Targeted RNA condensation in living cells via genetically encodable triplet repeat tags.

Living systems contain various membraneless organelles that segregate proteins and RNAs via liquid-liquid phase separation. Inspired by nature, many protein-based synthetic compartments have been engineered in vitro and in living cells. Here, we introduce a genetically encoded CAG-repeat RNA tag to reprogram cellular condensate formation and recruit various non-phase-transition RNAs for cellular modulation. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes and densities of these cellular RNA condensates. The cis- and trans-regulation functions of these CAG-repeat tags in cellular RNA localization, life time, RNA-protein interactions and gene expression have also been investigated. Considering the importance of RNA condensation in health and disease, we expect that these genetically encodable modular and self-assembled tags can be widely used for chemical biology and synthetic 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.

A portable electrochemical DNA sensor for sensitive and tunable detection of piconewton-scale cellular forces.

Cell-generated forces are a key player in cell biology, especially during cellular shape formation, migration, cancer development, and immune response. A new type of label-free smartphone-based electrochemical DNA sensor is developed here for cellular force measurement. When cells apply tension forces to the DNA sensors, the rapid rupture of DNA duplexes allows multiple redox reporters to reach the electrode and generate highly sensitive electrochemical signals. The sensitivity of these portable sensors can be further enhanced by incorporating a CRISPR-Cas12a system. Meanwhile, the threshold force values of these DNA-based sensors can be rationally tuned based on the force application geometries and also DNA intercalating agents. Overall, these highly sensitive, portable, cost-efficient, and easy-to-use electrochemical sensors can be powerful tools for detecting different cell-generated molecular forces.

Targeted RNA Condensation in Living Cells via Genetically Encodable Triplet Repeat Tags.

Living systems contain various functional membraneless organelles that can segregate selective proteins and RNAs via liquid-liquid phase separation. Inspired by nature, many synthetic compartments have been engineered in vitro and in living cells, mostly focused on protein-scaffolded systems. Herein, we introduce a nature-inspired genetically encoded RNA tag to program cellular condensate formations and recruit non-phase-transition target RNAs to achieve functional modulation. In our system, different lengths of CAG-repeat tags were tested as the self-assembled scaffold to drive multivalent condensate formation. Various selective target messenger RNAs and noncoding RNAs can be compartmentalized into these condensates. With the help of fluorogenic RNA aptamers, we have systematically studied the formation dynamics, spatial distributions, sizes, and densities of these cellular RNA condensates. The regulation functions of these CAG-repeat tags on the cellular RNA localization, lifetime, RNA-protein interactions, and gene expression have also been investigated. Considering the importance of RNA condensation in both health and disease conditions, these genetically encodable modular and self-assembled tags can be potentially widely used for chemical biology and synthetic biology studies.

Imaging and detecting intercellular tensile forces in spheroids and embryoid bodies using lipid-modified DNA probes.

Cells continuously experience and respond to different physical forces that are used to regulate their physiology and functions. Our ability to measure these mechanical cues is essential for understanding the bases of various mechanosensing and mechanotransduction processes. While multiple strategies have been developed to study mechanical forces within two-dimensional (2D) cell culture monolayers, the force measurement at cell-cell junctions in real three-dimensional (3D) cell models is still pretty rare. Considering that in real biological systems, cells are exposed to forces from 3D directions, measuring these molecular forces in their native environment is thus highly critical for the better understanding of different development and disease processes. We have recently developed a type of DNA-based molecular probe for measuring intercellular tensile forces in 2D cell models. Herein, we will report the further development and first-time usage of these molecular tension probes to visualize and detect mechanical forces within 3D spheroids and embryoid bodies (EBs). These probes can spontaneously anchor onto live cell membranes via the attached lipid moieties. By varying the concentrations of these DNA probes and their incubation time, we have first characterized the kinetics and efficiency of probe penetration and loading onto tumor spheroids and stem cell EBs of different sizes. After optimization, we have further imaged and measured E-cadherin-mediated forces in these 3D spheroids and EBs for the first time. Our results indicated that these DNA-based molecular tension probes can be used to study the spatiotemporal distributions of target mechanotransduction processes. These powerful imaging tools may be potentially applied to fill the gap between ongoing research of biomechanics in 2D systems and that in real 3D cell complexes.

Double-stranded RNA induction asa potential dynamic biomarkerfor DNA-demethylating agents.

