Tailed-Hoogsteen Triplex DNA Silver Nanoclusters Emit Red Fluorescence upon Target miRNA Sensing.

MicroRNAs (miRNAs) are small RNA molecules, typically 21‒22 nucleotides in size, which play a crucial role in regulating gene expression in most eukaryotes. Their significance in various biological processes and disease pathogenesis has led to considerable interest in their potential as biomarkers for diagnosis and therapeutic applications. In this study, a novel method for sensing target miRNAs using Tailed-Hoogsteen triplex DNA-encapsulated Silver Nanoclusters (DNA/AgNCs) is introduced. Upon hybridization of a miRNA with the tail, the Tailed-Hoogsteen triplex DNA/AgNCs exhibit a pronounced red fluorescence, effectively turning on the signal. It is successfully demonstrated that this miRNA sensor not only recognized target miRNAs in total RNA extracted from cells but also visualized target miRNAs when introduced into live cells, highlighting the advantages of the turn-on mechanism. Furthermore, through gel-fluorescence assays and small-angle X-ray scattering (SAXS) analysis, the turn-on mechanism is elucidated, revealing that the Tailed-Hoogsteen triplex DNA/AgNCs undergo a structural transition from a monomer to a dimer upon sensing the target miRNA. Overall, the findings suggest that Tailed-Hoogsteen triplex DNA/AgNCs hold great promise as practical sensors for small RNAs in both in vitro and cell imaging applications.

Energy Transfer Between i-Motif DNA Encapsulated Silver Nanoclusters and Fluorescein Amidite Efficiently Visualizes the Redox State of Live Cells.

The redox regulation, maintaining a balance between oxidation and reduction in living cells, is vital for cellular homeostasis, intricate signaling networks, and appropriate responses to physiological and environmental cues. Here, a novel redox sensor, based on DNA-encapsulated silver nanoclusters (DNA/AgNCs) and well-defined chemical fluorophores, effectively illustrating cellular redox states in live cells is introduced. Among various i-motif DNAs, the photophysical property of poly-cytosines (C20)-encapsulated AgNCs that sense reactive oxygen species (ROS) is adopted. However, the sensitivity of C20/AgNCs is insufficient for evaluating ROS levels in live cells. To overcome this drawback, the ROS sensing mechanism of C20/AgNCs through gel electrophoresis, mass spectrometry, and small-angle X-ray scattering is primarily defined. Then, by tethering fluorescein amidite (FAM) and Cyanine 5 (Cy5) dyes to each end of the C20/AgNCs sensor, an Energy Transfer (ET) between AgNCs and FAM is achieved, resulting in intensified green fluorescence upon ROS detection. Taken together, the FAM-C20/AgNCs-Cy5 redox sensor enables dynamic visualization of intracellular redox states, yielding insights into oxidative stress-related processes in live cells.

Locking-to-unlocking system is an efficient strategy to design DNA/silver nanoclusters (AgNCs) probe for human miRNAs.

MicroRNAs (miRNAs), small non-coding RNA molecules, are important biomarkers for research and medical purposes. Here, we describe the development of a fast and simple method using highly fluorescent oligonucleotide-silver nanocluster probes (DNA/AgNCs) to efficiently detect specific miRNAs. Due to the great sequence diversity of miRNAs in humans and other organisms, a uniform strategy for miRNA detection is attractive. The concept presented is an oligonucleotide-based locking-to-unlocking system that can be endowed with miRNA complementarity while maintaining the same secondary structure. The locking-to-unlocking system is based on fold-back anchored DNA templates that consist of a cytosine-rich loop for AgNCs stabilization, an miRNA recognition site and an overlap region for hairpin stabilization. When an miRNA is recognized, fluorescence in the visible region is specifically extinguished in a concentration-dependent manner. Here, the exact composition of the fold-back anchor for the locking-to-unlocking system has been systematically optimized, balancing propensity for loop-structure formation, encapsulation of emissive AgNCs and target sensitivity. It is demonstrated that the applied strategy successfully can detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines.

DNA/RNA chimera templates improve the emission intensity and target the accessibility of silver nanocluster-based sensors for human microRNA detection.

In recent years microRNAs (miRNAs) have been established as important biomarkers in a variety of diseases including cancer, diabetes, cardiovascular disease, aging, Alzheimer's disease, asthma, autoimmune disease and liver diseases. As a consequence, a variety of monitoring methods for miRNAs have been developed, including a fast and simple method for miRNA detection by exploitation of the unique photoluminescence of DNA-templated silver nanoclusters (DNA/AgNCs). To increase the versatility of the AgNC-based method, we have adopted DNA/RNA chimera templates for AgNC-based probes, allowing response from several human miRNAs which are hardly detectable with DNA-based probes. Here, we demonstrate in detail the power of DNA/RNA chimera/AgNC probes in detecting two human miRNAs, let-7a and miR-200c. The DNA/RNA chimera-based probes are highly efficient to determine the level of miRNAs in several human cell lines.

