Smart Hairpins@MnO2 Nanosystem Enables Target-Triggered Enzyme-Free Exponential Amplification for Ultrasensitive Imaging of Intracellular MicroRNAs in Living Cells.

Sensitive and specific imaging of microRNA (miRNA) in living cells is of great value for disease diagnosis and monitoring. Hybridization chain reaction (HCR) and DNAzyme-based methods have been considered as powerful tools for miRNA detection, with low efficient intracellular delivery and limited amplification efficiency. Herein, we propose a Hairpins@MnO2 nanosystem for intracellular enzyme-free exponential amplification for miRNA imaging. The enzyme-free exponential amplification is based on the synergistic cross-activation between HCR and DNAzymes. The MnO2 nanosheets were employed as the carrier of three kinds of hairpin DNA probes and further provided appropriate Mn2+ as DNAzyme cofactors in the living cell. Upon entering cells and in the presence of highly expressed glutathione (GSH) in tumors, MnO2 is reduced to release Mn2+ and the three kinds of hairpin DNA probes. In the presence of target miRNA, the released hairpin DNA H1 and H2 probes self-assemble via HCR into the wire-shaped active Mn2+-based DNAzymes which further catalyze the cleavage of H3 to generate numerous new triggers to reversely stimulate HCR amplifiers, thus offering tremendously amplified Förster resonance energy transfer readout. The method has a detection limit of 33 fM, which is 2.4 × 104 times lower than that of the traditional HCR system. The developed method also has a high specificity; even miRNAs with a single base difference can be distinguished. Live cell imaging experiments confirmed that this Hairpins@MnO2 nanosystem allows accurate differentiation of miRNA expression of cancer cells and normal cells. The method holds great potential in biological research of nucleic acids.

A novel nomogram to predict mortality in patients with stroke: a survival analysis based on the MIMIC-III clinical database.

BACKGROUND: Stroke is a disease characterized by sudden cerebral ischemia and is the second leading cause of death worldwide. We aimed to develop and validate a nomogram model to predict mortality in intensive care unit patients with stroke. METHODS: All data involved in this study were extracted from the Medical Information Mart for Intensive Care III database (MIMIC-III). The data were analyzed using multivariate Cox regression, and the performance of the novel nomogram, which assessed the patient's overall survival at 30, 180, and 360 days after stroke, was evaluated using Harrell's concordance index (C-index) and the area under the receiver operating characteristic curve. A calibration curve and decision curve were introduced to test the clinical value and effectiveness of our prediction model. RESULTS: A total of 767 patients with stroke were randomly divided into derivation (n = 536) and validation (n = 231) cohorts at a 7:3 ratio. Multivariate Cox regression showed that 12 independent predictors, including age, weight, ventilation, cardiac arrhythmia, metastatic cancer, explicit sepsis, Oxford Acute Severity of Illness Score or OASIS score, diastolic blood pressure, bicarbonate, chloride, red blood cell and white blood cell counts, played a significant role in the survival of individuals with stroke. The nomogram model was validated based on the C-indices, calibration plots, and decision curve analysis results. CONCLUSIONS: The plotted nomogram accurately predicted stroke outcomes and, thus may contribute to clinical decision-making and treatment as well as consultation services for patients.

Breast cancer plasma biopsy by in situ determination of exosomal microRNA-1246 with a molecular beacon.

Liquid biopsy is becoming an innovative tool in precision oncology owing to its noninvasive identification of biomarkers circulating in the body fluid at various time points for continuous and real-time analysis of disease progression. MicroRNAs in blood exosomes are identified as a new promising class of potential biomarkers for cancer diagnostics and prognostics. Conventional detection of blood exosomal microRNAs need multiple-step, complicated, costly, and time-consuming sample preparation of exosomes isolation and RNA extract, which affect the accuracy and reproducibility of analytical results. In this work, we set up an in situ quantitative analysis of human plasma exosomal miR-1246 by a probe of 2'-O-methyl and phosphorothioate modified molecular beacon. The probe has outstanding nuclease resistance in highly active RNase A/T1/I, which makes it stable for direct application in blood samples. With rapid rupture of exosomes membrane by Triton X-100, the probe can enter exosomes to specifically target miR-1246 exhibiting quantitative fluorescent signals. Using the output signals as a diagnostic marker, we differentiated 33 breast cancer patients from 37 healthy controls with 97.30% sensitivity and 93.94% specificity at the best cutoff. The blood biopsy is simple without extracting plasma exosomes and their nucleic acids content, time-saving in about 2 h of total analysis process, and microvolumes needed for plasma sample, suggesting its good potential to clinical application.

