Stepwise molding of a highly selective ribonucleopeptide receptor

The structural characteristics of RNA-peptide (RNP) complexes are suitable for molding of a ligand-binding pocket of the RNP complex in a stepwise manner. The first step involves molding of the RNA subunit by in vitro selection of an RNP pool originating from an RNA library and the peptide, as previously reported for the construction of an ATP-binding RNP complex from an RRE RNA-Rev peptide complex. The second step involves selection from an RNP library consisting of Rev peptides with randomized amino acid residues and the RNA subunit selected in the first molding. The ATP-binding pocket produced by sequential molding of RNA and peptide subunits shows higher affinity to ATP and a distinct specificity for ATP versus dATP as compared to the ATP-binding RNP receptor in which only the RNA subunit has been molded. The second step selection from the peptide-based RNP library allows expansion of the ATP recognition surface, consisting of both RNA and peptide subunits, to enhance the affinity and selectivity to discriminate ATP against dATP. Our approach of stepwise molding offers the advantage of increasing the diversity of the RNP library by utilizing characteristics of different biopolymers. The ribonucleopeptide-based, multi-subunit approach is also extendable to other biomacromolecular assemblies, which may yield artificial receptors and enzymes with increased specificity and more diverse chemical activities.     

Context-dependent fluorescence detection of a phosphorylated tyrosine residue by a ribonucleopeptide

Tools for selective recognition and sensing of specific phosphorylated tyrosine residues on the protein surface are essential for understanding signal transduction cascades in the cell. A stable complex of RNA and peptide, a ribonucleopeptide (RNP), provides effective approaches to tailor RNP receptors and fluorescent RNP sensors for small molecules. In vitro selection of an RNA-derived pool of RNP afforded RNP receptors specific for a phosphotyrosine residue within a defined amino-acid sequence Gly-Tyr-Ser-Arg. The RNP receptor for the specific phosphotyrosine residue was successfully converted to a fluorescent RNP sensor for sequence-specific recognition of a phosphorylated tyrosine by screening a pool of fluorescent phosphotyrosine-binding RNPs generated by a combination of the RNA subunits of phosphotyrosine-binding RNPs and various fluorophore-modified peptide subunits. The phosphotyrosine-binding RNP receptor and fluorescent RNP sensor constructed from the RNP receptor not only discriminated phosphotyrosine against tyrosine, phosphoserine, or phosphothreonine, but also showed specific recognition of amino acid residues surrounding the phosphotyrosine residue. A fluorescent RNP sensor for one of the tyrosine phosphorylation sites of p100 coactivator showed a binding affinity to the target site ~95-fold higher than the other tyrosine phosphorylation site. The fluorescent RNP sensor has an ability to function as a specific fluorescent sensor for the phosphorylated tyrosine residue within a defined amino-acid sequence in HeLa cell extracts.     

Structural aspects for the recognition of ATP by ribonucleopeptide receptors

A modular structure of ribonucleopeptide (RNP) affords a framework to construct macromolecular receptors and fluorescent sensors. We have isolated ATP-binding RNP with the minimum of nucleotides for ATP binding, in which the RNA consensus sequence is different from those reported for RNA aptamers against the ATP analogues. The three-dimensional structure of the substrate-binding complex of RNP was studied to understand the ATP-binding mechanism of RNP. A combination of NMR measurements, enzymatic and chemical mapping, and nucleotide mutation studies of the RNP-adenosine complex show that RNP interacts with the adenine ring of adenosine by forming a U:A:U triple with two invariant U nucleotides. The observed recognition mode for the adenine ring is different from those of RNA aptamers for ATP derivatives reported previously. The RNP-adenosine complex is folded into a particular structure by formation of the U:A:U triple and a Hoogsteen type A:U base pair. This recognition mechanism was successfully utilized to convert the substrate-binding specificity of RNP from ATP- to GTP-binding with a C(+):G:C triple recognition mode.     

Ratiometric fluorescence and visual sensing of ATP based on gold nanocluster-encapsulated metal-organic framework with a smartphone

Adenosine triphosphate (ATP) plays an important role in various biological processes and the ATP level is closely associated with many diseases. Herein, we designed a novel dual-emissive fluorescence nanoplatform for ATP sensing based on red emissive europium metal-organic framework (Eu-MOF) and blue emissive gold nanoclusters (AuNCs). The presence of ATP causes the decomposition of Eu-MOF owing to strong affinity of Eu³⁺ with ATP. As a result, the red emission of Eu-MOF decreases while the blue emission of AuNCs remains unchanged. The distinct red/blue emission intensity change enables the establishment of a ratiometric fluorescent and visual sensor of ATP. Moreover, a fluorescent paper-based sensor was fabricated with the ratiometric ATP probes, which enabled easy-to-use and visual detection of ATP in serum samples with a smartphone.     

Detection of DNA Hybridization via Fluorescence Intensity Variations of ZnSe-DNA Quantum Dot Biosensors

ZnSe quantum dots (QDs) that were capped with 11-mercaptoundecanoic acid (MUA) and conjugated to amino-modified ssDNA molecules exhibited variations in fluorescence emission intensity upon hybridization with complementary ssDNA in solution, a phenomenon that can be exploited for rapid detection of free ssDNA sequences. Conjugation of MUA-capped ZnSe QDs to amino-modified ssDNA molecules resulted in increased fluorescence emission intensity and stability at room temperature. Increasing the length of the ssDNA, that was conjugated to the QDs, resulted in increased fluorescence emission intensity up to a length of about 50 nucleotide bases, beyond which the peak emission intensity reached a plateau. Hybridization of QD-ssDNA conjugates with complementary ssDNA, either in free form or bound to QDs from the same population, resulted in additional fluorescence emission intensity amplification. A small red shift was observed when three-dimensional QD-dsDNA-QD structures were formed. The QD-ssDNA sensors with single ssDNA molecule per QD were developed and used for rapid quantitative detection of fully or partially complementary free ssDNA sequences in aqueous solution. Partial hybridization of the QD-ssDNA sensors with short ssDNA targets resulted in smaller QD emission intensity amplification, when compared to full hybridization. A QD-ssDNA sensor containing a sequence corresponding to the hemoglobin beta gene was used to detect and discriminate between free ssDNA targets consisting of a complementary ssDNA sequence and targets containing a single-base mutation that can cause sickle-cell anemia. Such QD-based biosensors can form the basis for rapid separation-free assays that can be used to detect target biomolecules in solution.     

Anthrone-spirolactam and quinoline hybrid based sensor for selective fluorescent detection of Fe3+ ions

The synthesis of a novel, and highly selective Fe³⁺ ion sensor based on anthrone-spirolactam and its quinoline hybrid ligand is reported. The designed ligand displayed selective detection of Fe³⁺ ions with enhanced fluorescence emission. The complexation of Fe³⁺ ion led to a red shift of 32 nm from 420 nm to 452 nm, and a several fold increase in intensity with fluorescent green emission. The complexation (detection) of Fe³⁺ ions with ligand resulted in chelation enhanced fluorescence and intramolecular charge transfer through the inhibition of C=N isomerization. This hybrid sensor shows high sensitivity and selectivity, spontaneous response, and works on a wide pH range a minimum detection limit of 6.83 × 10⁻⁸ M. Importantly, the sensor works through the fluorescence turn-on mechanism that overcomes the paramagnetic effect of Fe³⁺ ions. The binding mechanism between the ligand and the Fe³⁺ ions was established from the Job's plot method, optical studies, Fourier transfor infrared spectroscopy, NMR titration, fluorescence life-time studies, and density functional theory optimization. The sensor displayed excellent results in the quantification of Fe³⁺ ions from real water samples. Furthermore, due to its biocompatibility nature, fluorescent spotting of Fe³⁺ ions in live cells revealed its bioimaging applications.     

ATP detection using a label-free DNA aptamer and a cationic tetrahedralfluorene

A simple and sensitive method for ATP detection using a label-free DNA aptamer as the recognition element and ethidium bromide (EB) as the signal reporter is reported. The ATP-binding aptamer undergoes a conformational switch from the aptamer duplex to the aptamer/target complex upon target binding, which induces the fluorescence change of intercalated EB emission. Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between the ATP aptamer and ATP. Using EB alone as a signal reporter, the ATP detection limit was estimated to be approximately 0.2 mM. When a light harvesting cationic tetrahedralfluorene was used as an energy donor to sensitize the intercalated EB emission, a 10-fold increase in detection limit and a 2-fold increase in detection selectivity was demonstrated. The sensitivity and selectivity of the tetrahedralfluorene sensitized assay is comparable to or better than most fluorescent ATP assays with multiple labels.     

