RNAs have highly complex and dynamic cellular localization patterns. Technologies for imaging RNA in living cells are important for uncovering their function and regulatory pathways. One approach for ...imaging RNA involves genetically encoding fluorescent RNAs using RNA mimics of green fluorescent protein (GFP). These mimics are RNA aptamers that bind fluorophores resembling those naturally found in GFP and activate their fluorescence. These RNA-fluorophore complexes, including Spinach, Spinach2, and Broccoli, can be used to tag RNAs and to image their localization in living cells. In this article, we describe the generation and optimization of these aptamers, along with strategies for expanding the spectral properties of their associated RNA-fluorophore complexes. We also discuss the structural basis for the fluorescence and photophysical properties of Spinach, and we describe future prospects for designing enhanced RNA-fluorophore complexes with enhanced photostability and increased sensitivity.
Riboswitches are natural ligand-sensing RNAs typically that are found in the 5′ UTRs of mRNA. Numerous classes of riboswitches have been discovered, enabling mRNA to be regulated by diverse and ...physiologically important cellular metabolites and small molecules. Here we describe Spinach riboswitches, a new class of genetically encoded metabolite sensor derived from naturally occurring riboswitches. Drawing upon the structural switching mechanism of natural riboswitches, we show that Spinach can be swapped for the expression platform of various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. In the case of the thiamine 5′-pyrophosphate (TPP) riboswitch from the Escherichia coli thiM gene encoding hydroxyethylthiazole kinase, we show that insertion of Spinach results in an RNA sensor that exhibits fluorescence upon binding TPP. This TPP Spinach riboswitch binds TPP with affinity and selectivity similar to that of the endogenous riboswitch and enables the discovery of agonists and antagonists of the TPP riboswitch using simple fluorescence readouts. Furthermore, expression of the TPP Spinach riboswitch in Escherichia coli enables live imaging of dynamic changes in intracellular TPP concentrations in individual cells. Additionally, we show that other riboswitches that use a structural mechanism similar to that of the TPP riboswitch, including the guanine and adenine riboswitches from the Bacillus subtilis xpt gene encoding xanthine phosphoribosyltransferase, and the S-adenosyl-methionine-I riboswitch from the B . subtilis yitJ gene encoding methionine synthase, can be converted into Spinach riboswitches. Thus, Spinach riboswitches constitute a novel class of RNA-based fluorescent metabolite sensors that exploit the diversity of naturally occurring ligand-binding riboswitches.
Significance Developing sensors to image cellular metabolites and signaling molecules in living cells is challenging. Here we describe Spinach riboswitches, a novel class of genetically encoded metabolite sensor based on riboswitches, a group of naturally occurring ligand-binding RNAs. Spinach riboswitches use Spinach, an RNA aptamer that binds and activates the fluorescence of an otherwise nonfluorescent small-molecule fluorophore. Drawing upon structural insights into the mechanism of structural switching in riboswitches, we show that Spinach can be swapped into various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. Expression of Spinach riboswitches in cells allows metabolite levels to be imaged in real time in live bacterial cells. Spinach riboswitches thus provide a novel approach to image cellular metabolites in living cells.
Cell types, both healthy and diseased, can be classified by inventories of their cell-surface markers. Programmable analysis of multiple markers would enable clinicians to develop a comprehensive ...disease profile, leading to more accurate diagnosis and intervention. As a first step to accomplish this, we have designed a DNA-based device, called “Nano-Claw”. Combining the special structure-switching properties of DNA aptamers with toehold-mediated strand displacement reactions, this claw is capable of performing autonomous logic-based analysis of multiple cancer cell-surface markers and, in response, producing a diagnostic signal and/or targeted photodynamic therapy. We anticipate that this design can be widely applied in facilitating basic biomedical research, accurate disease diagnosis, and effective therapy.
Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. ...Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains.
DNA and RNA nanotechnology has been used for the development of dynamic molecular devices. In particular, programmable enzyme-free nucleic acid circuits, such as catalytic hairpin assembly, have been ...demonstrated as useful tools for bioanalysis and to scale up system complexity to an extent beyond current cellular genetic circuits. However, the intracellular functions of most synthetic nucleic acid circuits have been hindered by challenges in the biological delivery and degradation. On the other hand, genetically encoded and transcribed RNA circuits emerge as alternative powerful tools for long-term embedded cellular analysis and regulation. Herein, we reported a genetically encoded RNA-based catalytic hairpin assembly circuit for sensitive RNA imaging inside living cells. The split version of Broccoli, a fluorogenic RNA aptamer, was used as the reporter. One target RNA can catalytically trigger the fluorescence from tens-to-hundreds of Broccoli. As a result, target RNAs can be sensitively detected. We have further engineered our circuit to allow easy programming to image various target RNA sequences. This design principle opens the arena for developing a large variety of genetically encoded RNA circuits for cellular applications.