Experimental Study on Effects of Adipose-Derived Stem Cell-Seeded Silk Fibroin Chitosan Film on Wound Healing of a Diabetic Rat Model.

BACKGROUND: Wound healing is a complex process that relies on growth factors and stimulation of angiogenesis. Tissue engineering materials composed of adipose-derived stem cells (ADSCs) and silk fibroin (SF)/chitosan (CS) may be able to solve this problem. The aim of this study was to investigate the wound-healing potential of ADSC-seeded SF/CS in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Thirty-six male Sprague-Dawley rats were purchased and randomly assigned into 3 groups: a control group (no graft), a group treated with SF/CS film graft, and a group treated with ADSC-seeded SF/CS graft. The number of animals in each group was 12. Diabetes was induced by an intraperitoneal injection of streptozotocin. A cutaneous wound was incised at the dorsal region of all the experimental animals. The ADSCs were labeled with CM-Dil fluorescent staining. Wound healing was assessed for all animal groups by observing the rate of wound closure and hematoxylin and eosin staining. The expression of epidermal growth factor, transforming growth factor-ß, and vascular endothelial growth factor at the wound sites was studied by enzyme-linked immunosorbent assay to evaluate the effect of growth factors secreted by ADSCs. The differentiation of ADSCs was analyzed by immunofluorescence staining. RESULTS: The ADSC-seeded SF/CS film treatment significantly increased the rates of wound closure in treated animals, and hence wound healing was drastically enhanced for ADSC-SF/CS treatment groups compared with control groups and SF/CS film treatment group. Histological observations showed the condition of wound healing. Enzyme-linked immunosorbent assay and immunofluorescence staining observations showed the secretion and differentiation of ADSCs, respectively. CONCLUSIONS: Our analyses clearly suggested that it is feasible and effective to enhance wound healing in a diabetic rat model with ADSC-seeded SF/CS film.

[Predictive value of cord blood 25(OH)D3 for early infantile atopic dermatitis].

OBJECTIVE: To explore the predictive value of cord blood 25(OH)D3 [25(OH)D3] for infantile atopic dermatitis (AD), and to provide a reference for primary prevention of early infantile AD. METHODS: The neonates born from July to September, 2015 were enrolled. The cord blood samples were collected at birth to measure the level of 25(OH)D3. Outpatient follow-up was conducted for all the infants at 6 weeks, 3 months, and 6 months after birth. A survey was performed to investigate the incidence of AD. RESULTS: A total of 67 neonates completed a 6-month follow-up. The incidence of AD was 34% (23/67), and 91% (21/23) of these cases occurred in the first month after birth. The 23 AD children had a significantly lower cord 25(OH)D3 level than those without AD (P<0.05). The children with a cord 25(OH)D3 level <30 nmol/L showed a significantly higher incidence of AD than those with a cord 25(OH)D3 level ≥30 nmol/L (P<0.05). The receiver operating characteristic (ROC) analysis showed that the area under the ROC curve of cord 25(OH)D3 in predicting AD was 0.648 (standard error: 0.075; 95%CI: 0.502-0.795). Its sensitivity, specificity, positive predictive value, and negative predictive value were 52.2%, 79.5%, 57.1%, and 76.1%, respectively. Logistic regression analysis showed that low cord 25(OH)D3 level, preference for seafood during pregnancy, atopic family history, and mixed feeding were risk factors for infantile AD (P<0.05). CONCLUSIONS: Cord 25(OH)D3 level is inversely associated with the risk of infantile AD, but it has a low diagnostic value for this disease.

