RESUMEN
INTRODUCTION: Single-cell imaging-based assays are an area of active and growing investment in drug discovery and development. This approach offers researchers the capability to interrogate rare subpopulations of cells with minimal sample consumption and multiplexed readouts. Recent technological advances in the optical interrogation and manipulation of single cells have substantially increased the throughput and sensitivity of these assays. Areas covered: In this review, the authors focus on three classes of single-cell imaging-based analyses: single-cell microscopy combined with microfluidics, mass spectrometric imaging for subcellular compound localization, and imaging mass cytometry (IMC). They provide an overview of each technology and recent examples of their utility in advancing drug discovery, based on the potential for scalability, multiplexing, and capability to generate definitive data on cellular heterogeneity and target engagement. Expert opinion: Understanding target engagement and heterogeneity at the single-cell level will enable the development of safer and more effective therapies, particularly for new modalities like CAR-T cell therapies and gene editing approaches (AAV, CRISPR). Successful adoption of new single-cell imaging-based approaches in drug discovery will require tandem investment in advanced computational analysis and bioinformatic approaches, due to the complexity and multivariate nature of single-cell imaging data.
Asunto(s)
Desarrollo de Medicamentos/métodos , Descubrimiento de Drogas/métodos , Análisis de la Célula Individual/métodos , Animales , Biología Computacional/métodos , Humanos , Citometría de Imagen/métodos , Espectrometría de Masas/métodos , Microfluídica/métodos , Microscopía/métodosRESUMEN
Riboswitches are mRNA structural elements that act as intracellular sensors of small-molecule metabolites. By undergoing conformational changes capable of modulating translation or terminating transcription, riboswitches are able to play a role in regulating the concentration of essential metabolites in the cell. Computer-guided fluorescence experiments were carried out to interrogate molecular dynamics and conformational changes in the minimal riboswitch aptamer that binds 7-aminomethyl-7-deazaguanine (preQ1). Our combined experimental results and computational analysis suggest that the preQ1 riboswitch apo form is structured but shows no evidence of a ligand-binding pocket. Simulations of the apo and bound forms indicate a large conformational change is triggered by the breaking of the Watson-Crick base pairing of nucleotides G11 and C31 upon preQ1 removal, followed by collapse of the pocket due to interfering π-stacking. Computational predictions of local aptamer dynamics were validated by fluorescence experiments employing 2-aminopurine substitutions. In-line probing reactions confirmed that fluorophore-labeled riboswitches retain similar higher-order structural features as the unlabeled aptamer upon ligand binding, although their affinity for the ligand is reduced by the introduction of the fluorescent reporter.
Asunto(s)
Aptámeros de Nucleótidos/química , Simulación de Dinámica Molecular , Pirimidinonas/química , Pirroles/química , ARN/química , Riboswitch , 2-Aminopurina/química , Conformación de Ácido Nucleico , Espectrometría de Fluorescencia , Propiedades de SuperficieRESUMEN
There is a persistent need for small-molecule fluorescent labels optimized for single-molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red-shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed "DCDHF" fluorophores, for use in live-cell imaging based on the push-pull design: an amine donor group and a 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) acceptor group, separated by a pi-rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red-emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small-molecule fluorophores, which are needed for super-resolution imaging schemes that require active control (here turning-on) of single-molecule emission.
Asunto(s)
Colorantes Fluorescentes/química , Furanos/química , Nitrilos/química , Animales , Células CHO , Cricetinae , Cricetulus , Furanos/síntesis química , Conformación Molecular , Nitrilos/síntesis química , Péptidos/química , FotoquímicaRESUMEN
Study of long single-stranded telomeric DNA is important for a variety of basic science and biotechnological applications, yet few methods exist for synthesis and visualization of single copies of this DNA in solution at biologically relevant length scales necessary for assessment of heterogeneity in its structure and behavior. We have synthesized kilobase-long single-stranded human telomere mimics in situ by rolling circle replication (RCR) on a microscope coverslip surface and visualized individual strands by staining with SYBR Gold. Under buffer flow, differential extensibility and varying morphology of these long telomere-mimicking DNA sequences were observed at the single-molecule level in real time. Using this procedure, we detected striking differences in the extensibility of individual RCR products based on the human G-rich telomeric sequence in the presence and absence of short, complementary single-stranded oligonucleotides. We also apply this new mode of single-stranded DNA characterization to probe the interaction of kilobase-length telomere mimics with the small-molecule G-quadruplex-binding agent TMPyP4.