A Light‐Triggerable Nanoparticle Library for the Controlled Release of Non‐Coding RNAs

RNA‐based therapies offer a wide range of therapeutic interventions including the treatment of skin diseases; however, the strategies to efficiently deliver these biomolecules are still limited due to obstacles related to the cellular uptake and cytoplasmic delivery. Herein, we report the synthesis of a triggerable polymeric nanoparticle (NP) library composed of 160 formulations, presenting physico‐chemical diversity and differential responsiveness to light. Six formulations were more efficient (up to 500 %) than commercially available lipofectamine in gene‐knockdown activity. These formulations showed differential internalization by skin cells and the endosomal escape was rapid (minutes range). The NPs were effective in the release of siRNA and miRNA. Acute skin wounds treated with the top hit NP complexed with miRNA‐150‐5p healed faster than wounds treated with scrambled miRNA. Light‐activatable NPs offer a new strategy to topically deliver non‐coding RNAs.     

Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro‐RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE‐LIF) using fluorescence‐labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA‐499 was found when hybridization was conducted at 40°C in 50 mM Tris‐acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA‐499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA‐499 was completed within 1 h using CE‐LIF. These results showed the potential of CE for fast, specific, and sensitive high‐throughput analysis of low‐abundance miRNAs in cell extracts, biofluids, and tissues.     

Rapid Detection of Exosomal MicroRNAs Using Virus‐Mimicking Fusogenic Vesicles

Exosomal microRNAs (miRNAs) are important biomarkers for clinical diagnosis and disease treatment monitoring. However, most approaches for exosomal miRNA detection are time‐consuming, laborious, and expensive. Herein, we report a virus‐mimicking fusogenic vesicle (Vir‐FV) that enables rapid, efficient, and high‐throughput detection of exosomal miRNAs within 2 h. Fusogenic proteins on Vir‐FVs can specifically target the sialic‐acid‐containing receptors on exosomes, inducing efficient fusion of Vir‐FVs and exosomes. Upon vesicle content mixing, the molecular beacons encapsulated in Vir‐FVs specifically hybridize with the target miRNAs in the exosomes, generating fluorescence. Combined with flow cytometry, the Vir‐FVs can not only detect exosomal miRNAs but also distinguish tumor exosomes from normal exosomes by sensing the tumor‐related miRNAs, paving the way towards the rapid and efficient detection of exosomal miRNAs for diagnosis and prognosis prediction of diseases.     

Rational Engineering of a Dynamic,Entropy‐Driven DNA Nanomachine for Intracellular MicroRNA Imaging

We rationally engineered an elegant entropy‐driven DNA nanomachine with three‐dimensional track and applied it for intracellular miRNAs imaging. The proposed nanomachine is activated by target miRNA binding to drive a walking leg tethered to gold nanoparticle with a high density of DNA substrates. The autonomous and progressive walk on the DNA track via the entropy‐driven catalytic reaction of intramolecular toehold‐mediated strand migration leads to continuous disassembly of DNA substrates, accompanied by the recovery of fluorescence signal due to the specific release of a dye‐labeled substrate from DNA track. Our nanomachine outperforms the conventional intermolecular reaction‐based gold nanoparticle design in the context of an improved sensitivity and kinetics, attributed to the enhanced local effective concentrations of working DNA components from the proximity‐induced intramolecular reaction. Moreover, the nanomachine was applied for miRNA imaging inside living cells.     

Toehold‐initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

The ability to quantitate and visualize microRNAs (miRNAs) in situ in single cells would greatly facilitate the elucidation of miRNA‐mediated regulatory circuits and their disease associations. A toehold‐initiated strand‐displacement process was used to initiate rolling circle amplification of specific miRNAs, an approach that achieves both stringent recognition and in situ amplification of the target miRNA. This assay, termed toehold‐initiated rolling circle amplification (TIRCA), can be utilized to identify miRNAs at physiological temperature with high specificity and to visualize individual miRNAs in situ in single cells within 3 h. TIRCA is a competitive candidate technique for in situ miRNA imaging and may help us to understand the role of miRNAs in cellular processes and human diseases in more detail.     

Intracellular Entropy‐Driven Multi‐Bit DNA Computing for Tumor Progression Discrimination

Tumor progressions such as metastasis are complicated events that involve abnormal expression of different miRNAs and enzymes. Monitoring these biomolecules in live cells with computational DNA nanotechnology may enable discrimination of tumor progression via digital outputs. Herein, we report intracellular entropy‐driven multivalent DNA circuits to implement multi‐bit computing for simultaneous analysis of intracellular telomerase and microRNAs including miR‐21 and miR‐31. These three biomolecules can trigger respective DNA strand displacement recycling reactions for signal amplification. They are visualized by fluorescence imaging, and their signal outputs are encoded as multi‐bit binary codes for different cell types. The results can discriminate non‐tumorigenic, malignant and metastatic breast cells as well as respective tumors. This DNA computing circuit is further performed in a microfluidic chip to differentiate rare co‐cultured cells, which holds a potential for the analysis of clinical samples.     