Lanthanum-Based Metal-Organic Frameworks for Specific Detection of Sudan Virus RNA Conservative Sequences down to Single-Base Mismatch.

Reactions of La(NO3)3·6H2O with the polar, tritopic quaternized carboxylate ligands N-carboxymethyl-3,5-dicarboxylpyridinium bromide (H3CmdcpBr) and N-(4-carboxybenzyl)-3,5-dicarboxylpyridinium bromide (H3CbdcpBr) afford two water-stable metal-organic frameworks (MOFs) of {[La4(Cmdcp)6(H2O)9]}n (1, 3D) and {[La2(Cbdcp)3(H2O)10]}n (2, 2D). MOFs 1 and 2 absorb the carboxyfluorescein (FAM)-tagged probe DNA (P-DNA) and quench the fluorescence of FAM via a photoinduced electron transfer (PET) process. The nonemissive P-DNA@MOF hybrids thus formed in turn function as sensing platforms to distinguish conservative linear, single-stranded RNA sequences of Sudan virus with high selectivity and low detection limits of 112 and 67 pM, respectively (at a signal-to-noise ratio of 3). These hybrids also exhibit high specificity and discriminate down to single-base mismatch RNA sequences.

Genetically Determined Circulating Saturated and Unsaturated Fatty Acids and the Occurrence and Exacerbation of Chronic Obstructive Pulmonary Disease-A Two-Sample Mendelian Randomization Study.

Research on dietary fatty acids (FAs) and lung health has reported skeptical findings. This study aims to examine the causal relationship between circulating FAs and Chronic Obstructive Pulmonary Disease (COPD) onset and exacerbation, using a two-sample Mendelian Randomization (MR) analysis. Strong and independent genetic variants of FAs were obtained from the UK Biobank of European ancestry. The exposure traits included saturated FA (SFA), poly- and mono-unsaturated FA (PUFA and MUFA), omega-3 and omega-6 PUFA, docosahexaenoic acid (DHA), and linoleic acid (LA), all expressed as total FA (TFA) percentages. Summary statistics for COPD outcomes were obtained from the FinnGen consortium including COPD, COPD hospitalization, COPD/asthma-related infections, COPD-related respiratory insufficiency, and COPD/asthma/interstitial lung disease (ILD)-related pneumonia. The inverse-variance weighted (IVW) was the primary MR approach. MR-Egger regression and MR-PRESSO were utilized to evaluate heterogeneity and pleiotropy. MR-PRESSO tests suggested no obvious horizontal pleiotropy. MR results by the IVW approach indicated that the genetically high SFA/TFA levels were associated with an increased risk of COPD/asthma/ILD-related pneumonia (OR: 1.275, 95%CI: 1.103-1.474, p for FDR = 0.002). No significant relationship was observed between other types of FAs and COPD outcomes. Our MR analysis suggests that there is weak evidence that the genetically predicted high SFA/TFA was associated with an increased risk of pneumonia.

Optimizing electrochemical performance of Na0.67Ni0.17Co0.17Mn0.66O2 with P2 structure via preparing concentration-gradient particles for sodium-ion batteries.

P2-type layered oxides for rechargeable sodium-ion batteries have drawn a lot of attention because of their excellent electrochemical performance. However, these types of cathodes usually suffer from poor cyclic stability. To overcome this disadvantage, in this work, novel ball-shaped concentration-gradient oxide Na0.67Ni0.17Co0.17Mn0.66O2 with P2 structure modified by Mn-rich surface is successfully prepared using co-precipitation method. The concentration of Mn increased from the inner core to the surface, endowing the material with an excellent cyclic stability. The cathode exhibits enhanced electrochemical properties than that of the sample synthesized by solid-state method and concentration-constant material. It shows 143.2 mAh/g initial discharge capacity and retains 131 mAh/g between 2 V and 4.5 V after 100 rounds. The significant improvement in the electrochemical properties of the sample benefits from the unique concentration-gradient structure, and the Mn-rich surface that effectively stabilizes the basic P2 structure. The relatively higher Ni content in the core leads to a slight improvement in the discharge capacity of the sample. This strategy may provide new insights for preparing layered cathodes for sodium-ion batteries with high electrochemical performance.

