-eye readout.

Reduction in the contact time of impacting droplets by decorating a rectangular ridge on superhydrophobic surfaces

Increasing attentions have been paid to the reduction in contact time between rebounding droplets and solid surfaces because of its potential applications in self-cleaning, anti-icing, anti-dew, and so forth. Owing to the axisymmetric spreading/retraction dynamics of impacting droplets on flat surfaces, there is a minimum contact time. Recently, adding a cylindrical wire or a triangular ridge on superhydrophobic surfaces has been proven to be an effective approach to break through this limit. Here, via lattice Boltzmann simulations, we show that droplets impacting superhydrophobic surfaces decorated with a macro rectangular ridge can also yield a significantly reduced contact time. Based on the simulated snapshots of impact dynamics, we reveal that the presence of the rectangular ridge can induce a tunable non-axisymmetric spreading/retraction dynamics. We found that once an impacting droplet is split into two fragments by the ridge, the contact time will be lower than the limit achieved on the flat surfaces with the same contact angle. We observe three different retraction ways for the split fragments depending on impact Weber number (We) as well as ridge height and width. When the formed fragments only retract once before they rebound from the surface, a lower contact time can be achieved. However, if they retract twice or re-coalesce after retracting once, the contact time will increase. We demonstrate that a maximum 48% reduction in contact time can be reached on the ridged surface at We = 29.6.

Reduction of non-specific toxicity of immunotoxin by intein mediated reconstitution on target cells

Recombinant immunotoxins are chimeric proteins composed of a targeting peptide that binds to a specific tumor antigen and a toxin protein killing target cells. Recombinant immunotoxin exhibits potent cancer inhibiting effects both in vivo and in vitro. However, the non-specific toxicity causes severe syndromes limiting their clinical application. To reduce toxicity caused by recombinant immunotoxins in general, we divided an immunotoxin into two nontoxic segments that may restore toxic bioactivity on tumor cell surface based on the intein mediated trans-splicing reaction. Both split and reconstituted immunotoxins were tested for their biological activities. We found that the reconstituted immunotoxin retained antigen specificity and affinity toward cancer cells overexpressing HER2/neu. After being internalized into HER2/neu positive cells, the reconstituted immunotoxin showed comparable cytotoxicity as the original immunotoxin, while the split immunotoxin fragments showed no toxic activity to cells with or without HER2/neu expression. This approach can potentially be used under clinical settings to reduce non-specific toxicity by administering patients with inactive immunotoxin fragments. Cytotoxic effect only occurs at tumor sites where the inactive fragments bind, trans-splice and become active toxin.

Branched Hybridization Chain Reaction Circuit for Ultrasensitive Localizable Imaging of mRNA in Living Cells.

Hybridization chain reaction (HCR) circuits are valuable approaches to monitor low-abundance mRNA, and current HCR is still subjected to issues such as limited amplification efficiency, compromised localization resolution, or complicated designs. We report a novel branched HCR (bHCR) circuit for efficient signal-amplified imaging of mRNA in living cells. The bHCR can be realized using a simplified design by hierarchically coupling two HCR circuits with two split initiator fragments of the secondary HCR circuit incorporated in the probes for the primary HCR circuit. The bHCR circuit enables one to generate a hyperbranched assembly seeded from a single target initiator, affording the potential for localizing single target molecules in live cells. In vitro assays show that bHCR offers very high amplification efficiency and specificity in single mismatch discrimination with a detection limit of 500 fM. Live cell studies reveal that bHCR displays intense fluorescence spots indicating mRNA localization in living cells with improved contrast. The bHCR method can provide a useful platform for low-abundance biomarker detection and imaging for cell biology and diagnostics.

Bimolecular Fluorescence Complementation to Visualize Protein-Protein Interactions in Human Cells Based on Gateway Cloning Technology.

Bimolecular fluorescence complementation (BiFC) is a powerful and sensitive tool to discover new protein-protein interactions (PPIs). It enables visualization and localization of protein-protein interactions (PPIs) in living cells. The idea behind BiFC is to split a fluorescent protein, for example yellow fluorescent protein (YFP), into two parts that are unable to emit fluorescent signal on their own. Therefore, in order to regain fluorescence the split protein fragments must establish close proximity. This is accomplished by fusing the split fragments to proteins that are postulated to interact, and expressing them in living cells. Subsequently, detection of fluorescence indicates interaction of given proteins. Since complementation is practically irreversible it can capture weak and transient interactions. Using suitable vectors for human protein expression, thus avoiding viral cell transfection, we introduced Gateway-based cloning features to the BiFC system, thereby enabling time efficient vector construction in order to maximize the full potential of the BiFC approach to investigate many protein-protein interactions in a high-throughput fashion. This protocol explains steps in a typical protein-protein interaction survey, from the vector selection, cell transfection, and visualization of the fluorescent signal.