A Rapid,Amplification‐Free,and Sensitive Diagnostic Assay for Single‐Step Multiplexed Fluorescence Detection of MicroRNA

The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short‐sequence nucleic acids next‐generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single‐step, single‐temperature, and amplification‐free assay provides a unique combination of performance parameters compared to state‐of‐the‐art miRNA detection technologies. Precise multiplexed quantification of miRNA‐20a, ‐20b, and ‐21 at concentrations between 0.05 and 0.5 nM in a single 150 μL sample and detection limits between 0.2 and 0.9 nM in 7.5 μL serum samples demonstrate the feasibility of both high‐throughput and point‐of‐care clinical diagnostics.     

Multiplexed detection methods for profiling microRNA expression in biological samples

The recent discovery of short, non-protein coding RNA molecules, such as microRNA molecules (miRNAs), that can control gene expression has unveiled a whole new layer of complexity in the regulation of cell function. Since 2001, there has been a surge of interest in understanding the regulatory role of the hundreds to thousands of miRNAs expressed in both plants and animals. Significant progress in this area requires the development of quantitative bioanalytical methods for the rapid, multiplexed detection of all miRNAs that are present in a particular cell or tissue sample. In this Minireview, we discuss some of the latest methods for high-throughput miRNA profiling and the unique technological challenges that must be surmounted in this endeavor.     

A Covalent Approach for Site‐Specific RNA Labeling in Mammalian Cells

Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site‐ and sequence‐specific covalent labeling of RNAs in mammalian cells by using tRNA^Ile2‐agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide‐bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site‐specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA.     

A New One-Pot Fluorescence Derivatization Strategy for Highly Sensitive MicroRNA Analysis

MicroRNAs (miRNAs) modulate the expression of over 30 % of mammalian genes during development and apoptosis, and abnormal expression of miRNAs may lead to a range of human pathologies. Therefore, analysis of miRNAs is valuable for disease diagnostics. In this work, a novel one-pot fluorescence derivatization strategy was developed for miRNA analysis. The mechanism of the derivatization reaction was explored by using instrumental methods, including liquid chromatography, fluorescence spectroscopy, and mass spectrometry. Highly fluorescent N⁶-ethenoadenine (ϵ-adenine) was formed and detached from the miRNA sequence through the reaction of adenine in nucleic acids with 2-chloroacetaldehyde (CAA) at 100 °C. This is the first experimental evidence that the cooperation of formed ϵ-adenine and water-mediated hydrogen-bond interaction between the proton at the 2′- and the oxyanion at 3′-positions stabilized the oxocarbenium significantly, which makes the depurination and derivatization of miRNA highly effective. Based on this derivatization strategy, a facile and sensitive high-performance liquid chromatography method was developed for quantitative assay of miRNAs. In combination with magnetic solid-phase extraction (MSPE), the HPLC method was shown to be useful for the determination of microRNAs at sub-picomolar level in serum samples.     

A computational model for non-conserved mature miRNAs from the rice genome

Several computational approaches employ the high complementarity of plant miRNAs to target mRNAs as a filter to recognize miRNA. Numerous non-conserved miRNAs are known with more recent evolutionary origin as a result of target gene duplication events. We present here a computational model with knowledge inputs from reported non-conserved mature miRNAs of Oryza sativa (rice). Sequence- and structure-based approaches were used to retrieve miRNA features based on rice Argonaute protein and develop a multiple linear regression (MLR) model (r² = 0.996, q²_cv = 0.989) which scored mature miRNAs as predicted by the MaturePred program. The model was validated by scoring test set (q² = 0.990) and computationally predicted mature miRNAs as external test set (q²_test = 0.895). This strategy successfully enhanced the confidence of retrieving most probable non-conserved miRNAs from the rice genome. We anticipate that this computational model would recognize unknown non-conserved miRNA candidates and nurture the current mechanistic understanding of miRNA sorting to unveil the role of non-conserved miRNAs in gene silencing.     

Fluorescence Signal Amplification Strategies Based on DNA Nanotechnology for miRNA Detection

In this review,the most recent progresses in the field of fluorescence signal amplification strategies based on DNA nanotechnology for miRNA are summarized.The types of signal amplification are given and the principles of amplification strategies are explained,including rolling circle amplification(RCA),catalytic hairpin assembly(CHA),hybridization chain reaction(HCR)and DNA walker.Subsequently,the application of these signal amplification methods in biosensing and bioimaging are covered and described.Finally,the challenges and the outlook of fluorescence signal amplification methods for miRNA detection are briefly commented.