Hypomethylating agents (HMAs), such as azacitidine and decitabine, induce cancer cell death by demethylating DNAs to promote the expression of tumor-suppressor genes. HMAs also reactivate the transcription of endogenous double-stranded RNAs (dsRNAs) that trigger the innate immune response and subsequent apoptosis via viral mimicry. However, the expression patterns of endogenous dsRNAs and their relevance in the efficacy of HMAs remain largely uninvestigated. Here, we employ amidine-conjugated spiropyran (Am-SP) to examine the dynamic expression pattern of total dsRNAs regulated by HMAs. By analyzing the bone-marrow aspirates of myelodysplastic syndrome or acute myeloid leukemia patients who received the HMAs, we find a dramatic increase in total dsRNA levels upon treatment only in patients who later benefited from the therapy. We further apply our approach in solid tumor cell lines and show that the degree of dsRNA induction correlates with the effectiveness of decitabine in most cases. Notably, when dsRNA induction is accompanied by increased expression of nc886 RNA, decitabine becomes ineffective. Collectively, our study establishes the potential application of monitoring the total dsRNA levels by a small molecule as an analytical method and a dynamic marker to predict the clinical outcome of the HMA therapy.

Mitochondrial double-stranded RNAs as a pivotal mediator in the pathogenesis of SjÓ§gren's syndrome.

SjÓ§gren's syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (I-IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. Here, we investigate the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating I-IFNs and other glandular phenotypes of SS. We find that mt-dsRNAs are elevated in the saliva and tears of SS patients (n = 73 for saliva and n = 16 for tears) and in salivary glands of non-obese diabetic mice with salivary dysfunction. Using the in-house-developed 3D culture of immortalized human salivary gland cells, we show that stimulation by exogenous dsRNAs increase mt-dsRNAs, activate the innate immune system, trigger I-IFNs, and promote glandular phenotypes. These responses are mediated via the Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT) pathway. Indeed, a small chemical inhibitor of JAK1 attenuates mtRNA elevation and immune activation. We further show that muscarinic receptor ligand acetylcholine ameliorates autoimmune characteristics by preventing mt-dsRNA-mediated immune activation. Last, direct suppression of mt-dsRNAs reverses the glandular phenotypes of SS. Altogether, our study underscores the significance of mt-dsRNA upregulation in the pathogenesis of SS and suggests mt-dsRNAs as propagators of a pseudo-viral signal in the SS target tissue.

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.

Chemo- and biosensing applications of spiropyran and its derivatives - A review.

Spiropyrans are a class of photochromic compounds that feature two main isoforms, a closed spiropyran (SP) and an open merocyanine (MC), which are inter-switchable with UV and visible light. The double bond containing MC isomer has several resonance forms with different optical spectra. Consequently, the overall spectral characteristic of the spiropyran solution depends on the relative contribution of each resonance form. In addition, the reversible changes of the absorbance or fluorescence spectra of MC are dictated by the environment. By utilizing these properties, spiropyran and its derivatives have been employed to monitor a variety of molecules and ions that can alter the resonance forms of MC. In this review, we provide a comprehensive discussion on a range of species that can be detected by spiropyran including metal ions, anions, acids, solvents, and even biomolecules, the major analytical detection mechanisms involved, and the novelty of such techniques. This study establishes spiropyran as a versatile platform for the detection of both organic and inorganic target molecules.

Spiropyran as a potential molecular diagnostic tool for double-stranded RNA detection.

BACKGROUND: Long double-stranded RNAs (dsRNAs) are duplex RNAs that can induce immune response when present in mammalian cells. These RNAs are historically associated with viral replication, but recent evidence suggests that human cells naturally encode endogenous dsRNAs that can regulate antiviral machineries in cellular contexts beyond immune response. RESULTS: In this study, we use photochromic organic compound spiropyran to profile and quantitate dsRNA expression. We show that the open form of spiropyran, merocyanine, can intercalate between RNA base pairs, which leads to protonation and alteration in the spectral property of the compound. By quantifying the spectral change, we can detect and quantify dsRNA expression level, both synthetic and cellular. We further demonstrate that spiropyrans can be used as a molecular diagnostic tool to profile endogenously expressed dsRNAs. Particularly, we show that spiropyrans can robustly detect elevated dsRNA levels when colorectal cancer cells are treated with 5-aza-2'-deoxycytidine, an FDA-approved DNA-demethylating agent used for chemotherapy, thus demonstrating the use of spiropyran for predicting responsiveness to the drug treatment. CONCLUSION: As dsRNAs are signature of virus and accumulation of dsRNAs is implicated in various degenerative disease, our work establishes potential application of spiropyrans as a simple spectral tool to diagnose human disease based on dsRNA expression.