RNA Condensate as a Versatile Platform for Improving Fluorogenic RNA Aptamer Properties and Cell Imaging.

Fluorogenic RNA aptamers are valuable tools for cell imaging, but they still suffer from shortcomings such as easy degradation, limited photostability, and low fluorescence enhancement. Molecular crowding conditions enable the stabilization of the structure, promotion of folding, and improvement of activity of functional RNA. Based on artificial RNA condensates, here we present a versatile platform to improve fluorogenic RNA aptamer properties and develop sensors for target analyte imaging in living cells. Using the CUG repeat as a general tag to drive phase separation, various fluorogenic aptamer-based RNA condensates (FLARE) were prepared. We show that the molecular crowding of FLARE can improve the enzymatic resistance, thermostability, photostability, and binding affinity of fluorogenic RNA aptamers. Moreover, the FLARE systems can be modularly engineered into sensors (FLARES), which demonstrate enhanced brightness and sensitivity compared to free sensors dispersed in homogeneous solution. This scalable design principle provides new insights into RNA aptamer property regulation and cellular imaging.

Leveraging DNA-Based Nanostructures for Advanced Error Detection and Correction in Data Communication.

This study demonstrates the implementation of the Hamming code using DNA-based nanostructures for error detection and correction in communication systems. The designed DNA nanostructures conduct logical operations to compute check codes and identify and correct erroneous data based on fluorescence signals. The execution of intricate DNA logic operations requires individuals with specialized training. By interpretation of the fluorescence signals generated by the DNA nanostructures, binary language can be extracted, effectively protecting data security. The findings highlight the potential of DNA as a versatile platform for reliable data transmission.

Catalytic hairpin assembly-assisted dual-signal amplification platform for ultrasensitive detection of tumor markers and intelligent diagnosis of gastric cancer.

Quantification of extracellular tumor markers has shown great promise for non-invasive cancer diagnosis. Combined detection of multiple tumor markers instead of a single one is valuable for accurate diagnosis. Here, we integrate CRISPR-Cas12a with DNA catalytic hairpin assembly (CHA) to doubly amplify the output signal for detecting microRNA-182 (miR-182), which is overexpressed by gastric cancer patients. Additionally, we develop a CHA system with self-replicating capacity (SRCHA) to realize dual-signal amplification for the detection of carcinoembryonic antigen (CEA), a broad-spectrum tumor marker. The proposed cascade amplification strategies enable ultrasensitive detection of miR-182 and CEA with low LODs of 0.063 fM and 4.8 pg mL-1, respectively. Moreover, we design a ternary "AND" logic gate using different concentrations of miR-182 and CEA as inputs, which demonstrates intelligent diagnosis of gastric cancer staging with a high accuracy of 93.3% in a clinical cohort of 30 individuals. Overall, our study expands the application of CRISPR-Cas12a in biosensing and provides a new diagnostic strategy for non-invasive liquid biopsy of gastric cancer before resorting to a traumatic tissue biopsy.

A strand displacement signal amplification-assisted fluorescence assay for sensitive detection of microRNA.

MicroRNA is a vital biomarker because of its abnormal expression in the emergence and development of diseases, especially in cancers. Herein, a label-free fluorescent sensing platform is proposed for detecting microRNA-21, coupled with the cascade toehold-mediated strand displacement reaction and magnetic beads. Target microRNA-21 acts as an initiator to trigger the cascade toehold-mediated strand displacement reaction and it outputs double-stranded DNA. After magnetic separation, the double-stranded DNA is intercalated by SYBR Green I, resulting in an amplified fluorescent signal. Under the optimal conditions, a wide linear range (0.5-60 nmol/L) and low limits of detection (0.19 nmol/L) are exhibited. What's more, the biosensor shows great specificity and reliability between microRNA-21 and other microRNAs involved in cancer (microRNA-34a, microRNA-155, microRNA-10b, and let-7a). Owing to the properties of fabulous sensitivity, high selectivity, and simplicity of operator, the proposed method paves a promising way for microRNA-21 detection in cancer diagnosis and biological research.

Biosynthetic Nanobubble-Mediated CRISPR/Cas9 Gene Editing of Cdh2 Inhibits Breast Cancer Metastasis.