Two web-based dynamically interactive nomograms and risk stratification systems for predicting survival outcomes and guiding treatment in non-metastatic nasopharyngeal carcinoma.

BACKGROUND: A nomogram is a valuable and easily accessible tool for individualizing cancer prognosis. This study aims to establish and validate two prognostic nomograms for long-term overall survival (OS) and cancer-specific survival (CSS) in non-metastatic nasopharyngeal carcinoma (NPC) patients and to investigate the treatment options for the nomogram-based risk stratification subgroups. METHODS: A total of 3959 patients with non-metastatic NPC between 2004 and 2015 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. The patients were randomly allocated to the training and validation cohorts in a 7:3 ratio. Prognostic nomograms were constructed to estimate OS and CSS by integrating significant variables from multivariate Cox regression employing a backward stepwise method. We examined the correlation indices (C-index) and areas under the curves (AUC) of time-dependent receiver operating characteristic curves to assess the discriminative ability of our survival models. The comprehensive enhancements of predictive performance were evaluated with net reclassification operating improvement (NRI) and integrated discrimination improvement (IDI). Reliability was validated using calibration plots. Decision curve analysis (DCA) was used to estimate clinical efficacy and capability. Finally, the nomogram-based risk stratification system used Kaplan-Meier survival analysis and log-rank tests to examine differences between subgroups. RESULTS: The following independent parameters were significant predictors for OS: sex, age, race, marital status, histological type, median household income, AJCC stage tumor size, and lymph node size. Except for the race variables mentioned above, the rest were independent prognostic factors for CSS. The C-index, AUC, NRI, and IDI indicated satisfactory discriminating properties. The calibration curves exhibited high concordance with the exact outcomes. Moreover, the DCA demonstrated performed well for net benefits. The prognosis significantly differed between low- and high-risk patients (p < 0.001). In a treatment-based stratified survival analysis in risk-stratified subgroups, chemotherapy benefited patients in the high-risk group compared to radiotherapy alone. Radiotherapy only was recommended in the low-risk group. CONCLUSIONS: Our nomograms have satisfactory performance and have been validated. It can assist clinicians in prognosis assessment and individualized treatment of non-metastatic NPC patients.

Ankylosing spondylitis and psychiatric disorders in European population: a Mendelian randomization study.

Background: Epidemiologic evidence has demonstrated a correlation between ankylosing spondylitis and psychiatric disorders. However, little is known about the common genetics and causality of this association. This study aimed to investigate the common genetics and causality between ankylosing spondylitis (AS) and psychiatric disorders. Methods: A two-sample Mendelian Randomization (MR) analysis was carried out to confirm causal relationships between ankylosing spondylitis and five mental health conditions including major depressive disorder (MDD), anxiety disorder (AXD), schizophrenia (SCZ), bipolar disorder (BIP), and anorexia nervosa (AN). Genetic instrumental variables associated with exposures and outcomes were derived from the largest available summary statistics of genome-wide association studies (GWAS). Bidirectional causal estimation of MR was primarily obtained using the inverse variance weighting (IVW) method. Other MR methods include MR-Egger regression, Weighted Median Estimator (WME), Weighted Mode, Simple Mode, and Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO). Sensitivity analyses are conducted to estimate the robustness of MR results. Results: The findings suggest that AS may be causally responsible for the risk of developing SCZ (OR = 1.18, 95% confidence interval = (1.06, 1.31), P = 2.58 × 10-3) and AN (OR = 1.32, 95% confidence interval = (1.07, 1.64), P = 9.43 × 10-3). In addition, MDD, AXD, SCZ, AN, and BIP were not inversely causally related to AS (all p > 0.05). Conclusion: Our study provides fresh insights into the relationship between AS and psychiatric disorders (SCZ and AN). Furthermore, it may provide new clues for risk management and preventive interventions for mental disorders in patients with AS.

