We describe a method for imaging individual mRNA molecules in fixed cells by probing each mRNA species with 48 or more short, singly labeled oligonucleotide probes. This makes each mRNA molecule visible as a computationally identifiable fluorescent spot via fluorescence microscopy. We demonstrate simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, mammalian cell lines and neurons.

RNA transport granules deliver translationally repressed mRNAs to synaptic sites in dendrites, where synaptic activity promotes their localized translation. Although the identity of many proteins that make up the neuronal granules is known, the stoichiometry of their core component, the mRNA, is poorly understood. By imaging nine different dendritically localized mRNA species with single-molecule sensitivity and subdiffraction-limit resolution in cultured hippocampal neurons, we show that two molecules of the same or different mRNA species do not assemble in common structures. Even mRNA species with a common dendritic localization element, the sequence that is believed to mediate the incorporation of these mRNAs into common complexes, do not colocalize. These results suggest that mRNA molecules traffic to the distal reaches of dendrites singly and independently of others, a model that permits a finer control of mRNA content within a synapse for synaptic plasticity.long-term potentiation | mRNA localization | RNA granules | single-molecule imaging | synaptic transmission

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases he a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibioties were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

We describe a multiplexing technology, named Evalution, based on novel digitally encoded microparticles in microfluidic channels. Quantitative multiplexing is becoming increasingly important for research and routine clinical diagnostics, but fast, easy-to-use, flexible and highly reproducible technologies are needed to leverage the advantages of multiplexing. The presented technology has been tailored to ensure (i) short assay times and high reproducibility thanks to reaction-limited binding regime, (ii) dynamic control of assay conditions and real-time binding monitoring allowing optimization of multiple parameters within a single assay run, (iii) compatibility with various immunoassay formats such as coflowing the samples and detection antibodies simultaneously and hence simplifying workflows, (iv) analyte quantification based on initial binding rates leading to increased system dynamic range and (v) high sensitivity via enhanced fluorescence collection. These key features are demonstrated with assays for proteins and nucleic acids showing the versatility of this technology.

Protein sensing in a nanofluidic environment dramatically shortens immunoassay time-to-result through the combined action of efficient mass transfer and short diffusion distances. Here, we report on a fully automated point-of-care in vitro diagnostic platform based on disposable capsule containing nanofluidic sensors in which fluorescent immunoassays are performed. Performances of the system were established with three model assays for ferritin, immunoglobulin E (IgE) and pancreatic stone protein (PSP). The described system has the typical high capture efficiency of nano-confined spaces combined with forced-flow, which induce a constant maximal concentration gradient on the sensor. Remarkably, analytes are detected in zeptomole quantities from a drop of blood. Dose-response curves show that high precision and accuracy are achieved in the clinically relevant assay ranges. Moreover, accuracy of the system is excellent agreement with laboratory reference method, as illustrated in a method comparison with total IgE as a model. While several academic proof-of-concepts have already described the possibility to exploit the properties of fluids at the nanoscale to develop immunoassay, the transition of these models to a product fulfilling requirements for use at the point-of-care in terms of operability, affordability, reliability and analytic performances remains a challenging endeavor. This study demonstrates that nanofluidic-based immunoassays can efficiently quantify protein biomarkers in the femto-and picomolar range within ultra-short assay time, high precision and accuracy on a closed, small, easyto-operate platform.