In-solution multiplex miRNA detection using DNA-templated silver nanocluster probes.

MicroRNAs (miRNAs) are small regulatory RNAs (size ∼21nt to ∼25nt) that can be used as biomarkers of disease diagnosis, and efforts have been directed towards the invention of a rapid, simple and sequence-selective detection method for miRNAs. We recently developed a DNA/silver nanoclusters (AgNCs)-based turn-off fluorescence method in the presence of target miRNA. To further advance our method toward multiplex miRNA detection in solution, the design of various fluorescent DNA/AgNCs probes was essential. Therefore, tethering of DNA-12nt scaffolds with 9 different AgNCs emitters to target-sensing DNA sequences was investigated. Interestingly, for the creation of spectrally different DNA/AgNCs probes, not only were the emitters encapsulated in 9 different DNA-12nt scaffolds necessary but the tethered target-sensing DNA sequences are also crucial to tune the fluorescence across the visible to infra-red region. In this study, we obtained three spectrally distinctive emitters of each DNA/AgNCs probes such as green, red, and near-infrared (NIR) fluorescence. Using these DNA/AgNCs probes, we here show a proof of concept for a rapid, one-step, in-solution multiplex miRNA detection method.

Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes.

MicroRNAs (miRNAs) are small regulatory RNAs (size ~21 nt to ~25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded.

Noncanonical Head-to-Head Hairpin DNA Dimerization Is Essential for the Synthesis of Orange Emissive Silver Nanoclusters.

DNA secondary structures, such as dimers and hairpins, are important for the synthesis of DNA template-embedded silver nanoclusters (DNA/AgNCs). However, the arrangement of AgNCs within a given DNA template and how the AgNC influences the secondary structure of the DNA template are still unclear. Here, we introduce a noncanonical head-to-head hairpin DNA nanostructure that is driven by orange-emissive AgNCs. Through detailed in-gel analysis, sugar backbone switching, inductively coupled plasma mass spectrometry, small-angle X-ray scattering, and small angle neutron scattering, we show that the orange-emissive AgNCs mediate cytosine-Ag-cytosine bridging between two six-cytosine loop (6C-loop) hairpin DNA templates. Unlike green, red, or far-red emissive AgNCs, which are embedded inside a hairpin and duplex DNA template, the orange-emissive AgNCs are localized on the interface between the two 6C-loop hairpin DNA templates, thereby linking them. Moreover, we found that deoxyribose in the backbone of the 6C-loop at the third and fourth cytosines is crucial for the formation of the orange-emissive AgNCs and the head-to-head hairpin DNA structure. Taken together, we suggest that the specific wavelength of AgNCs fluorescence is determined by the mutual interaction between the secondary or tertiary structures of DNA- and AgNC-mediated intermolecular DNA cross-linking.

The structural shift of a DNA template between a hairpin and a dimer tunes the emission color of DNA-templated AgNCs.

The scaffolding DNA sequence and the size of silver nanoclusters (AgNCs), confined in a DNA template are the key parameters in determining the fluorescent properties of DNA-stabilized silver nanoclusters (DNA/AgNCs). In addition, we suggest here that the structural shift of a DNA hairpin-dimer is as important as the DNA sequence in determining the emission wavelength of DNA/AgNCs. Furthermore, we show that the structural shift post AgNC formation can be triggered by incubation time and pre-AgNC formation under salt conditions. As an important factor in predicting the emission properties of DNA/AgNCs, the modulation of DNA secondary structures with either sequence changes or ionic conditions can be applied for the dual-color detection system of a target molecule. Particularly, the dual-color detection method may increase the reliability of DNA/AgNC sensors for miRNAs.

MicroRNA Biomarkers in Neurodegenerative Diseases and Emerging Nano-Sensors Technology.

MicroRNAs (miRNAs) are essential small RNA molecules (20-24 nt) that negatively regulate the expression of target genes at the post-transcriptional level. Due to their roles in a variety of biological processes, the aberrant expression profiles of miRNAs have been identified as biomarkers for many diseases, such as cancer, diabetes, cardiovascular disease and neurodegenerative diseases. In order to precisely, rapidly and economically monitor the expression of miRNAs, many cutting-edge nanotechnologies have been developed. One of the nanotechnologies, based on DNA encapsulated silver nanoclusters (DNA/AgNCs), has increasingly been adopted to create nanoscale bio-sensing systems due to its attractive optical properties, such as brightness, tuneable emission wavelengths and photostability. Using the DNA/AgNCs sensor methods, the presence of miRNAs can be detected simply by monitoring the fluorescence alteration of DNA/AgNCs sensors. We introduce these DNA/ AgNCs sensor methods and discuss their possible applications for detecting miRNA biomarkers in neurodegenerative diseases.