RESUMEN
Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3'-OH unprotected cleavable fluorescent 2'-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor(R) 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.
Asunto(s)
Desoxirribonucleótidos/síntesis química , Colorantes Fluorescentes/química , Análisis de Secuencia de ADN , Desoxirribonucleótidos/química , Desoxirribonucleótidos/clasificación , Microscopía Fluorescente , Compuestos Orgánicos/químicaRESUMEN
Understanding cellular signaling mediated by cell surface receptors is key to modern biomedical research and drug development. The discovery of a growing number of potential molecular targets and therapeutic compounds requires downscaling and accelerated functional screening. Receptor-mediated cellular responses are typically investigated on single cells or cell populations. Here, we show how to monitor cellular signaling reactions at a yet unreached miniaturization level. On the basis of our observations, cytochalasin induces mammalian cells to extrude from their plasma membrane submicrometer-sized native vesicles. They comprise functional cell surface receptors correctly exposing their extracellular ligand binding sites on the outer vesicle surface and retaining cytosolic proteins in the vesicle interior. As a prototypical example, ligand binding to the ionotropic 5-HT(3) receptor and subsequent transmembrane Ca(2+) signaling were monitored in single attoliter vesicles. Thus, native vesicles are the smallest autonomous containers capable of performing cellular signaling reactions under physiological conditions. Because a single cell delivers about 50 native vesicles, which can be isolated and addressed as individuals, our concept allows multiple functional analyses of individual cells having a limited availability and opens new vistas for miniaturized bioanalytics.