Exchanging and Releasing Information in Synthetic Digital Polymers Using a Strand-Displacement Strategy.

Toehold-mediated strand displacement (TMSD) was tested as a tool to edit information in synthetic digital polymers. Uniform DNA-polymer biohybrid macromolecules were first synthesized by automated phosphoramidite chemistry and characterized by HPLC, mass spectrometry, and polyacrylamide gel electrophoresis (PAGE). These precursors were diblock structures containing a synthetic poly(phosphodiester) (PPDE) segment covalently attached to a single-stranded DNA sequence. Three types of biohybrids were prepared herein: a substrate containing an accessible toehold as well as input and output macromolecules. The substrate and the input macromolecules contained noncoded PPDE homopolymers, whereas the output macromolecule contained a digitally encoded segment. After hybridization of the substrate with the output, incubation in the presence of the input led to efficient TMSD and the release of the digital segment. TMSD can therefore be used to erase or rewrite information in self-assembled biohybrid superstructures. Furthermore, it was found in this work that the conjugation of DNA single strands to synthetic segments of chosen lengths greatly facilitates the characterization and PAGE visualization of the TMSD process.

Droplet Surface Immunoassay by Relocation (D-SIRe) for High-Throughput Analysis of Cytosolic Proteins at the Single-Cell Level.

Enzyme-linked immunosorbent assay (ELISA) is a central analytic method in biological science for the detection of proteins. Introduction of droplet-based microfluidics allowed the development of miniaturized, less-consuming, and more sensitive ELISA assays by coencapsulating the biological sample and antibody-functionalized particles. We report herein an alternative in-droplet immunoassay format, which avoids the use of particles. It exploits the oil/aqueous-phase interface as a protein capture and detection surface. This is achieved using tailored perfluorinated surfactants bearing azide-functionalized PEG-based polar headgroups, which spontaneously react when meeting at the droplet formation site, with strained alkyne-functionalized antibodies solubilized in the water phase. The resulting antibody-functionalized inner surface can then be used to capture a target protein. This surface capture process leads to concomitant relocation at the surface of a labeled detection antibody and in turn to a drastic change in the shape of the fluorescence signal from a convex shape (not captured) to a characteristic concave shape (captured). This novel droplet surface immunoassay by fluorescence relocation (D-SIRe) proved to be fast and sensitive at 2.3 attomoles of analyte per droplet. It was further demonstrated to allow detection of cytosolic proteins at the single bacteria level.

FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride.

Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh-throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.

µIVC-Useq: a microfluidic-assisted high-throughput functionnal screening in tandem with next generation sequencing and artificial neural network to rapidly characterize RNA molecules.

The function of an RNA is intimately linked to its three-dimensional structure. X-ray crystallography or NMR allow the fine structural characterization of small RNA (e.g., aptamers) with a precision down to atomic resolution. Yet, these technics are time consuming, laborious and do not inform on mutational robustness and the extent to which a sequence can be modified without altering RNA function, an important set of information to assist RNA engineering. On another hand, thought powerful, in silico predictions still lack the required accuracy. These limitations can be overcome by using high-throughput microfluidic-assisted functional screening technologies, as they allow exploring large mutant libraries in a rapid and cost-effective manner. Among them, we recently introduced the microfluidic-assisted In Vitro Compartmentalization (µIVC), an efficient screening strategy in which reactions are performed in picoliter droplets at rates of several thousand per second. We later improved µIVC efficiency by using in tandem with high throughput sequencing, thought a laborious bioinformatic step was still required at the end of the process. In the present work, we strongly increased the automation level of the pipeline by implementing an artificial neural network enabling unsupervised bioinformatic analysis. We demonstrate the efficiency of this "µIVC-Useq" technology by rapidly identifying a set of sequences readily accepted by a key domain of the light-up RNA aptamer SRB-2. This work not only shed some new light on the way this aptamer can be engineered, but it also allowed us to easily identify new variants with an up-to 10-fold improved performance.

Real-time tracking of root hair nucleus morphodynamics using a microfluidic approach.

Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RHs show a fast apical growth, constituting a unique single cell model system for analysing cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, although high-resolution imaging is still lacking to enable an investigation of the accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) that enables us to capture high resolution confocal imaging of Arabidopsis RH development with real-time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, to illustrate the possibilities offered by the CMD, we have compared the real-time variations in the circularity, area and aspect ratio of nuclei moving in growing and mature RHs. Interestingly, we observed higher aspect ratios in the nuclei of mature RHs, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development.

