Ligand Dilution Analysis Facilitates Aptamer Binding Characterization at the Single-Molecule Level

Cell-specific aptamers offer a powerful tool to study membrane receptors at the single-molecule level. Most target receptors of aptamers are highly expressed on the cell surface, but difficult to analyze in situ because of dense distribution and fast velocity. Therefore, we herein propose a random sampling-based analysis strategy termed ligand dilution analysis (LDA) for easily implemented aptamer-based receptor study. Receptor density on the cell surface can be calculated based on a regression model. By using a synergistic ligand dilution design, colocalization and differentiation of aptamer and monoclonal antibody (mAb) binding on a single receptor can be realized. Once this is accomplished, precise binding site and detailed aptamer-receptor binding mode can be further determined using molecular docking and molecular dynamics simulation. The ligand dilution strategy also sets the stage for an aptamer-based dynamics analysis of two- and three-dimensional motion and fluctuation of highly expressed receptors on the live cell membrane.     

Repetitive reversible labeling of proteins at polyhistidine sequences for single-molecule imaging in live cells.

Sensitive live-cell fluorescence microscopy and single-molecule imaging are severely limited by rapid photobleaching of fluorescent probes. Herein, we show how to circumvent this problem using a novel, generic labeling strategy. Small nickel-nitrilotriacetate fluorescent probes are reversibly bound to oligohistidine sequences of exposed proteins on cell surfaces, permitting selective observation of the proteins by fluorescence microscopy. Photobleached probes are removed by washing and replaced by new fluorophores, thus enabling repetitive acquisition of single-molecule trajectories on the same cell and allowing variation of experimental conditions between acquisitions. This method offers free choice of fluorophores while being minimally perturbing. The strength of the method is demonstrated by labeling engineered polyhistidine sequences of the serotonin-gated 5-HT(3) receptor on the surface of live mammalian cells. Single-molecule microscopy reveals pronounced heterogeneous mobility patterns of the 5-HT(3) receptor. After activating the receptor with serotonin, the number of immobile receptors increases substantially, which might be important for receptor regulation at synapses.     

Molecular Targeting of Epidermal Growth Factor Receptor (EGFR) and Vascular Endothelial Growth Factor Receptor (VEGFR)

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) are two extensively studied membrane-bound receptor tyrosine kinase proteins that are frequently overexpressed in many cancers. As a result, these receptor families constitute attractive targets for imaging and therapeutic applications in the detection and treatment of cancer. This review explores the dynamic structure and structure-function relationships of these two growth factor receptors and their significance as it relates to theranostics of cancer, followed by some of the common inhibition modalities frequently employed to target EGFR and VEGFR, such as tyrosine kinase inhibitors (TKIs), antibodies, nanobodies, and peptides. A summary of the recent advances in molecular imaging techniques, including positron emission tomography (PET), single-photon emission computerized tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and optical imaging (OI), and in particular, near-IR fluorescence imaging using tetrapyrrolic-based fluorophores, concludes this review.     

In vivo Fluorescence Imaging of Tumors using Molecular Aptamers Generated by Cell‐SELEX

Poor sensitivity and low specificity of current molecular imaging probes limit their application in clinical settings. To address these challenges, we used a process known as cell‐SELEX to develop unique molecular probes termed aptamers with the high binding affinity, sensitivity, and specificity needed for in vivo molecular imaging inside living animals. Importantly, aptamers can be selected by cell‐SELEX to recognize target cells, or even surface membrane proteins, without requiring prior molecular signature information. As a result, we are able to present the first report of aptamers molecularly engineered with signaling molecules and optimized for the fluorescence imaging of specific tumor cells inside a mouse. Using a Cy5‐labeled aptamer TD05 (Cy5‐TD05) as the probe, the in vivo efficacy of aptamer‐based molecular imaging in Ramos (B‐cell lymphoma) xenograft nude mice was tested. After intravenous injection of Cy5‐TD05 into mice bearing grafted tumors, noninvasive, whole‐body fluorescence imaging then allowed the spatial and temporal distribution to be directly monitored. Our results demonstrate that the aptamers could effectively recognize tumors with high sensitivity and specificity, thus establishing the efficacy of these fluorescent aptamers for diagnostic applications and in vivo studies requiring real‐time molecular imaging.     