Differential receptors create patterns diagnostic for ATP and GTP

Herein we report the combination of a library of resin-bound sensors along with a multicomponent sensor array. This novel combinatorial array sensor system shows selectivity for nucleotide phosphates in solution. The design of the anchored receptor includes a 1,3,5-trisubstituted-2,4,6-triethylbenzene scaffold coupled with peptide libraries. Each chemosensor is placed into a micromachined cavity within a silicon wafer, and the optical changes observed by a charged-coupled device result in near-real-time digital analysis of solutions. A colorimetric displacement assay was performed, and time-dependent imaging studies of the selected sensing ensembles result in a differential responses upon addition of adenosine 5'-triphosphate (ATP), adenosine 5'-monophosphate (AMP), or guanosine 5'-triphosphate (GTP). An advantage to this approach is that it creates an array of sensors that gives a fingerprint response for each analyte. Principal component analysis indicates that the library of chemosensors can differentiate between ATP, GTP, and AMP. On the basis of factor loading values, individual sensors from the library were sequenced to elucidate their chemical composition.     

Light emitting diode excitation emission matrix fluorescence spectroscopy

An excitation emission matrix (EEM) fluorescence instrument has been developed using a linear array of light emitting diodes (LED). The wavelengths covered extend from the upper UV through the visible spectrum: 370-640 nm. Using an LED array to excite fluorescence emission at multiple excitation wavelengths is a low-cost alternative to an expensive high power lamp and imaging spectrograph. The LED-EEM system is a departure from other EEM spectroscopy systems in that LEDs often have broad excitation ranges which may overlap with neighboring channels. The LED array can be considered a hybrid between a spectroscopic and sensor system, as the broad LED excitation range produces a partially selective optical measurement. The instrument has been tested and characterized using fluorescent dyes: limits of detection (LOD) for 9,10-bis(phenylethynyl)-anthracene and rhodamine B were in the mid parts-per-trillion range; detection limits for the other compounds were in the low parts-per-billion range (< 5 ppb). The LED-EEMs were analyzed using parallel factor analysis (PARAFAC), which allowed the mathematical resolution of the individual contributions of the mono- and dianion fluorescein tautomers a priori. Correct identification and quantitation of six fluorescent dyes in two to six component mixtures (concentrations between 12.5 and 500 ppb) has been achieved with root mean squared errors of prediction (RMSEP) of less than 4.0 ppb for all components.     

Fluorescence wavelength, intensity and lifetime for multidimensional transduction of selective interactions of acetylcholine receptor by lipid membranes

Concurrent analysis of the fluorescence intensity, at different emission wavelengths, of lipid vesicles containing acetylcholine receptor (AChR) labelled with a nitrobenzoxadiazole (NBD) moiety shows that selective interactions with the agonist carbamylcholine can be detected reproducibly by a self-calibration method with muM detection limits. Concurrent analysis of the fluorescence intensity and lifetime of the new probe 4-dicyanomethylene-1,2,3,4-tetrahydromethylquinoline (DCQ) shows that general alterations of lipid membrane structure induced by temperature variation in the head-group region of lipid vesicles can be determined. A general approach to detection of selective interactions is introduced by observation of fluorescence intensity and lifetime changes of the probe NBD-phosphatidyl ethanolamine dispersed in lipid membranes containing unlabelled AChR. Detection and differentiation of selective interactions between carbamylcholine and the antagonist alpha-bungarotoxin are possible by correlation with intensity and lifetime at different emission wavelengths.     

In vitro selection of ATP-binding receptors using a ribonucleopeptide complex

A recently described three-dimensional structure of the ribosome provides a sense of remarkable diversity of RNA-protein complexes. We have designed a new class of scaffold for artificial receptors, in which a short peptide and RNA with a randomized nucleotide region form a stable and specific complex. The randomized nucleotide region was placed next to the HIV-1 Rev response element to enable the formation of "ribonucleopeptide" pools in the presence of the Rev peptide. In vitro selection of RNA oligonucleotides from the randomized pool afforded a ribonucleopeptide receptor specific for ATP. The ATP-binding ribonucleopeptide did not share the known consensus nucleotide sequence for ATP aptamers and completely lost its ATP-binding ability in the absence of the Rev peptide. The ATP-binding activity of the ribonucleopeptide was increased by a substitution of the N-terminal amino acid of the Rev peptide. These results demonstrate directly that the peptide is incorporated in the functional structure of RNA and suggest that amino acids outside the RNA-binding region of the peptide modulate the ATP-binding of ribonucleopeptide. Our approach would provide an alternative strategy for the design of "tailor-made" ribonucleopeptide receptors and enzymes.     