The epithelial-mesenchymal transition (EMT), a process in which epithelial cells undergo a series of biochemical changes to acquire a mesenchymal phenotype, has been linked to tumor metastasis. Here, we present a novel strategy for knocking out the EMT-related Cdh2 gene, which encodes N-cadherin through CRISPR/Cas9-mediated gene editing by an ultrasound combined with biosynthetic nanobubbles (Gas Vesicles, GVs). Polyethyleneimine were employed as a gene delivery vector to deliver sgRNA into 4T1 cells that stably express the Cas9 protein, resulting in the stable Cdh2 gene- knockout cell lines. The Western blotting assay confirmed the absence of an N-cadherin protein in these Cdh2 gene-knockout 4T1 cell lines. Significantly reduced tumor cell migration was observed in the Cdh2 gene-knockout 4T1 cells in comparison with the wild-type cells. Our study demonstrated that an ultrasound combined with GVs could effectively mediate CRISPR/Cas9 gene editing of a Cdh2 gene to inhibit tumor invasion and metastasis.

Background-suppressed tumor-targeted photoacoustic imaging using bacterial carriers.

Photoacoustic (PA) imaging offers promise for biomedical applications due to its ability to image deep within biological tissues while providing detailed molecular information; however, its detection sensitivity is limited by high background signals that arise from endogenous chromophores. Genetic reporter proteins with photoswitchable properties enable the removal of background signals through the subtraction of PA images for each light-absorbing form. Unfortunately, the application of photoswitchable chromoproteins for tumor-targeted imaging has been hampered by the lack of an effective targeted delivery scheme; that is, photoswitchable probes must be delivered in vivo with high targeting efficiency and specificity. To overcome this limitation, we have developed a tumor-targeting delivery system in which tumor-homing bacteria (Escherichia coli) are exploited as carriers to affect the point-specific delivery of genetically encoded photochromic probes to the tumor area. To improve the efficiency of the desired background suppression, we engineered a phytochrome-based reporter protein (mDrBphP-PCMm/F469W) that displays higher photoswitching contrast than those in the current state of the art. Photoacoustic computed tomography was applied to achieve good depth and resolution in the context of in vivo (mice) imaging. The present system effectively integrates a genetically encoded phytochrome-based reporter protein, PA imaging, and synthetic biology (GPS), to achieve essentially background-suppressed tumor-targeted PA monitoring in deep-seated tissues. The ability to image tumors at substantial depths may enable target-specific cancer diagnoses to be made with greater sensitivity, fidelity, and specificity.

Machine learning-assisted array from fluorescent conjugated microporous polymers for multiple explosives recognition.

The fluorescent properties of conjugated microporous polyphenylene (CMPs) were tuned through a wide range by inclusion of small amount of comonomer as chromophore in the network. The multi-color CMPs were used for explosives sensing and demonstrated broad sensitivity (ranging from -0.01888 µM-1 to -0.00467 µM-1) and LODs (ranging from 31.0 nM to 125.3 nM) against thirteen explosive compounds including nitroaromatics (NACs), nitramines (NAMs) and nitrogen-rich heterocycles (NRHCs). The CMPs were also developed as a sensor array for discrimination of thirteen explosives, specifically including NT, p-DNB, DNT, TNT, TNP, TNR, RDX, HMX, CL-20, FOX-7, NTO, DABT and DHT. By using classical statistical method "Linear Discriminant Analysis (LDA)", the thirteen explosives at a fixed concentration were completely discriminated and unknown test samples were indentied with 88% classification accuracy. Moreover, explosives in different concentrations and the mixtures of explosives were also successfully classified. Compared with LDA, Machine Learning algorithms have significant advantages in analyzing the array-based sensing data. Different Machine Learning models for pattern recognition have also been implemented and discussed here and much higher accuracy (96% for "neural network") can be achieved in predicting unknown test samples after training.

Promoting the effect of microbubble-enhanced ultrasound on hyperthermia in rabbit liver.

PURPOSE: The heat-sink effect is one reason for the insufficient temperature increase in hyperthermia (HT) treatment for cancer. Microbubbles (MBs) nucleate inertial cavitation under therapeutic ultrasound (TUS) exposure, which form microbubble-enhanced ultrasound (MEUS), which results in blocking blood perfusion in the targeted liver tissues. This study aimed to determine if synergistic effects exist during HT in the liver when combined with MEUS. METHODS: Forty rabbits with surgically exposed livers were randomly divided into TUS + MB + HT, MB + HT, normal saline + HT, and MB + sham groups (n = 10 in each group). Liver perfusion was evaluated using contrast-enhanced ultrasound. The temperatures of the liver tissues were monitored using thermocouples. Pathological changes were determined by hematoxylin and eosin (H&E) staining. Serum hepatic transaminases were evaluated. RESULTS: MEUS pretreatment almost completely blocked the perfusion of targeted areas. The TUS + MB + HT and MB + HT groups showed significantly higher temperatures in treated areas than those in the other groups. However, the TUS + MB + HT group exhibited a more stable and regular increase in temperatures in the fitting curves compared with the MB + HT group. H&E staining revealed swelling hepatocytes, hemorrhage, and thrombosis in the portal area in the TUS + MB + HT group. CONCLUSION: MEUS reduced the blood perfusion in the targeted liver tissues, and, therefore, overcame the heat-sink effect during the HT procedure in rabbits. MEUS pretreatment might have the potential to enhance the therapeutic effect of HT.