Rational Design of Self-Quenched Rhodamine Dimers as Fluorogenic Aptamer Probes for Live-Cell RNA Imaging.

With the growing interest in the understanding of the importance of RNAs in health and disease, detection of RNAs in living cells is of high importance. Fluorogenic dyes that light up specifically selected RNA aptamers constitute an attractive direction in the design of RNA imaging probes. In this work, based on our recently proposed concept of a fluorogenic dimer, we aim to develop a robust molecular tool for intracellular RNA imaging. We rationally designed a fluorogenic self-quenched dimer (orange Gemini, o-Gemini) based on rhodamine and evaluated its capacity to light up its cognate aptamer o-Coral in solution and live cells. We found that the removal of biotin from the dimer slightly improved the fluorogenic response without losing the affinity to the cognate aptamer (o-Coral). On the other hand, replacing sulforhodamine with a carboxyrhodamine produced drastic improvement of the affinity and the turn-on response to o-Coral and, thus, a better limit of detection. In live cells expressing o-Coral-tagged RNAs, the carboxyrhodamine analogue of o-Gemini without a biotin unit displayed a higher signal as well as faster internalization into the cells. We suppose that less hydrophilic carboxyrhodamine compared to sulforhodamine can more readily penetrate through the cell plasma membrane and, together with its higher affinity to o-Coral, provide the observed improvement in the imaging experiments. The promiscuity of the o-Coral RNA aptamer to the fluorogenic dimer allowed us to tune a fluorogen chemical structure and thus drastically improve the fluorescence response of the probe to o-Coral-tagged RNAs.

A dimerization-based fluorogenic dye-aptamer module for RNA imaging in live cells.

Live-cell imaging of RNA has remained a challenge because of the lack of naturally fluorescent RNAs. Recently developed RNA aptamers that can light-up small fluorogenic dyes could overcome this limitation, but they still suffer from poor brightness and photostability. Here, we propose the concept of a cell-permeable fluorogenic dimer of self-quenched sulforhodamine B dyes (Gemini-561) and the corresponding dimerized aptamer (o-Coral) that can drastically enhance performance of the current RNA imaging method. The improved brightness and photostability, together with high affinity of this complex, allowed direct fluorescence imaging in live mammalian cells of RNA polymerase III transcription products as well as messenger RNAs labeled with a single copy of the aptamer; that is, without tag multimerization. The developed fluorogenic module enables fast and sensitive detection of RNA inside live cells, while the proposed design concept opens the route to new generation of ultrabright RNA probes.

The nature of the purine at position 34 in tRNAs of 4-codon boxes is correlated with nucleotides at positions 32 and 38 to maintain decoding fidelity.

Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.

Droplet-Based Microfluidic Chip Design, Fabrication, and Use for Ultrahigh-Throughput DNA Analysis and Quantification.

DNA is widely used as a biomarker of contamination, infection, or disease, which has stimulated the development of a wide palette of detection and quantification methods. Even though several analytical approaches based on isothermal amplification have been proposed, DNA is still mainly detected and quantified by quantitative PCR (qPCR). However, for some analyses (e.g., in cancer research) qPCR may suffer from limitations arising from competitions between highly similar template DNAs, the presence of inhibitors, or suboptimal primer design. Nevertheless, digitalizing the analysis (i.e., individualizing DNA molecules into compartments prior to amplifying them in situ) allows to address most of these issues. By its capacity to generate and manipulate millions of highly similar picoliter volume water-in-oil droplets, microfluidics offers both the required miniaturization and parallelization capacity, and led to the introduction of digital droplet PCR (ddPCR). This chapter aims at introducing the reader to the basic principles behind ddPCR while also providing the key guidelines to fabricate, set up, and use his/her own ddPCR platform. We further provide procedures to detect and quantify DNA either purified in solution or directly from individualized cells. This approach not only gives access to DNA absolute concentration with unrivaled sensitivity, but it may also be the starting point of more complex in vitro analytical pipelines discussed at the end of the chapter.

Structure and functional reselection of the Mango-III fluorogenic RNA aptamer.

Several turn-on RNA aptamers that activate small-molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to the nucleobase planes of those motifs. The improved iMango-III and Mango-III(A10U) fluoresce ~50% brighter than enhanced green fluorescent protein, making them suitable tags for live cell RNA visualization.

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

Biosensors 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.

Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI.

Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach, comprising five mutations for which the exact role has not yet been deciphered. In this work, we cocrystallized a reengineered version of iSpinach in complex with the DFHBI and solved the X-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach sheds some light on the possible function of the different beneficial mutations carried by iSpinach.

Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.

iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications.

Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme.

Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions.

In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities. While Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an efficient approach to generate new RNA aptamers, it is less suited for the isolation of efficient ribozymes as it does not select directly for the catalysis. In vitro compartmentalization (IVC) in aqueous droplets in emulsions allows catalytic RNAs to be selected under multiple-turnover conditions but suffers severe limitations that can be overcome using the droplet-based microfluidics workflow described in this paper. Using microfluidics, millions of genes in a library can be individually compartmentalized in highly monodisperse aqueous droplets and serial operations performed on them. This allows the different steps of the evolution process (gene amplification, transcription, and phenotypic assay) to be uncoupled, making the method highly flexible, applicable to the selection and evolution of a variety of RNAs, and easily adaptable for evolution of DNA or proteins. To demonstrate the method, we performed cycles of random mutagenesis and selection to evolve the X-motif, a ribozyme which, like many ribozymes selected using SELEX, has limited multiple-turnover activity. This led to the selection of variants, likely to be the optimal ribozymes that can be generated using point mutagenesis alone, with a turnover number under multiple-turnover conditions, k(ss) cat, ∼ 28-fold higher than the original X-motif, primarily due to an increase in the rate of product release, the rate-limiting step in the multiple-turnover reaction.

Light-Up RNA Aptamers and Their Cognate Fluorogens: From Their Development to Their Applications.

An RNA-based fluorogenic module consists of a light-up RNA aptamer able to specifically interact with a fluorogen to form a fluorescent complex. Over the past decade, significant efforts have been devoted to the development of such modules, which now cover the whole visible spectrum, as well as to their engineering to serve in a wide range of applications. In this review, we summarize the different strategies used to develop each partner (the fluorogen and the light-up RNA aptamer) prior to giving an overview of their applications that range from live-cell RNA imaging to the set-up of high-throughput drug screening pipelines. We then conclude with a critical discussion on the current limitations of these modules and how combining in vitro selection with screening approaches may help develop even better molecules.

Activity-Fed Translation (AFT) Assay: A New High-Throughput Screening Strategy for Enzymes in Droplets.

There is an increasing demand for the development of sensitive enzymatic assays compatible with droplet-based microfluidics. Here we describe an original strategy, activity-fed translation (AFT), based on the coupling of enzymatic activity to in vitro translation of a fluorescent protein. We show that methionine release upon the hydrolysis of phenylacetylmethionine by penicillin acylase enabled in vitro expression of green fluorescent protein. An autocatalytic setup where both proteins are expressed makes the assay highly sensitive, as fluorescence was detected in droplets containing single PAC genes. Adding a PCR step in the droplets prior to the assay increased the sensitivity further. The strategy is potentially applicable for any activity that can be coupled to the production of an amino acid, and as the microdroplet volume is small the use of costly reagents such as in vitro expression mixtures is not limiting for high-throughput screening projects.

Direct fluoride monitoring using a fluorogenic RNA-based biosensor.

Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.

High-Throughput Development and Optimization of RNA-Based Fluorogenic Biosensors of Small Molecules Using Droplet-Based Microfluidics.

Small-molecule sensing is a major issue as they can serve both in fundamental science and as makers of various diseases, contaminations, or even environment pollution. RNA aptamers are single-stranded nucleic acids that can adopt different conformations and specifically recognize a wide range of ligands, making them good candidates to develop biosensors of small molecules. Recently, light-up RNA aptamers have been introduced and used as starting building blocks of RNA-based fluorogenic biosensors. They are typically made of three domains: a reporter domain (a light-up aptamer), connected to a sensor domain (another aptamer) via a communication module. The latter is instrumental as being in charge of information transmission between the sensor and the reporting domains. Here we present an ultrahigh-throughput screening procedure to develop RNA-based fluorogenic biosensors by selecting optimized communication modules through an exhaustive functional exploration of every possible sequence permutation using droplet-based microfluidics and next-generation sequencing.