Associations of Maternal Serum Iodine Concentration with Obstetric Complications and Birth Outcomes-Longitudinal Analysis Based on the Huizhou Mother-Infant Cohort, South China.

This study aimed to explore the temporal associations between maternal serum iodine concentration (SIC) and common pregnancy outcomes in Chinese women. Eligible singleton pregnant women aged 20-34 years were selected, and their fasting blood samples were collected during early (T1, n = 1101) and mid-pregnancy (T2, n = 403) for SIC testing by inductively coupled plasma mass spectrometry. Multivariable linear regression indicated that log10SIC at T1 (ß = -0.082), T2 (ß = -0.198), and their % change (ß = -0.131) were inversely associated with gestational weight gain (GWG, all p < 0.05). Maternal log10SIC at both T1 (ß = 0.077) and T2 (ß = 0.105) were positively associated with the Apgar score at 1 min (both p < 0.05). Women in the third quartile (Q3) of SIC at T1 had a lower risk of small for gestational age (SGA, OR = 0.405, 95% CI: 0.198-0.829) compared with those in Q4. Restricted cubic spline regression suggested a U-shaped association between SIC and SGA risk, and SIC above 94 µg/L at T1 was the starting point for an increased risk of SGA. The risk of premature rupture of membrane (PROM) increased by 96% (OR = 1.960, 95% CI: 1.010-3.804) in Q4 compared to that in Q1. Our longitudinal data from an iodine-replete region of China indicated that high maternal SIC could restrict GWG and improve Apgar scores at delivery, but might increase the risk of SGA and PROM.

Reference Intervals of Serum Iodine Concentration in Chinese Pregnant Women.

The study aims to establish trimester-specific reference ranges for serum iodine (SI) in Chinese pregnant women and explore its associations with maternal and infantile thyroid function. Apparently healthy pregnant women were enrolled during their first antenatal visit. Fasting venous and spot urine samples were collected for determining serum and urinary iodine (UI) levels by a validated inductively coupled plasma mass spectrometry. Serum free triiodothyronine (FT3), free thyroxine (FT4), thyrotropin (TSH), and neonatal TSH levels were tested by electro-chemiluminescent assay. The reference ranges of SI were established by percentile method and reported as 2.5-97.5%. ROC analysis was applied to compare the discriminative ability of SI, UI, and UI to urinary creatine ratio (UI /UCr) in early pregnancy for various thyroid conditions. The trimester-specific reference ranges of SI for Chinese pregnant women were 60.91-114.53 µg/L for the first trimester (T1, n = 1029), 54.57-103.42 µg/L for the second trimester (T2, n = 379), and 52.03-110.40 µg/L for the third trimester (T3, n = 455). Maternal SI at T1 but not UI and UI/UCr was significantly correlated with FT3 (r = 0.393, P < 0.001), FT4 (r = 0.637, P < 0.001), and TSH (r = -0.299, P<0.001). Maternal SI change% from T1 to T2 (but not SI change% from T1 to T3) had marginal correlation with neonatal TSH (r=-0.106, P=0.046). ROC analysis showed that maternal SI at T1 had better predictability for several thyroid conditions than UIC and UI/UCr.

[Anti-inflammatory effect and mechanism of artemisinin and dihydroartemisinin].

OBJECTIVE: To study and compare the anti-inflammatory effect and molecular mechanism of artemisinin and dihydroartemisinin. METHOD: Mouse mononuclear macrophage RAW264.7 cells were stimulated to release inflammatory mediators such as TNF-alpha, IL-6 and NO, in order to assess the drugs' inhibitory effect on macrophage's release of above inflammatory mediators. The levels of TNF-alpha and IL-6 were determined by ELISA and the cytotoxicity was determined by MTT method. The protein expression of iNOS, COX-2 and beta-actin were tested by Western blot. The enzymatic activity of COX-2 was determined by colorimetric method. RESULT: Dihydroartemisinin significantly inhibited LPS-induced release of TNF-alpha, IL-6 and NO from RAW264.7 in mice with the concentration range of 12.5 - 100 micromol x L(-1), and showed good dose dependence. Artemisinin only inhibited the IL-6 release to a certain extent. CONCLUSION: Dihydroartemisinin inhibits macrophages from releasing inflammatory factors TNF-alpha and IL-6 and inflammatory mediators NO by down-regulating iNOS protein. Artemisinin may help dihydroartemisinin to show its anti-inflammatory effect through metabolism.