A benzaldehyde-indole fused chromophore-based fluorescent probe for double-response to cyanide and hypochlorite in living cells.

With the rapid development of various industries, cyanide (CN-) and hypochlorite (ClO-) have a tremendously adverse effect on the health of humans and animals. In this study, a fluorescent probe HHTB based on a benzaldehyde-indole fused chromophore was designed to detect cyanide and hypochlorite simultaneously. The synthesized probe was found to have strong anti-interference ability. In addition, the designed probe could respond rapidly to ClO- in just 80 s, while the color changed visibly from red to colorless. Moreover, the response time to CN- was longer (about 160 s), with the apparent color change from red to light red. The ratiometric and colorimetric absorbance variation of HHTB was due to the nucleophilic attack of CN- on the indole CN functional group and the strong oxidization of ClO- which destroyed the CC bonds and the conjugation systems. Furthermore, the probe HHTB responding to ClO- and CN- presented high sensitivity, as the calculated detection limits were 1.18 nM and 1.40 nM, respectively. The probe was also found to have low biological toxicity and was used in living cells successfully. Therefore, it has good application prospect in the field of cell imaging and biomedicine. The binding mechanism of HHTB-CN and the reaction mechanism of HHTB and ClO- were further elucidated by a series of experiments.

Fluorescent nucleotide-lanthanide nanoparticles for highly selective determination of picric acid.

A new method based on coordination polymer nanoparticles (CPNs) derived from nucleotides and Tb3+ ions (GMP/Tb) for the selective and sensitive determination of aqueous 2,4,6-trinitrophenol (TNP) (picric acid) is established. The fluorescence of GMP/Tb nanoparticles is effectively quenched by TNP via photo-induced charge transfer (PCT), thus achieving its selectivity toward TNP over other nitroaromatic explosives. The decreased fluorescence of GMP/Tb shows a good linear relationship to the concentrations of TNP ranging from 5.0 to 40.0 µM, and the limit of detection is 26.0 nM (5.96 ppb). The proposed GMP/Tb probe also achieves satisfactory results in real samples. The obtained recoveries of this method in river water samples are in the range 93.15-106.10%. The relative standard deviation (RSD) are 0.57 to 1.01% based on three repeated determinations. This fabricated detector provides a feasible path for determination of ppb-level TNP in natural water samples, which can help humans to avoid TNP-contaminated drinking water. Graphical abstract.

G-quadruplex and duplex DNA binding studies of novel Ruthenium(II) complexes containing ascididemin ligands.

In this paper, three new Ruthenium(II) polypyridyl complexes containing ascididemin (ASC) as main ligand have been synthesized and characterized. Their interactions with different G-quadruplex (Htelo, c-myc and c-kit) (Htelo: human telomeric DNA, c-myc: cellular-myelocytomatosis viral oncogene, c-kit: oncogene c-kit promoter sequences) and duplex (ds26) DNA sequences were comparatively studied with the free ligand ASC by a series of spectroscopic techniques including UV-vis (ultraviolet-visible) spectroscopy, FID (fluorescent intercalator displacement) assay, and FRET (fluorescence resonance energy transfer) melting assay. Molecular docking studies were also performed to support the binding mode of the compounds with G-quadruplex DNA. Results indicated that [Ru(bpy)2ASC]·(PF6)2 (1), [Ru(phen)2ASC]·(PF6)2 (2), [Ru(tatp)2ASC]·(PF6)2 (3) (bpy = 2,2'­bipyridine, phen = 1,10­phenanthroline, tatp = 1,4,8,9­tetra­aza­triphenylene) and ASC can effectively bind G-quadruplex and duplex DNA and stabilization ability lies in the order 3 > 2 > 1 > ASC. Complex 3 was determined to be the most promising candidate for further in vitro studies and potential anticancer drug.

Pullulan-derived nanocomposite hydrogels for wastewater remediation: Synthesis and characterization.