The ability to self-assemble one-dimensional DNA building blocks into two- and three-dimensional nanostructures via DNA/RNA nanotechnology has led to broad applications in bioimaging, basic ...biological mechanism studies, disease diagnosis, and drug delivery. However, the cellular uptake of most nucleic acid nanostructures is dependent on passive delivery or the enhanced permeability and retention effect, which may not be suitable for certain types of cancers, especially for treatment in vivo. To meet this need, we have constructed a multifunctional aptamer-based DNA nanoassembly (AptNA) for targeted cancer therapy. In particular, we first designed various Y-shaped functional DNA domains through predesigned base pair hybridization, including targeting aptamers, intercalated anticancer drugs, and therapeutic antisense oligonucleotides. Then these functional DNA domains were linked to an X-shaped DNA core connector, termed a building unit, through the complementary sequences in the arms of functional domains and connector. Finally, hundreds (∼100–200) of these basic building units with 5′-modification of acrydite groups were further photo-cross-linked into a multifunctional and programmable aptamer-based nanoassembly structure able to take advantage of facile modular design and assembly, high programmability, excellent biostability and biocompatibility, as well as selective recognition and transportation. With these properties, AptNAs were demonstrated to have specific cytotoxic effect against leukemia cells. Moreover, the incorporation of therapeutic antisense oligonucleotides resulted in the inhibition of P-gp expression (a drug efflux pump to increase excretion of anticancer drugs) as well as a decrease in drug resistance. Therefore, these multifunctional and programmable aptamer-based DNA nanoassemblies show promise as candidates for targeted drug delivery and cancer therapy.
Genetically encoded biosensors are useful tools for detecting the presence and levels of diverse biomolecules in living cells. However, low-abundance targets are difficult to detect because they are ...often unable to bind and activate enough biosensors to detect using standard microscopic imaging approaches. Here we describe a type of RNA-based biosensor, an RNA integrator, which enables detection of low-abundance targets in vitro and in living cells. The RNA integrator is an RNA sequence comprising a ribozyme and an unfolded form of the fluorogenic aptamer Broccoli. Upon binding its target, the ribozyme undergoes cleavage and releases Broccoli, which subsequently folds and becomes fluorescent. Importantly, each target molecule can bind and induce cleavage of multiple copies of the integrator sensor, resulting in an amplified signal. We show that this approach can be generalized to numerous different ribozyme types for the detection of various small molecules.
Regulatable nanorobots: A DNA‐based walking device was designed that is capable of autonomous locomotion, with light control of initiation, termination, and velocity (see picture). This device is ...reminiscent of cellular protein motors in nature, especially those of green plants.
An azobenzene-containing surfactant was synthesized for the phase transfer of α-cyclodextrin (α-CD)-capped gold nanoparticles between water and toluene phases by host–guest chemistry. With the use of ...the photoisomerization of azobenzene, the reversible phase transfer of gold nanoparticles was realized by irradiation with UV and visible light. Furthermore, the phase transfer scheme was applied for the quenching of a reaction catalyzed by gold nanoparticles, as well as the recovery and recycling of the gold nanoparticles from aqueous solutions. This work will have significant impact on materials transfer and recovery in catalysis and biotechnological applications.
Strand displacement cascades are commonly used to make dynamically assembled structures. Particularly, the concept of “toehold-mediated DNA branch migration reactions” has attracted considerable ...attention in relation to dynamic DNA nanostructures. However, it is a challenge to obtain and control the formation of pure 1:1 ratio DNA duplexes with toehold structures. Here, for the first time, we report a photocontrolled toehold formation method, which is based on the photocleavage of 2-nitrobenzyl linker-embedded DNA hairpin precursor structures. UV light irradiation (λ ≈ 365 nm) of solutions containing these DNA hairpin structures causes the complete cleavage of the nitrobenzyl linker, and pure 1:1 DNA duplexes with toehold structures are easily formed. Our experimental results indicate that the amount of toehold can be controlled by simply changing the dose of UV irradiation and that the resulting toehold structures can be used for subsequent toehold-mediated DNA branch migration reactions, e.g., DNA hybridization chain reactions. This newly established method will find broad application in the construction of light-powered, controllable, and dynamic DNA nanostructures or large-scale DNA circuits.
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