Conformational flexibility of ribosomal decoding-site RNA monitored by fluorescent pteridine base analogues

Conformational transitions in ribosomal decoding-site RNA have previously been monitored by replacement of flexible adenine bases with fluorescent 2-aminopurine (2AP). Here, we have studied pteridine base analogues 3-methylisoxanthopterin (3MI) and 6-methylisoxanthopterin (6MI) as fluorescent labels for the investigation of conformational changes in the decoding site that are triggered by ligand binding. The higher quantum yield of 3MI and 6MI along with their relatively red-shifted excitation and emission wavelengths renders the pteridines interesting alternatives to 2AP for the establishment of ligand-binding assays for RNA based on fluorescence labelling.     

Dual fluorophore PNA FIT-probes - extremely responsive and bright hybridization probes for the sensitive detection of DNA and RNA

Fluorescently labeled oligonucleotides are commonly employed as probes to detect specific DNA or RNA sequences in homogeneous solution. Useful probes should experience strong increases in fluorescent emission upon hybridization with the target. We developed dual labeled peptide nucleic acid probes, which signal the presence of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. This enabled the very sensitive detection of a DNA target (40 pM LOD), which was detectable at less than 0.1% of the beacon concentration. In contrast to existing DNA-based molecular beacons, this PNA-based method does not require a stem sequence to enforce dye-dye communication. Rather, the method relies on the energy transfer between a "smart" thiazole orange (TO) nucleotide, which requires formation of the probe-target complex in order to become fluorescent, and terminally appended acceptor dyes. To improve upon fluorescence responsiveness the energy pathways were dissected. Hydrophobic, spectrally mismatched dye combinations allowed significant (99.97%) decreases of background emission in the absence of a target. By contrast, spectral overlap between TO donor emission and acceptor excitation enabled extremely bright FRET signals. This and the large apparent Stokes shift (82 nm) suggests potential applications in the detection of specific RNA targets in biogenic matrices without the need of sample pre-processing prior to detection.     

A new determining method of copper(<Emphasis Type="Bold">II</Emphasis>) ions at ng ml<Superscript>−1</Superscript> levels based on quenching of the water-soluble nanocrystals fluorescence

CdTe nanocrystals (NCs) were prepared and modified in aqueous solution by using 3-mercaptopropionic acid (MPA). Compared with previous fluorescent sensors based on organic fluorophores, these NCs have the following merits. First, they are sensitive and the synthetic procedure is simple; second, they have narrow, tunable, symmetric emission spectra and are photochemically stable. The applicability of these NCs to the determination of Cu(II) ions was studied successfully. Maximum fluorescence intensity was produced at pH 7.8, with excitation and emission wavelengths at 365 and 532 nm, respectively. Under optimal conditions, linear relationships were found between the relative fluorescence intensity and the concentration of Cu(II) ions in the range 0–256 ng ml^–1 and the limit of detection was 0.19 ng ml^–1. This method was applied to determine real samples successfully. In addition, the quenching mechanism is also described.     

基于DPA识别基团的锌离子荧光传感器

锌离子在生物体系中扮演着重要角色,其分析和检测在疾病诊断和医疗检测等方面具有重要价值。用于 Zn²⁺ 检测的荧光传感器具有检测方便、灵敏度高等优点,引起了广泛关注。典型的荧光传感器通常是由识别基团和作为报告单元的荧光团通过间隔基团或直接连接而组成的。识别基团是荧光传感器的作用核心,在高选择性识别过程中起着至关重要的作用。自 1996 年第一次接在荧光素上以来,DPA (N,N-二(2-吡啶甲基)胺, di-2-picolylamine) 基团在锌离子传感器的设计中得到了广泛应用。本文综述了近年来文献中报道的基于 DPA 识别基团的锌离子传感器,介绍了锌离子荧光传感器的合成方法与识别原理,最后简单介绍了锌离子传感器中其它几种常见的识别基团。     

Transition-metal-based Chemosensing Ensembles: ATP Sensing in Physiological Conditions

Sensing of biologically relevant anionic substrates in physiological conditions, employing the strategy of the chemosensing ensembles, is reported. Coordination of a fluorescent indicator to a dicopper(II) polyazamacrocyclic receptor ([Cu²(L)]) results in the collapse of its fluorescence emission. Competitive binding of substrates for the receptor releases the indicator in solution, with full emission recovery. The spectral changes obtained for some indicators and substrates were analysed to determine their respective association constants for the receptor. Discrimination of micromolar ATP quantities from other interferents (small inorganic anions and well-known neurotransmitters) is improved by a judicious choice of the indicator, the resulting ATP sensor promising interesting biological applications.     

Design of a hybrid biosensor for enhanced phosphopeptide recognition based on a phosphoprotein binding domain coupled with a fluorescent chemosensor

Protein-based fluorescent biosensors with sufficient sensing specificity are useful analytical tools for detection of biologically important substances in complicated biological systems. Here, we present the design of a hybrid biosensor, specific for a bis-phosphorylated peptide, based on a natural phosphoprotein binding domain coupled with an artificial fluorescent chemosensor. The hybrid biosensor consists of a phosphoprotein binding domain, the WW domain, into which has been introduced a fluorescent stilbazole having Zn(II)-dipicolylamine (Dpa) as a phosphate binding motif. It showed strong binding affinity and high sensing selectivity toward a specific bis-phosphorylated peptide in the presence of various phosphate species such as the monophosphorylated peptide, ATP, and others. Detailed fluorescence titration experiments clearly indicate that the binding-induced fluorescence enhancement and the sensing selectivity were achieved by the cooperative action of both binding sites of the hybrid biosensor, i.e., the WW domain and the Zn(II)-Dpa chemosensor unit. Thus, it is clear that the tethered Zn(II)-Dpa-stilbazole unit operated not only as a fluorescence signal transducer, but also as a sub-binding site in the hybrid biosensor. Taking advantage of its selective sensing property, the hybrid biosensor was successfully applied to real-time and label-free fluorescence monitoring of a protein kinase-catalyzed phosphorylation.     

A New Fluorimetric Method for the Determination of Ascorbic Acid

Abstract

The fluorescent reaction between ascorbic acid(AA) and 2-cyanoacetamide was studied. The experimental results showed that AA can react with 2-cyanoacetamide at pH 12.9–13.3, and form a fluorescent product, which emitted strong fluorescence. The fluorescence intensity was measured at excitation and emission wavelengths of 329 and 380nm, respectively. A linear relationship was obtained between the fluorescence intensity and AA concentration in the range of 0.1–50μg/ml, the regression coefficient is 0.9994, and the detection limit of AA is 0.03μg/ml. (signal-to-noise=3).     

Green and orange CdTe quantum dots as effective pH-sensitive fluorescent probes for dual simultaneous and independent detection of viruses

One of the most highlighted and fastest moving interfaces of nanotechnology is the application of quantum dots (QDs) in biology. The unparalleled advantages of the size-tunable fluorescent emission and the simultaneous excitation at a single wavelength make QDs the great possibility for use in optical encoding detection. In this paper, we report that green and orange CdTe QDs as convenient, cheap, reversible, and effective pH-sensitive fluorescent probes could monitor the proton (H+) flux driven by ATP synthesis for dual simultaneous and independent detection of viruses on the basis of antibody-antigen reactions. A new kind of biosensor (consisting of the mixture of green-QDs-labeled chromatophores and orange-QDs-labeled chromatophores) fluorescent measurement system was established for rapid, simultaneous, and independent detection of two different kinds of viruses (i.e., H9 avian influenza virus and MHV68 virus). It is crucial to find that the green and orange QDs labeled biosensors coexisting in the detection system can work independently and do not interfere with each another in the fluorescence assays. In addition, a primary steady electric double layer (EDL) model for the QDs biosensors was proposed to illustrate the mechanism of simultaneous and independent detection of the biosensors. We believe that the pH-sensitive CdTe QDs based detection system, described in this paper, is an important step toward optical encoding and has a great potential for simultaneous and independent qualitative and quantitative multiple detection systems.     

A nanoscale vesicular polydiacetylene sensor for organic amines by fluorescence recovery

We report a nanoscale lipid membrane-based sensor of conjugated polydiacetylene (PDA) vesicles for fluorescence detection of organic amines. The vesicle sensor was constructed by incorporation of a BODIPY fluorescent dye into the PDA vesicles. The fluorescent properties of the resulting vesicles can be manipulated by adjusting lipid components, and are controlled by environmental and solution conditions. The fluorescence of the BODIPY dye was significantly quenched in the polymerization of diacetylene lipid vesicles by a UV irradiation process. However, it was sufficiently recovered by external stimuli such as a hike of solution pH. The fluorescence recovery process was reversible, and a decrease in solution pH resulted in repeated quenching. The reported system transforms an external stimulus into a large fluorescence intensity change, demonstrating great potential in developing new signal reporting method for biosensor design. The quench-recovery phenomenon of the BODIPY-PDA is believed to be related to the energy transfer between the dye and the PDA conjugate backbone. The vesicle sensor was applied for detecting an organic amine, triethylamine (TEA) and a large linear relationship was obtained between the increase in fluorescence intensity and the concentrations of TEA. The detection limit of TEA by vesicle sensors using fluorescence recovery was found to be 10 μM.