Polysaccharide is a renewable, biocompatible and biodegradable material that has received considerable attention. In this work, a series of polysaccharide gels were synthesized from the chemical cross-linking of pullulan with diglycerols. The effects of the diglycerol chain length on the performance of the gels were evaluated by XRD, TGA, SEM, rheological testings and swelling measurements. Overall, increasing the chain length resulted in a smaller pore size and stronger mechanical strength. Tetramethylene glycol diglycidyl ether (longest chain length)-derived Gel-T, which had the best performance (acceptable porous structure, good swelling ability and strong rigidity), was used to produce a nanocomposite hydrogel with montmorillonite (MMT). The incorporation of MMT led to a decrease in gel swelling and an increase in gel strength. The obtained nanocomposite system exhibited excellent adsorption properties (80 mg/g) towards crystal violet, and the adsorption behaviours were well represented by the pseudo-second-order kinetic and Langmuir isotherm models. Altogether, this study provides a better understanding of the structure-function relationships of pullulan-constructed hydrogel materials and will help to design more practical adsorbents for dye removal.

Regulation of multi-factors (tail/loop/link/ions) for G-quadruplex enantioselectivity of Δ- and Λ- [Ru(bpy)2(dppz-idzo)]2.

Chiral recognition of DNA molecules is important because much evidence has indicated that transformations of chirality and diverse conformations of DNA are involved in a series of key biological events. Among these, enrichment of G-quadruplexes (GQs) in the genome, and the exploration of their multiple structures, has aroused great interest. Herein, we compared nearly 100 different sequences with 3'-tail sequences of variable length or different linkers or diverse loops and mutative ionic concentrations. All sequences were capable of forming stable GQs, with fluorescence signal enhancement upon binding with Δ- and Λ- [Ru(bpy)2(dppz-idzo)]2+ (Δ/Λ-1). Our results show that multiple factors, including the 3'-tail length, linkers, loop length and ionic concentration, regulate the enantioselectivity of GQs. Furthermore, molecular docking simulations revealed that chiral recognition of GQs depends on the binding site. To the best of our knowledge, this is the first systematic study regarding the regulation of multi-factors for GQ selectivity of chiral Ru-complexes. These results will serve as a useful reference for enantioselective recognition of genomic GQs and may facilitate the development of chiral anticancer agents for targeting GQs.

Coordination polymer nanoparticles from nucleotide and lanthanide ions as a versatile platform for color-tunable luminescence and integrating Boolean logic operations.

Novel supramolecular coordination polymer nanoparticles (CPNs) were synthesized via the self-assembly of guanosine monophosphate (GMP) and lanthanide ions (Ln3+, including Tb3+, Eu3+ and Ce3+) in aqueous solution. These CPNs (GMP/Tb3+, GMP/Eu3+ and GMP/Ce3+) have an identical coordination environment but exhibit completely different luminescence properties responding to external stimuli such as dipicolinic acid (DPA), ethylene diamine tetraacetic acid (EDTA), pH and metal ions, which has inspired us to tune the emission color of the CPNs and perform multiple logic operations. Firstly, color-tunable luminescence from red to green can be easily achieved by modulating the doping ratio of Tb3+ and Eu3+ into GMP. Notably, trichromatic white light emitting CPNs can be successfully realized by simultaneously doping Tb3+, Eu3+ and Ce3+ into the host or just adjusting the pH of the solution. What's more, by employing GMP/Tb3+ CPNs as a logic operator, we have achieved the implementation of multilayered gate cascades (INH-INH, NOR-OR). When GMP/Eu3+ CPNs served as a logic operator, the logic elements can be integrated as another combinatorial gate (AND-INH). Moreover, by employing the red emission of Eu3+ and blue emission of GMP as the dual-output signal transducer, a set of parallel logic gates was established successfully. These results help elucidate the design rules by which simple logic can be integrated to construct cascaded logic gates and expand the applications of CPNs in light-emitting diode (LED) lamps and biological systems.

Integration of G-quadruplex and DNA-templated Ag NCs for nonarithmetic information processing.