A universal catalytic hairpin assembly system for direct plasma biopsy of exosomal PIWI-interacting RNAs and microRNAs.

PIWI-interacting RNAs (piRNAs) are a complex class of small non-coding RNAs which specifically interact with the PIWI protein to play important roles in germline development and somatic tissues. Aberrant expressions of piRNAs have been recently found in a variety of malignant tumors and associated with cancer hallmarks. However, current methods of analyzing piRNAs are limited to reverse transcription quantitative polymerase chain reaction and next generation sequencing. In this study, we have developed a universal catalytic hybridization assembly system (uniCHA) to quantify piRNAs as well as microRNAs. The system simply comprises two universal hairpin DNA strands and one starting hairpin DNA which can be tailored by a simple rule to bind different piRNA and miRNA targets. The uniCHA system was proved to be able to analyze various piRNAs and miRNAs at the same reaction condition with low leakage and high sensitivity of pM level. With this system, we have detected piR-651 and miR-1246 in 106 particles µL-1 MCF-7 cell-secreted exosomes, and successfully performed a direct plasma biopsy to diagnose breast cancer with sensitivity and specificity both at 100% in cohorts of 21 breast cancer patients and 13 healthy controls. This universal biosensing system provides a simple and efficient strategy in analyzing multiple piRNA/miRNA biomarkers in complicated biological samples, indicating its potential of clinical application in cancer diagnostics.

An intramolecular DNAzyme-based amplification for miRNA analysis with improving reaction kinetics and high sensitivity.

Sensitive, specific and rapid methods for detecting microRNAs (miRNAs) play critical roles in disease diagnosis and therapy. Enzyme-free amplification techniques based on DNAzyme assembly have recently been developed for the highly specific miRNA analysis. However, traditional DNAzyme-based assembly (free DNAzyme) amplifiers is mainly dependent on the target-induced split DNAzyme fragments to assemble into activated DNAzyme structures, which have made a compromise between the sensitivity and specificity due to the random diffusion of dissociative probes in a bulk solution with poor kinetics. Herein, based on a rationally designed DNA probe, we developed an intramolecular DNAzyme assembly (intra-DNAzyme) method to overcome these challenges. The miR-373 is used as model analyte for our current proof-of-concept experiments. Compared with the free-DNAzyme method, our method showed significantly improved analytical performance in terms of dynamic range, assay sensitivity and speed. This method can detect miR-373 specifically with a detection limit as low as 4.3 fM, which is about 83.7 times lower than the previous free-DNAzyme method. This intra-DNAzyme strategy would be of great value in both basic research and clinical diagnosis.

Circulating Epstein-Barr virus DNA associated with hepatic impairment and its diagnostic and prognostic role in patients with gastric cancer.

Epstein-Barr virus (EBV) infection may affect all tissues and organs of the body. Little is known about the impact of this entity on its systematic incorporation in patients with gastric cancer (GC). This study enrolled a total of 113 GC patients with EBV infection (EBVaGC) and 167 GC patients without EBV infection (EBVnGC). It was found that the CRP levels (indicative of inflammatory status) were significantly increased in EBVaGC compared with those in EBVnGC (12.11 mg/L vs. 5.72 mg/L, P = 0.008), but WBC and neutrophils counts were similar in both groups (P > 0.05). Consistent elevations in the levels of liver enzymes, ALP and GGT, with incompatible alterations in ALT or AST were observed in EBVaGC. Slightly prolonged coagulation indices, PT and INR, and decreased albumin consistently suggested impaired synthesis capability of the liver in EBVaGC (all P < 0.05). The level of circulating EBV DNA was positively correlated with the level of GGT, tumor marker CA72-4 and the lymphocyte infiltration in tumor tissues (all P < 0.05). Of note, the EBV associated high-lymphocyte infiltrated tissues presented rich CD8 + T cells. Circulating EBV DNA further showed a predictive role in distinguishing EBVaGC from EBVnGC (AUC 0.79, 95% CI 0.73 to 0.85, P < 0.001), and was associated closely with better overall survival (HR 0.45, 95% CI 0.21 to 0.96, P = 0.039). EBV infection in patients with gastric cancer may be linked to hepatic impairment and immune response. Circulating cell-free EBV DNA is not only a biomarker for the screening of an EBV-related GC subtype but is also an independently prognosis factor for the long-term survival benefit in GC patients.