E. coli-Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins.

Ubiquitylation is an eukaryotic signal that regulates most cellular pathways. However, four major hurdles pose challenges to study ubiquitylation: (1) high redundancy between ubiquitin (Ub) cascades, (2) ubiquitylation is tightly regulated in the cell, (3) the transient nature of the Ub signal, and (4) difficulties to purify functional ubiquitylation apparatus for in vitro assay. Here, we present systems that express functional Ub cascades in E. coli, which lacks deubiquitylases, Ub-dependent degradations, and control mechanisms for ubiquitylation. Therefore, expression of an ubiquitylation cascade results in the accumulation of stable ubiquitylated protein that can be genetically selected or purified, thus circumventing the above challenges. Co-expression of split antibiotic resistance protein fragments tethered to Ub and ubiquitylation targets along with ubiquitylation enzymes (E1, E2, and E3) gives rise to bacterial growth on selective media. We show that ubiquitylation rate is highly correlated with growth efficiency. Hence, genetic libraries and simple manipulations in the selection system facilitate the identification and characterization of components and interfaces along Ub cascades. The bacterial expression system also facilitates the detection of ubiquitylated proteins. Furthermore, the expression system allows affinity chromatography-based purification of milligram quantities of ubiquitylated proteins for downstream biochemical, biophysical, and structural studies.

In Situ Spatial Complementation of Aptamer‐Mediated Recognition Enables Live‐Cell Imaging of Native RNA Transcripts in Real Time

AbstractDirect cellular imaging of the localization and dynamics of biomolecules helps to understand their function and reveals novel mechanisms at the single‐cell resolution. In contrast to routine fluorescent‐protein‐based protein imaging, technology for RNA imaging remains less well explored because of the lack of enabling technology. Herein, we report the development of an aptamer‐initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)‐mimicking turn‐on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA. When genetically encoded in cells, endogenous mRNA molecules recruited Split‐Broccoli and brought the two fragments into spatial proximity, which formed a fluorophore‐binding site in situ and turned on fluorescence. Significantly, we demonstrated the use of AiFC for high‐contrast and real‐time imaging of endogenous RNA molecules in living mammalian cells. We envision wide application and practical utility of this enabling technology to in vivo single‐cell visualization and mechanistic analysis of macromolecular interactions.

In Situ Spatial Complementation of Aptamer-Mediated Recognition Enables Live-Cell Imaging of Native RNA Transcripts in Real Time.

Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their function and reveals novel mechanisms at the single-cell resolution. In contrast to routine fluorescent-protein-based protein imaging, technology for RNA imaging remains less well explored because of the lack of enabling technology. Herein, we report the development of an aptamer-initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)-mimicking turn-on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA. When genetically encoded in cells, endogenous mRNA molecules recruited Split-Broccoli and brought the two fragments into spatial proximity, which formed a fluorophore-binding site in situ and turned on fluorescence. Significantly, we demonstrated the use of AiFC for high-contrast and real-time imaging of endogenous RNA molecules in living mammalian cells. We envision wide application and practical utility of this enabling technology to in vivo single-cell visualization and mechanistic analysis of macromolecular interactions.

Fluorescent and colorimetric dual-mode aptasensor for thrombin detection based on target-induced conjunction of split aptamer fragments

Since the lack of detection diversity of the single-signal readout strategy, it is urgent to develop fast and multisignal assay strategies. A highly selective and sensitive assay method with colorimetric and fluorometric dual signals readouts is presented in this paper. It is based on the principle that the target induced conjunction of split aptamer fragments assembled on the surface of Au nanoparticles (AuNPs). In the presence of targets, the color of solution changed from wine red to blue and can be measured both visual inspection and spectrophotometry because of the aggregation of AuNPs. At the same time, the report probes which are original hybrid with the anchoring aptamer fragments on the AuNPs surface can be released and recovers the fluorescence. By use of this detection strategy, the limit of detection for thrombin (TMB), as a model of analyte, were 0.45 and 0.16nM, respectively. Furthermore, this protocol can discriminate TMB from other analogue with high selectivity and can be used to detect TMB in human serum samples. The results came from the two signals were well consistent with each other, which demonstrated that it has application potential for detection of TMB in complex matrix.