To create sophisticated molecular logic circuits from scratch, you may not believe how common the building blocks can be and how diverse and powerful such circuits can be when scaled up. Using the two simple building blocks of G-quadruplex and silver nanoclusters (Ag NCs), we experimentally construct a series of multifunctional, label-free, and multi-output logic circuits to perform nonarithmetic functions: a 1-to-2 decoder, a 4-to-2 encoder, an 8-to-3 encoder, dual transfer gates, a 2 : 1 multiplexer, and a 1 : 2 demultiplexer. Moreover, a parity checker which is capable of identifying odd and even numbers from natural numbers is constructed conceptually. Finally, a multi-valued logic gate (ternary inhibit gate) is readily achieved by taking this DNA/Ag NC system as a universal platform. All of the above logic circuits share the same building blocks, indicating the great prospects of the assembly of nanomaterials and DNA for biochemical logic devices. Considering its biocompatibility, the novel prototypes developed here may have potential applications in the fields of biological computers and medical diagnosis and serve as a promising proof of principle in the not-too-distant future.

Ultrasensitive and universal fluorescent aptasensor for the detection of biomolecules (ATP, adenosine and thrombin) based on DNA/Ag nanoclusters fluorescence light-up system.

We report here an ultrasensitive strategy based on the recognition-induced conformational alteration of aptamer and fluorescence turn-on abilities of guanine-rich (G-rich) DNA sequence in proximity to silver nanoclusters for adenosine triphosphate (ATP), adenosine (A) and thrombin (TB) detection. Herein, we designed two tailored DNA sequences noted as complementary DNA (abbreviated as c-DNA) and signal probe DNA (abbreviated as s-DNA), respectively. c-DNA is designed as a special structure consisting of a sequence complementary to aptamer at the 3'-end and a guanine-rich DNA sequence at the 5'-end; s-DNA contains a cytosine-rich sequence responsible for Ag NCs templated synthesis at the 3'-end and a link sequence (part of aptamer) complementary to partial of the c-DNA at the 5'-end. In the presence of target, the aptamer associated with the target, resulting in the formation of duplex DNA (dsDNA), the DNA-Ag NCs thereafter could close to the guanine-rich sequence, leading to enhanced fluorescence signal readout. The widespread application of the sensing system is achieved success in the detection of three biomolecules. ATP, adenosine and thrombin in the range of 0.5-8.0 µM, 0.5-7.0 µM and 50-900 nM could be linearly detected with the detection limits of 91.6 nM, 103.4 nM and 8.4 nM, respectively. This label-free and turn-on fluorescent sensing system employing the mechanism proposed here turns out to be sensitive, selective, and convenient for the detection of biomolecules without washing and separation steps.

A RET-supported logic gate combinatorial library to enable modeling and implementation of intelligent logic functions.

Boolean logic gates integrate multiple digital inputs into a digital output. Among these, logic gates based on nucleic acids have attracted a great deal of attention due to the prospect of controlling living systems in the way we control electronic computers. Herein, by employing Thioflavin T (ThT) as a signal transducer, we integrated multiple components based on RET (a type of proto-oncogene) into a logic gate combinatorial library, including basic logic gates (NOR, INHIBIT, IMPLICATION), a single three-input NOR gate, and combinatorial gates (INHIBIT-OR, NOT-AND-NOR). In this library, gates were connected in series where the output of the previous gate was the input for the next gate. Subsequently, by taking advantage of the library, some intelligent logic functions were realized. Expectedly, a biocomputing keypad-lock security system was designed by sequential logic operations. Moreover, a parity checker which can identify even numbers and odd numbers from natural numbers was established successfully. This work helps elucidate the design rules by which simple logic can be harnessed to produce diverse and complex calculations by rewiring communication between different gates. Together, our system may serve as a promising proof of principle that demonstrates increased computational complexity by linking multiple logic gates together.

A universal label-free fluorescent aptasensor based on Ru complex and quantum dots for adenosine, dopamine and 17ß-estradiol detection.

Based on specific aptamer binding properties, a strategy for adenosine, dopamine and 17ß-estradiol detection was realised by employing Ru complex and quantum dots (QDs) as fluorescence probes. Ru complex, which could quench the fluorescence of QDs, preferred to bind with aptamer DNA and resulted in the fluorescence rise of QDs. When the aptamer DNA was incubated with the target first, it could not bind with Ru complex and the fluorescence of QDs was quenched. Under the optimal condition, the fluorescence intensity was linearly proportional to the concentration of adenosine, dopamine and 17ß-estradiol with a limit of detection (LOD) of 101 nM, 19 nM and 37 nM, respectively. The experiments in fetal bovine serum were also carried out with good results. This universal method was rapid, label-free, low-cost, easy-operating and highly repeatable for the detection of adenosine, dopamine and 17ß-estradiol. Qualitative detection by naked eyes was also available without complex instruments. It could also be extended to detect various analytes, such as metal ions, proteins and small molecules by using appropriate aptamers.