A universal rolling circle amplification for label-free and highly specific nucleic acid sensing.

Traditional RCA methods face some drawbacks including limited specificity and amplification templates with sequence dependence. Herein, a universal RCA (URCA) strategy for label-free nucleic acid sensing with high specificity was proposed, which could be used for sensing of different nucleic acids without redesigning or synthesizing new amplification templates. The URCA strategy also showed high accuracy for miRNA analysis in practical samples.

A domino-like localized cascade toehold assembly amplification-based DNA nanowire for microRNA imaging in living cells.

High sensitivity and specificity imaging of miRNA in living cells plays an important role in understanding miRNA-related regulation and pathological research. Localized DNA circuits have shown good performance in reaction rate and sensitivity and have been proposed for sensitive imaging of miRNA in living cells. However, most reported localized DNA circuits have a high risk of derailment or a limited loading rate capacity, which hinder their further application. To solve these issues, we herein developed a domino-like localized cascade toehold assembly (LCTA) amplification-based DNA nanowire to achieve highly sensitive and highly specific imaging of miRNAs in living cells by using DNA nanowires as reactant delivery vehicles and confining both reactant probes in a compact space. The LCTA is constructed by interval hybridization of DNA double-stranded probe pairs to a DNA nanowire with multiplex footholds generated by alternating chain hybridization. Due to the localized effect, the LCTA showed high reaction kinetics and sensitivity, and the method could detect miRNAs as low as 51 pM. The LCTA was proven to be able to accurately distinguish the miRNA expression difference between normal cells and cancer cells. In particular, the developed LCTA could be used to construct an OR logic gate to simultaneously image the total amount of multiple miRNAs in living cells. We believe that the developed LCTA can be an effective intracellular nucleic acid imaging tool and can promote the development of nucleic acid-related clinical disease diagnosis and DNA logical sensors.

Label-free and highly sensitive APE1 detection based on rolling circle amplification combined with G-quadruplex.

The highly sensitive detection of low-abundant apurinic/apyrimidinic endonuclease 1 (APE1) activity is of great significance for early diagnosis of disease and pathological research. Many methods for detecting APE1 based on isothermal nucleic acids amplification have been developed for improving its sensitivity. However, some of these methods have certain limitations, such as multiple reaction steps, narrow linear range, and complicated processes for fluorescent labeling. Herein, we develop a highly sensitive and label-free APE1 fluorescence detection method based on rolling circle amplification combined with G-quadruplex (RCA-G4). A hairpin probe (HP) labeled with the AP site can be recognized and cleaved by APE1, leading to the release of the primer sequence, which triggered RCA to produce long chain amplification products with a great amount of repeated sequences. The formed amplicon contains a series G-quadruplex structure, which can be combined with Thioflavin T (ThT) to produce fluorescence and achieve high sensitivity label-free detection of APE1. Benefit from the high amplification efficiency of RCA and the high fluorescence quantum yield of G-quadruplex/ThT, a detection limit as low as 1.52 × 10-6 U/mL and the linear range from 2 × 10-6 to 10 U/mL were obtained. The developed RCA-G4 method can be successfully used to detect APE1 in serum samples with a recovery from 96.3% to 105.7%. We believe that this approach is expected to play an important role in APE1-related disease research and drug development.