Sigma受体显像剂

σ受体与细胞功能、生物过程以及许多疾病关系密切。σ1受体已成为发展精神分裂症、抑郁症、早老性痴呆和帕金森病等中枢神经系统（CNS）神经精神疾病药物新的靶点，而σ2受体是肿瘤增殖的生物标志。σ1受体显像剂的研制将为CNS神经精神疾病提供敏感特异的诊断方法，而σ2受体显像剂的研究将为肿瘤的早期诊断提供灵敏的分子探针。本文综述了σ受体显像剂的研究进展，结合σ受体显像剂的用途，详细讨论了不同σ受体显像剂的设计方法，并对其未来发展作了展望。     

Chemically induced photoswitching of fluorescent probes--a general concept for super-resolution microscopy

We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.     

DNA Aptamers in the Detection of Leukemia Cells by the Thickness Shear Mode Acoustics Method

By using the thickness shear mode acoustics method (TSM) and single-molecule force spectroscopy (SMFS) we studied the interactions between DNA aptamers (sgc8c) specific to the protein tyrosine kinase 7 (PTK7), which is localized in the membranes of leukemia lymphoblastics (MOLT-4), and lymphocyte (Jurkat) cell lines, as well with PTK7-negative U266 myeloid leukemia cells. The TSM method allowed the development of a highly sensitive, label-free biosensor for the detection leukemia cells with a limit of detection of (195±20) cells/mL. SMFS approved the high selectivity of the sgc8c aptamers to the PTK7 receptors at the cell surface and allowed determining the binding probability of the aptamers to the PTK7 receptors at different cell lines.     

Selection of DNA ligands for protein kinase C-delta

Protein kinase Cs are a family of serine and threonine kinases that mediate a wide variety of cellular signalling processes such as cell growth, differentiation, apoptosis and tumor development. We have selected high-affinity DNA aptamers for PKCdelta by capillary electrophoresis based SELEX (systematic evolution of ligands by exponential enrichment, CE-SELEX). We have demonstrated that fluorescently tagged PB9 aptamer can specifically recognize PKCdelta under in vitro conditions. The Kd of the aptamer-protein binding is 122 nM. These aptamers will enable us to apply fluorescently labelled probes to study the spatiotemporal dynamics and activation of individual endogenous PKC isoforms during various cell signalling processes.     

Visual detection of cancer cells by colorimetric aptasensor based on aggregation of gold nanoparticles induced by DNA hybridization

A simple but highly sensitive colorimetric method was developed to detect cancer cells based on aptamer–cell interaction. Cancer cells were able to capture nucleolin aptamers (AS 1411) through affinity interaction between AS 1411 and nucleolin receptors that are over expressed in cancer cells, The specific binding of AS 1411 to the target cells triggered the removal of aptamers from the solution. Therefore no aptamer remained in the solution to hybridize with complementary ssDNA-AuNP probes as a result the solution color is red. In the absence of target cells or the presence of normal cells, ssDNA-AuNP probes and aptamers were coexisted in solution and the aptamers assembled DNA-AuNPs, produced a purple solution. UV–vis spectrometry demonstrated that this hybridization-based method exhibited selective colorimetric responses to the presence or absence of target cells, which is detectable with naked eye. The linear response for MCF-7 cells in a concentration range from 10 to 10⁵ cells was obtained with a detection limit of 10 cells. The proposed method could be extended to detect other cells and showed potential applications in cancer cell detection and early cancer diagnosis.     

Single-molecule immunoassay and DNA diagnosis

Many assays relevant to disease diagnosis are based on electrophoresis, where the migration velocity is used for distinguishing molecules of different size or charge. However, standard gel electrophoresis is not only slow but also insensitive. We describe a single-molecule imaging procedure to measure the electrophoretic mobilities of up to 100000 distinct molecules every second. The results correlate well with capillary electrophoresis (CE) experiments and afford confident discrimination between normal (16.5 kbp) and abnormal (6.1 kbp) mitochondrial DNA fragments, or beta-phycoerythrin-labeled digoxigenin (BP-D) and its immunocomplex (anti-D-BP-D). This demonstrates that virtually all electrophoresis diagnostic protocols from slab gels to CE should be adaptable to single-molecule detection. This opens up the prossibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction (PCR) or other biological amplification.     

