Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More.

The central role of RNA in living systems made it highly desirable to have noninvasive and sensitive technologies allowing for imaging the synthesis and the location of these molecules in living cells. This need motivated the development of small pro-fluorescent molecules called "fluorogens" that become fluorescent upon binding to genetically encodable RNAs called "light-up aptamers." Yet, the development of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs. Then, the vast application potential of these molecules for live-cell RNA imaging and other applications is presented and discussed.journal article2020importe

Design and construction of a microfluidics workstation for high-throughput multi-wavelength fluorescence and transmittance activated droplet analysis and sorting

Droplet microfluidics has revolutionized quantitative high-throughput bioassays and screening, especially in the field of single-cell analysis where applications include cell characterization, antibody discovery and directed evolution. However, droplet microfluidic platforms capable of phenotypic, fluorescence-based readouts and sorting are still mostly found in specialized labs, because their setup is complex. Complementary to conventional FACS, microfluidic droplet sorters allow the screening of cell libraries for secreted factors, or even for the effects of secreted or surface-displayed factors on a second cell type. Furthermore, they also enable PCR-activated droplet sorting for the isolation of genetic material harboring specific markers. In this protocol, we provide a detailed step-by-step guide for the construction of a high-throughput droplet analyzer and sorter, which can be accomplished in similar to 45 working hours by nonspecialists. The resulting instrument is equipped with three lasers to excite the fluorophores in droplets and photosensors that acquire fluorescence signals in the blue (425-465 nm), green (505-545 nm) and red (580-630 nm) spectrum. This instrument also allows transmittance-activated droplet sorting by analyzing the brightfield light intensity transmitting through the droplets. The setup is validated by sorting droplets containing fluorescent beads at 200 Hz with 99.4% accuracy. We show results from an experiment where droplets hosting single cells were sorted on the basis of increased matrix metalloprotease activity as an application of our workstation in single-cell molecular biology, e.g., to analyze molecular determinants of cancer metastasis.LBM

Optimization of fluorogenic RNA-based biosensors using droplet-based microfluidic ultrahigh-throughput screening

International audienceBiosensors are biological molecules able to detect and report the presence of a target molecule by the emission of a signal. Nucleic acids are particularly appealing for the design of such molecule since their great structural plasticity makes them able to specifically interact with a wide range of ligands and their structure can rearrange upon recognition to trigger a reporting event. A biosensor is typically made of three main domains: a sensing domain that is connected to a reporting domain via a communication module in charge of transmitting the sensing event through the molecule. The communication module is therefore an instrumental element of the sensor. This module is usually empirically developed through a trial-and-error strategy with the testing of only a few combinations judged relevant by the experimenter. In this work, we introduce a novel method combining the use of droplet-based microfluidics and next generation sequencing. This method allows to functionally characterize up to a million of different sequences in a single set of experiments and, by doing so, to exhaustively test every possible sequence permutations of the communication module. Here, we demonstrate the efficiency of the approach by isolating a set of optimized RNA biosensors able to sense theophylline and to convert this recognition into fluorescence emission

Technological and computational advances driving high-throughput oncology

Engineering and computational advances have opened many new avenues in cancer research, particularly when being exploited in interdisciplinary approaches. For example, the combination of microfluidics, novel sequencing technologies, and computational analyses has been crucial to enable single-cell assays, giving a detailed picture of tumor heterogeneity for the very first time. In a similar way, these `tech' disciplines have been elementary for generating large data sets in multidimensional cancer `omics' approaches, cell-cell interaction screens, 3D tumor models, and tissue level analyses. In this review we summarize the most important technology and computational developments that have been or will be instrumental for transitioning classical cancer research to a large datadriven, high-throughput, high-content discipline across all biological scales.LBM

Technological and computational advances driving high-throughput oncology

Engineering and computational advances have opened many new avenues in cancer research, particularly when being exploited in interdisciplinary approaches. For example, the combination of microfluidics, novel sequencing technologies, and computational analyses has been crucial to enable single-cell assays, giving a detailed picture of tumor heterogeneity for the very first time. In a similar way, these 'tech' disciplines have been elementary for generating large data sets in multidimensional cancer 'omics' approaches, cell-cell interaction screens, 3D tumor models, and tissue level analyses. In this review we summarize the most important technology and computational developments that have been or will be instrumental for transitioning classical cancer research to a large data-driven, high-throughput, high-content discipline across all biological scales

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells