Multiplexed sequential imaging in living cells with orthogonal fluorogenic RNA aptamer/dye pairs.

Detecting multiple targets in living cells is important in cell biology. However, multiplexed fluorescence imaging beyond two-to-three targets remains a technical challenge. Herein, we introduce a multiplexed imaging strategy, 'sequential Fluorogenic RNA Imaging-Enabled Sensor' (seqFRIES), which enables live-cell target detection via sequential rounds of imaging-and-stripping. In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study four fluorogenic RNA aptamer/dye pairs that can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can be completed in ∼20 min. Meanwhile, seqFRIES-mediated simultaneous detection of critical signalling molecules and mRNA targets was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for multiplexed and dynamic live-cell imaging and cell biology studies.

Digging into the biophysical features of cell membranes with lipid-DNA conjugates.

Lipid-DNA conjugates have emerged as highly useful tools to modify the cell membranes. These conjugates generally consist of a lipid anchor for membrane modification and a functional DNA nanostructure for membrane analysis or regulation. There are several unique properties of these lipid-DNA conjugates, especially including their programmability, fast and efficient membrane insertion, and precise sequence-specific assembly. These unique properties have enabled a broad range of biophysical applications on live cell membranes. In this review, we will mainly focus on recent tremendous progress, especially during the past three years, in regulating the biophysical features of these lipid-DNA conjugates and their key applications in studying cell membrane biophysics. Some insights into the current challenges and future directions of this interdisciplinary field have also been provided.

Advanced DNA Zipper Probes for Detecting Cell Membrane Lipid Domains.

The cell membrane is a complex mixture of lipids, proteins, and other components. By forming dynamic lipid domains, different membrane molecules can selectively interact with each other to control cell signaling. Herein, we report several new types of lipid-DNA conjugates, termed as "DNA zippers", which can be used to measure cell membrane dynamic interactions and the formation of lipid domains. Dependent on the choice of lipid moieties, cholesterol- and sphingomyelin-conjugated DNA zippers specifically locate in and detect membrane lipid-ordered domains, while in contrast, a tocopherol-DNA zipper can be applied for the selective imaging of lipid-disordered phases. These versatile and programmable probes can be further engineered into membrane competition assays to simultaneously detect multiple types of membrane dynamic interactions. These DNA zipper probes can be broadly used to study the correlation between lipid domains and various cellular processes, such as the epithelial-mesenchymal transition.

Imaging Membrane Order and Dynamic Interactions in Living Cells with a DNA Zipper Probe.

The cell membrane is a dynamic and heterogeneous structure composed of distinct sub-compartments. Within these compartments, preferential interactions occur among various lipids and proteins. Currently, it is still challenging to image these short-lived membrane complexes, especially in living cells. In this work, we present a DNA-based probe, termed "DNA Zipper", which allows the membrane order and pattern of transient interactions to be imaged in living cells using standard fluorescence microscopes. By fine-tuning the length and binding affinity of DNA duplex, these probes can precisely extend the duration of membrane lipid interactions via dynamic DNA hybridization. The correlation between membrane order and the activation of T-cell receptor signaling has also been studied. These programmable DNA probes function after a brief cell incubation, which can be easily adapted to study lipid interactions and membrane order during different membrane signaling events.

Quantitative and Multiplexed Fluorescence Lifetime Imaging of Intercellular Tensile Forces.

Mechanical interactions between cells have been shown to play critical roles in regulating cell signaling and communications. However, the precise measurement of intercellular forces is still quite challenging, especially considering the complex environment at cell-cell junctions. In this study, we report a fluorescence lifetime-based approach to image and quantify intercellular molecular tensions. Using this method, tensile forces among multiple ligand-receptor pairs can be measured simultaneously. We first validated our approach and developed lifetime measurement-based DNA tension probes to image E-cadherin-mediated tension on epithelial cells. These probes were then further applied to quantify the correlations between E-cadherin and N-cadherin tensions during an epithelial-mesenchymal transition process. The modular design of these probes can potentially be used to study the mechanical features of various physiological and pathological processes.

In Situ Genetically Cascaded Amplification for Imaging RNA Subcellular Locations.

In situ amplification methods, such as hybridization chain reaction, are valuable tools for mapping the spatial distribution and subcellular location of target analytes. However, the live-cell applications of these methods are still limited due to challenges in the probe delivery, degradation, and cytotoxicity. Herein, we report a novel genetically encoded in situ amplification method to noninvasively image the subcellular location of RNA targets in living cells. In our system, a fluorogenic RNA reporter, Broccoli, was split into two nonfluorescent fragments and conjugated to the end of two RNA hairpin strands. The binding of one target RNA can then trigger a cascaded hybridization between these hairpin pairs and thus activate multiple Broccoli fluorescence signals. We have shown that such an in situ amplified strategy can be used for the sensitive detection and location imaging of various RNA targets in living bacterial and mammalian cells. This new design principle provides an effective and versatile platform for tracking various intracellular analytes.

A Genetically Encoded RNA Photosensitizer for Targeted Cell Regulation.

Genetically encoded RNA devices have emerged for various cellular applications in imaging and biosensing, but their functions as precise regulators in living systems are still limited. Inspired by protein photosensitizers, we propose here a genetically encoded RNA aptamer based photosensitizer (GRAP). Upon illumination, the RNA photosensitizer can controllably generate reactive oxygen species for targeted cell regulation. The GRAP system can be selectively activated by endogenous stimuli and light of different wavelengths. Compared with their protein analogues, GRAP is highly programmable and exhibits reduced off-target effects. These results indicate that GRAP enables efficient noninvasive target cell ablation with high temporal and spatial precision. This new RNA regulator system will be widely used for optogenetics, targeted cell ablation, subcellular manipulation, and imaging.

Genetically Encoded Ratiometric RNA-Based Sensors for Quantitative Imaging of Small Molecules in Living Cells.

Precisely determining the intracellular concentrations of metabolites and signaling molecules is critical in studying cell biology. Fluorogenic RNA-based sensors have emerged to detect various targets in living cells. However, it is still challenging to apply these genetically encoded sensors to quantify the cellular concentrations and distributions of targets. Herein, using a pair of orthogonal fluorogenic RNA aptamers, DNB and Broccoli, we engineered a modular sensor system to apply the DNB-to-Broccoli fluorescence ratio to quantify the cell-to-cell variations of target concentrations. These ratiometric sensors can be broadly applied for live-cell imaging and quantification of metabolites, signaling molecules, and other synthetic compounds.

Visualizing Intercellular Tensile Forces by DNA-Based Membrane Molecular Probes.

Mechanical forces play critical roles in collective cell behaviors such as cell migration, proliferation, and differentiation. Extensive efforts have been made to measure forces between cells and extracellular matrices. However, force studies at cell-cell junctions remain a challenge. Herein, we reported a novel strategy to construct membrane DNA tension probes to visualize tensile forces at cell junctions. These lipid-modified probes can self-assemble onto cell membranes with high efficiency and stability. Upon experiencing tensile forces generated by neighboring cells, unfolding of the probes leads to a large increase in the fluorescence intensity. Compatible with readily accessible fluorescence microscopes, these easy-to-use membrane DNA tension probes can be broadly used to measure intercellular tensile forces.

Prewetting couples membrane and protein phase transitions to greatly enhance coexistence in models and cells.

Both membranes and biopolymers can individually separate into coexisting liquid phases. Here we explore biopolymer prewetting at membranes, a phase transition that emerges when these two thermodynamic systems are coupled. In reconstitution, we couple short poly-L-Lysine and poly-L-Glutamic Acid polyelectrolytes to membranes of saturated lipids, unsaturated lipids, and cholesterol, and detect coexisting prewet and dry surface phases well outside of the region of coexistence for each individual system. Notability, polyelectrolyte prewetting is highly sensitive to membrane lipid composition, occurring at 10 fold lower polymer concentration in a membrane close to its phase transition compared to one without a phase transition. In cells, protein prewetting is achieved using an optogenetic tool that enables titration of condensing proteins and tethering to the plasma membrane inner leaflet. Here we show that protein prewetting occurs for conditions well outside those where proteins condense in the cytoplasm, and that the stability of prewet domains is sensitive to perturbations of plasma membrane composition and structure. Our work presents an example of how thermodynamic phase transitions can impact cellular structure outside their individual coexistence regions, suggesting new possible roles for phase-separation-prone systems in cell biology.

Multiplexed Sequential Imaging in Living Cells with Orthogonal Fluorogenic RNA Aptamer/Dye Pairs.

Single-cell detection of multiple target analytes is an important goal in cell biology. However, due to the spectral overlap of common fluorophores, multiplexed fluorescence imaging beyond two-to-three targets inside living cells remains a technical challenge. Herein, we introduce a multiplexed imaging strategy that enables live-cell target detection via sequential rounds of imaging-and-stripping process, which is named as "sequential Fluorogenic RNA Imaging-Enabled Sensor" (seqFRIES). In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study five in vitro orthogonal fluorogenic RNA aptamer/dye pairs (>10-fold higher fluorescence signals), four of which can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can now be completed in ~20 min. Meanwhile, seqFRIES-mediated simultaneous detection of two critical signaling molecules, guanosine tetraphosphate and cyclic diguanylate, was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for highly multiplexed and dynamic cellular imaging and cell biology studies.

Current Methods for Detecting Cell Membrane Transient Interactions.

Short-lived cell membrane complexes play a key role in regulating cell signaling and communication. Many of these complexes are formed based on low-affinity and transient interactions among various lipids and proteins. New techniques have emerged to study these previously overlooked membrane transient interactions. Exciting functions of these transient interactions have been discovered in cellular events such as immune signaling, host-pathogen interactions, and diseases such as cancer. In this review, we have summarized current experimental methods that allow us to detect and analyze short-lived cell membrane protein-protein, lipid-protein, and lipid-lipid interactions. These methods can provide useful information about the strengths, kinetics, and/or spatial patterns of membrane transient interactions. However, each method also has its own limitations. We hope this review can be used as a guideline to help the audience to choose proper approaches for studying membrane transient interactions in different membrane trafficking and cell signaling events.

Lipid-Oligonucleotide Conjugates for Simple and Efficient Cell Membrane Engineering and Bioanalysis.

Cell membrane modification is important for tissue engineering, cell-based therapies, and cell biology studies. Recently, oligonucleotides have attracted considerable attention to remodel and functionalize live cell membranes. In particular, a type of amphiphilic lipid-oligonucleotide conjugates have been rationally designed and synthesized for this purpose. These conjugates have enabled a rapid, straightforward and efficient cell membrane modification. Taking advantage of the highly precise and programmable self-assembly of DNAs and RNAs, lipid-oligonucleotide conjugates have been used for membrane bioanalysis, therapeutics, building artificial membrane structures, and regulating cell-surface and cell-cell interactions. In this review, we have summarized the current knowledge in the design, synthesis, and regulating membrane properties of lipid-oligonucleotide conjugates. In addition, their state-of-the-art applications in cell membrane engineering and bioanalysis have been illustrated.

Paper-based fluorogenic RNA aptamer sensors for label-free detection of small molecules.

Sensors based on fluorogenic RNA aptamers have emerged in recent years. These sensors have been used for in vitro and intracellular detection of a broad range of biological and medical targets. However, the potential application of fluorogenic RNA-based sensors for point-of-care testing is still little studied. Here, we report a paper substrate-based portable fluorogenic RNA sensor system. Target detection can be simply performed by rehydration of RNA sensor-embedded filter papers. This affordable sensor system can be used for the selective, sensitive, and rapid detection of different target analytes, such as antibiotics and cellular signaling molecules. We believe that these paper-based fluorogenic RNA sensors show great potential for point-of-care testing of a wide range of targets from small molecules, nucleic acids, proteins, to various pathogens.

Quantifying tensile forces at cell-cell junctions with a DNA-based fluorescent probe.

Cells are physically contacting with each other. Direct and precise quantification of forces at cell-cell junctions is still challenging. Herein, we have developed a DNA-based ratiometric fluorescent probe, termed DNAMeter, to quantify intercellular tensile forces. These lipid-modified DNAMeters can spontaneously anchor onto live cell membranes. The DNAMeter consists of two self-assembled DNA hairpins of different force tolerance. Once the intercellular tension exceeds the force tolerance to unfold a DNA hairpin, a specific fluorescence signal will be activated, which enables the real-time imaging and quantification of tensile forces. Using E-cadherin-modified DNAMeter as an example, we have demonstrated an approach to quantify, at the molecular level, the magnitude and distribution of E-cadherin tension among epithelial cells. Compatible with readily accessible fluorescence microscopes, these easy-to-use DNA tension probes can be broadly used to quantify mechanotransduction in collective cell behaviors.

Efficient and selective DNA modification on bacterial membranes.

With highly precise self-assembly and programmability, DNA has been widely used as a versatile material in nanotechnology and synthetic biology. Recently, DNA-based nanostructures and devices have been engineered onto eukaryotic cell membranes for various exciting applications in the detection and regulation of cell functions. While in contrast, the potential of applying DNA nanotechnology for bacterial membrane studies is still largely underexplored, which is mainly due to the lack of tools to modify DNA on bacterial membranes. Herein, using lipid-DNA conjugates, we have developed a simple, fast, and highly efficient system to engineer bacterial membranes with designer DNA molecules. We have constructed a small library of synthetic lipids, conjugated with DNA oligonucleotides, and characterized their membrane insertion properties on various Gram-negative and Gram-positive bacteria. Simply after incubation, these lipid-DNA conjugates can be rapidly and efficiently inserted onto target bacterial membranes. Based on the membrane selectivity of these conjugates, we have further demonstrated their applications in differentiating bacterial strains and potentially in pathogen detection. These lipid-DNA conjugates are promising tools to facilitate the possibly broad usage of DNA nanotechnology for bacterial membrane analysis, functionalization, and therapy.

Ratiometric Fluorogenic RNA-Based Sensors for Imaging Live-Cell Dynamics of Small Molecules.

Imaging the cellular dynamics of metabolites and signaling molecules is critical for understanding various metabolism and signal transduction pathways. Genetically encoded RNA-based sensors are emerging powerful tools for this purpose. However, it was challenging to use these sensors to precisely determine the intracellular concentrations of target analytes. To solve this problem, we have recently developed ratiometric sensors using an orthogonal pair of RNA/fluorophore conjugates: Broccoli/DFHBI-1T (3,5-difluoro-4-hydroxybenzylidene-1-trifluoroethyl-imidazolinone) and DNB (dinitroaniline-binding aptamer)/SR-DN (sulforhodamine B-dinitroaniline). The cellular DNB-to-Broccoli fluorescence intensity ratio can be directly applied to quantify the target concentrations at the single-cell level. Unfortunately, due to the instability of the SR-DN dye, this ratiometric sensor is difficult to use for monitoring target dynamics. Herein, by replacing SR-DN with a stable TMR (tetramethylrhodamine)-DN dye, we developed a ratiometric sensor system based on Broccoli/DFHBI-1T and DNB/TMR-DN, which can be used for dynamic imaging in living cells. We believe these advanced genetically encoded ratiometric sensors can be widely used for intracellular studies of various target analytes.

A quantitative assessment of the dynamic modification of lipid-DNA probes on live cell membranes.

Synthetic lipid-DNA probes have recently attracted much attention for cell membrane analysis, transmembrane signal transduction, and regulating intercellular networks. These lipid-DNA probes can spontaneously insert onto plasma membranes simply after incubation. The highly precise and controllable DNA interactions have further allowed the programmable manipulation of these membrane-anchored functional probes. However, we still have quite limited understanding of how these lipid-DNA probes interact with cell membranes and also what parameters determine this process. In this study, we have systematically studied the dynamic process of cell membrane modification with a group of lipid-DNA probes. Our results indicated that the hydrophobicity of the lipid-DNA probes is strongly correlated with their membrane insertion and departure rates. Most cell membrane insertion stems from the monomeric form of probes, rather than the aggregates. Lipid-DNA probes can be removed from cell membranes through either endocytosis or direct outflow into the solution. As a result, long-term probe modifications on cell membranes can be realized in the presence of excess probes in the solution and/or endocytosis inhibitors. For the first time, we have successfully improved the membrane persistence of lipid-DNA probes to more than 24 h. Our quantitative data have dramatically improved our understanding of how lipid-DNA probes dynamically interact with cell membranes. These results can be further used to allow a broad range of applications of lipid-DNA probes for cell membrane analysis and regulation.