Aptamers-based sandwich assay for silver-enhanced fluorescence multiplex detection

In this work, aptamers-modified silver nanoparticles (AgNPs) were prepared as capture substrate, and fluorescent dyes-modified aptamers were synthesized as detection probes. The sandwich assay was based on dual aptamers, which was aimed to accomplish the highly sensitive detection of single protein and multiplex detection of proteins on one-spot. We found that aptamers-modified AgNPs based microarray was much superior to the aptamer based microarray in fluorescence detection of proteins. The result shows that the detection limit of the sandwich assay using AgNPs probes for thrombin or platelet-derived growth factor-BB (PDGF-BB) is 80 or 8 times lower than that of aptamers used directly. For multiplex detection of proteins, the detection limit was 625 pM for PDGF-BB and 21 pM for thrombin respectively. The sandwich assay based on dual aptamers and AgNPs was sensitive and specific.     

Long-Term,Single-Molecule Imaging of Proteins in Live Cells with Photoregulated Fluxional Fluorophores

Single-molecule localization microscopy (SMLM) can reveal nanometric details of biological samples, but its high phototoxicity hampers long-term imaging in live specimens. A significant part of this phototoxicity stems from repeated irradiations that are necessary for controlled switching of fluorophores to maintain the sparse labeling of the sample. Lower phototoxicity can be obtained using fluorophores that blink spontaneously, but controlling the density of single-molecule emitters is challenging. We recently developed photoregulated fluxional fluorophores (PFFs) that combine the benefits of spontaneously blinking dyes with photocontrol of emitter density. These dyes, however, were limited to imaging acidic organelles in live cells. Herein, we report a systematic study of PFFs that culminates in probes that are functional at physiological pH and operate at longer wavelengths than their predecessors. Moreover, these probes are compatible with HaloTag labeling, thus enabling timelapse, single-molecule imaging of specific protein targets for exceptionally long times.     

Anti-idiotype RNAs that mimic the leucine-rich nuclear export signal and specifically bind to CRM1/exportin 1

BACKGROUND: Anti-idiotype approaches are based on the assumption that an antibody recognising a ligand can be structurally related to the receptor. Recently we have generated anti-idiotype RNA aptamers designed to mimic the human immunodeficiency virus-1 (HIV-1) Rev nuclear export signal (NES). Nuclear injection of either NES-peptide conjugates or aptamer causes the inhibition of Rev-mediated export. This implied that NES mimics and export substrate might compete for binding to the NES receptor. The mechanism of inhibition, however, is unknown. RESULTS: The interaction between the export aptamer and CRM1 was characterised in vitro. The aptamer binds specifically to CRM1 and this interaction is sensitive to competition by Rev NES-peptide conjugates. The recognition domain of CRM1 has been mapped and includes residues found previously to affect binding of leptomycin B, a fungicide interfering with nuclear export. CONCLUSIONS: Inhibition of Rev-mediated export in vivo by export aptamers appears to result from the binding of the aptamers to the NES-recognition domain of CRM1. This observation demonstrates that anti-idiotype RNA can mimic faithfully structural and functional properties of a protein and can be used to map ligand-binding domains of receptors.     

基于Cell-SELEX的核酸适配体在生化分析与生物成像中的应用

基于Cell-SELEX的核酸适配体是指以活细胞为靶标物,通过指数富集的配基系统进化技术(Systematic evolution of ligands by exponential enrichment,SELEX)从人工合成的DNA/RNA文库中筛选得到的单链寡核苷酸.它能够与靶标细胞高亲和性、高特异性结合,具有分子量低、合成简单、化学稳定性好、免疫原性低、易于功能化修饰等优点,已广泛应用于生命科学研究领域.本文综述了基于Cell-SELEX技术筛选的核酸适配体在肿瘤细胞检测、分析和成像方面的研究进展,并对核酸适配体研究的发展前景和趋势进行了展望.     

Au:CdHgTe quantum dots for in vivo tumor-targeted multispectral fluorescence imaging

Near-infrared gold-doped CdHgTe quantum dots (QDs) with improved photoluminescence and biocompatibility were developed using an aqueous solution route with l-glutathione and l-cysteine as stabilizers. As-prepared Au:CdHgTe QDs were covalently linked to arginine–glycine–aspartic acid (RGD) peptide, anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), and anti- carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) MAb separately. Three Au:CdHgTe QD bioconjugates (QD800-RGD, QD820-anti-CEACAM1, and QD840-anti-EGFR) were successfully used as probes for in vivo tumor-targeted multispectral fluorescence imaging of xenografts. Fluorescence signals from the QD bioconjugates used to detect three tumor markers were spectrally unmixed, and their co-localization was analyzed. The results indicate that multiple tumor markers could be simultaneously detected by multispectral fluorescence imaging in vivo using QD bioconjugates as probes. This approach has excellent potential as an imaging method for the noninvasive exploration and detection of multiple tumor markers in vivo, thereby substantially aiding the diagnosis of cancer.     

Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads

G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. In order to study the complex biochemical signaling network coupling to the intracellular side of GPCRs, it is necessary to engineer and control the downstream signaling components, which is difficult to realize in living cells. We have developed a bioanalytical platform enabling the study of GPCRs in their native membrane transferred inside-out from live cells to lectin-coated beads, with both membrane sides of the receptor being accessible for molecular interactions. Using heterologously expressed adenosine A(2A) receptor carrying a yellow fluorescent protein, we showed that the tethered membranes comprised fully functional receptors in terms of ligand and G protein binding. The interactions between the different signaling partners during the formation and subsequent dissociation of the ternary signaling complex on single beads could be observed in real time using multicolor fluorescence microscopy. This approach of tethering inside-out native membranes accessible from both sides is straightforward and readily applied to other transmembrane proteins. It represents a generic platform suitable for ensemble as well as single-molecule measurements to investigate signaling processes at plasma membranes.     

Photochemical Mechanisms of Fluorophores Employed in Single-Molecule Localization Microscopy

Decoding cellular processes requires visualization of the spatial distribution and dynamic interactions of biomolecules. It is therefore not surprising that innovations in imaging technologies have facilitated advances in biomedical research. The advent of super-resolution imaging technologies has empowered biomedical researchers with the ability to answer long-standing questions about cellular processes at an entirely new level. Fluorescent probes greatly enhance the specificity and resolution of super-resolution imaging experiments. Here, we introduce key super-resolution imaging technologies, with a brief discussion on single-molecule localization microscopy (SMLM). We evaluate the chemistry and photochemical mechanisms of fluorescent probes employed in SMLM. This Review provides guidance on the identification and adoption of fluorescent probes in single molecule localization microscopy to inspire the design of next-generation fluorescent probes amenable to single-molecule imaging.     

Ultrabright Terbium Nanoparticles for FRET Biosensing and in Situ Imaging of Epidermal Growth Factor Receptors**

Lanthanide-doped nanoparticles (LnNPs) have become an important class of fluorophores for advanced biosensing and bioimaging. LnNPs that are photosensitized by surface-attached antenna ligands can possess exceptional brightness. However, their functional bioconjugation remains an important challenge for their translation into bioanalytical applications. To solve this problem, we designed a ligand that can be simultaneously applied as efficient light harvesting antenna for Tb surface ions and strong linker of biomolecules to the LnNPs surfaces. To demonstrate generic applicability of the photosensitized TbNP-bioconjugates, we applied them in two prototypical applications for biosensing and bioimaging. First, in-solution biorecognition was shown by time-resolved Förster resonance energy transfer (FRET) between streptavidin-functionalized TbNPs to biotinylated dyes (ATTO 610). Second, in situ detection of ligand–receptor binding on cells was accomplished with TbNP-antibody (Matuzumab) conjugates that could specifically bind to transmembrane epidermal growth factor receptors (EGFR). High specificity and sensitivity were demonstrated by time-gated imaging of EGFR on both strongly (A431) and weakly (HeLa and Cos7) EGFR-expressing cell lines, whereas non-expressing cell lines (NIH3T3) and EGFR-passivated A431 cells did not show any signals. Despite the relatively large size of TbNP-antibody conjugates, they could be internalized by A431 cells upon binding to extracellular EGFR, which showed their potential as bright and stable luminescence markers for intracellular signaling.     

Next‐Generation Sequencing as Input for Chemometrics in Differential Sensing Routines

Differential sensing (DS) methods traditionally use spatially arrayed receptors and optical signals to create score plots from multivariate data which classify individual analytes or complex mixtures. Herein, a new approach is described, in which nucleic acid sequences and sequence counts are used as the multivariate data without the necessity of a spatial array. To demonstrate this approach to DS, previously selected aptamers, identified from the literature, were used as semi‐specific receptors, Next‐Gen DNA sequencing was used to generate data, and cell line differentiation was the test‐bed application. The study of a principal component analysis loading plot revealed cross‐reactivity between the aptamers. The technique generates high‐dimensionality score plots, and should be applicable to any mixture of complex and subtly different analytes for which nucleic acid‐based receptors exist.