A genetic screen to discover SUMOylated proteins in living mammalian cells

Post-translational modification by the Small Ubiquitin-related Modifier (SUMO) is indispensable for diverse biological mechanisms. Although various attempts have been made to discover novel SUMO substrate proteins to unveil the roles of SUMOylation, the reversibility of SUMOylation, and the differences in the SUMOylation level still makes it difficult to explore infrequently-SUMOylated proteins in mammalian cells. Here, we developed a method to screen for mammalian SUMOylated proteins using the reconstitution of split fluorescent protein fragments in living mammalian cells. Briefly, the cells harboring cDNAs of SUMOylated proteins were identified by the reconstituted fluorescence emission and separated by cell sorting. The method successfully identified 36 unreported SUMO2-substrate candidates with distinct intracellular localizations and functions. Of the candidates, we found Atac2, a histone acetyltransferase, was SUMOylated at a lysine 408, and further modified by multiple SUMOs without isoform specificity. Because the present method is applicable to other SUMO isoforms and mammalian cell-types, it could contribute to a deeper understanding of the role of SUMOylation in various biological contexts.

Managing Fragmented Database Using BVAGQ-AR Replication Model

One of the mechanisms for managing data is replication since it improves data access and reliability. However, the amount of various data grows rapidly since technology is widely available at a low-cost. Problem arises when the database is packed with data, but it has lacked of knowledge. If the unreasonable data is used in database replication, it will cause waste of data storage and delay the time taken for a replication process. This paper proposes a new algorithm namely Binary Vote Assignment on Grid Quorum with Association Rule (BVAGQ-AR) to handle fragmented database replication. BVAGQ-AR algorithm is capable of partitioning the database into disjoint fragments. Handling fragmented database replication becomes challenging issue to administrator since the distributed database is disseminated into split partitions or fragments. This paper will discuss about how to build reliable system by using the proposed BVAGQ-AR algorithm for distributed database fragmentation. The result shows that managing fragmented database replication through proposed BVAGQ-AR algorithm able to preserve data consistency.

Fluorescence Resonance Energy Transfer-Based DNA Nanoprism with a Split Aptamer for Adenosine Triphosphate Sensing in Living Cells.

We have developed a DNA nanoprobe for adenosine triphosphate (ATP) sensing in living cells, based on the split aptamer and the DNA triangular prism (TP). In which nucleic acid aptamer was split into two fragments, the stem of the split aptamer was respectively labeled donor and acceptor fluorophores that underwent a fluorescence resonance energy transfer if two ATP molecules were bound as target molecule to the recognition module. Hence, ATP as a target induced the self-assembly of split aptamer fragments and thereby brought the dual fluorophores into close proximity for high fluorescence resonance energy transfer (FRET) efficiency. In the in vitro assay, an almost 5-fold increase in FA/FD signal was observed, the fluorescence emission ratio was found to be linear with the concentration of ATP in the range of 0.03-2 mM, and the nanoprobe was highly selective toward ATP. For the strong protecting capability to nucleic acids from enzymatic cleavage and the excellent biocompatibility of the TP, the DNA TP nanoprobe exhibited high cellular permeability, fast response, and successfully realized "FRET-off" to "FRET-on" sensing of ATP in living cells. Moreover, the intracellular imaging experiments indicated that the DNA TP nanoprobe could effectively detect ATP and distinguish among changes of ATP levels in living cells. More importantly, using of the split aptamer and the FRET-off to FRET-on sensing mechanism could efficiently avoid false-positive signals. This design provided a strategy to develop biosensors based on the DNA nanostructures for intracellular molecules analysis.

A Bimolecular Fluorescence Complementation Tool for Identification of Protein-Protein Interactions in Candida albicans.

Investigation of protein-protein interactions (PPI) in Candida albicans is essential for understanding the regulation of the signal transduction network that triggers its pathogenic lifestyle. Unique features of C. albicans, such as its alternative codon usage and incomplete meiosis, have enforced the optimization of standard genetic methods as well as development of novel approaches. Since the existing methods for detection of PPI are limited for direct visualization of the interacting complex in vivo, we have established a bimolecular fluorescence complementation (BiFC) assay in C. albicans, a powerful technique for studying PPI. We have developed an optimized set of plasmids that allows for N- and C-terminal tagging of proteins with split yeast-enhanced monomeric Venus fragments, so that all eight combinations of fusion orientations can be analyzed. With the use of our BiFC assay we demonstrate three interaction complexes in vivo, which were also confirmed by two-hybrid analysis. Our Candida-optimized BiFC assay represents a useful molecular tool for PPI studies and shows great promise in expanding our knowledge of molecular mechanisms of protein functions.

Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis.

Naturally split inteins have found widespread use in chemical biology due to their ability to drive the ligation of separately expressed polypeptides through a process termed protein trans-splicing (PTS). In this study, we harness PTS by rendering association of split intein fragments conditional upon the presence of a user-defined protease. We show that these intein “zymogens” can be used to create protein sensors and actuators that respond to the presence of various stimuli, including bacterial pathogens, viral infections, and light. We also show that this design strategy is compatible with several orthogonal split intein pairs, thereby opening the way to the creation of multiplexed sensor systems.