Thermus thermophilus Argonaute-Based Isothermal Amplification Assay for Ultrasensitive and Specific RNA Detection.

Recent advances in prokaryotic Argonaute proteins (pAgos) as potential genome-editing tools have provided new insights into the development of pAgos-based nucleic acid detection platforms. However, pAgos-based isothermal detection remains challenging. Here, we report a true isothermal amplification strategy, termed Thermus thermophilus Argonaute-based thermostable exponential amplification reaction (TtAgoEAR), to detect RNA with ultrasensitivity and single-nucleotide resolution at a constant temperature of 66 °C. We demonstrate the reliable detection of lncRNA, mRNA, and virus RNA with attomolar sensitivity and that TtAgoEAR can be applied to detect RNA targets in in cell lines, saliva, and tissues. We utilize this assay to distinguish pancreatic cancer cells carrying the mutation from wild-type cells with as little as 2 ng of RNA material. We also show that TtAgoEAR is easily adaptable to a lateral-flow-based readout. These results demonstrate that TtAgoEAR has great potential to facilitate reliable and easy RNA detection in point-of-care diagnosis and field analysis.

Evaluation of classification ability of logistic regression model on SERS data of miRNAs.

Logistic regression (LR) is a supervised multiple linear regression model, which uses linear weighted calculation for input to obtain weight coefficients of model. The surface enhanced Raman spectroscopy (SERS) technology greatly enhances the Raman signal of analyte. LR model was used to analyze the data of seven types of pancreatic cancer-related miRNAs obtained from commercial SERS substrate. The classification ability of the model on such data was observed under the configurations of different key parameters (classification mode, regularization method and loss function optimization way), and the effect of the two types of data formats were also evaluated. The results showed that though LR model used to classify this data did not perform well as expected, miRNA-191 and miRNA-4306 still had high recalls (sensitivity), which laid a theoretical foundation for the purpose of using LR model to identify these two miRNAs to jointly diagnose of pancreatic cancer at miRNA level.

One-step isothermal amplification strategy for microRNA specific and ultrasensitive detection based on nicking-assisted entropy-driven DNA circuit triggered exponential amplification reaction.

Sensitive and specific detection of microRNAs (miRNAs) is of critical significance for early diagnosis of cancers such as pancreatic cancer with atypical initial symptoms and high mortality. Despite exponential amplification reaction (EXPAR) is an attractive isothermal amplification method for detecting miRNAs, it faces the problems of the dependence difference and low specificity. To address such challenges, herein, a nicking-assisted entropy-driven DNA circuit triggered exponential amplification reaction (NAED-EXPAR) was firstly employed for ultrasensitive and specific detection of miRNA in "one-pot" manner at constant temperature. Nicking-assisted entropy-driven DNA circuit can specifically recognize the target miRNA, leading to continuous disassembly of DNA substrates via intramolecular toehold-mediated branch migration. During the reaction, the catalytic circuit can consume excess fuel DNA strands to produce a large number of primers. Then the newly formed primers can trigger EXPAR for highly efficient signal amplification. Mechanism analysis shows that the amplification efficiency of NAED-EXPAR is superior than that of single EXPAR. For miR-21, the detection limit of NAED-EXPAR can reach 100 aM, which is at least five orders of magnitude higher than the standard EXPAR that directly uses the target as primer. NAED-EXPAR shows improved specificity for identifying single nucleotide variations and enables sensitive and accurate analysis of miR-21 in human cancer cell lines. This method is expected to offer a new approach for the reliable quantification of miRNAs in complex biological matrices and provide valuable information for early cancer diagnosis.

A Novel and Rapid Serum Detection Technology for Non-Invasive Screening of Gastric Cancer Based on Raman Spectroscopy Combined With Different Machine Learning Methods.

Gastric cancer (GC) is the fifth most common cancer in the world and a serious threat to human health. Due to its high morbidity and mortality, a simple, rapid and accurate early screening method for GC is urgently needed. In this study, the potential of Raman spectroscopy combined with different machine learning methods was explored to distinguish serum samples from GC patients and healthy controls. Serum Raman spectra were collected from 109 patients with GC (including 35 in stage I, 14 in stage II, 35 in stage III, and 25 in stage IV) and 104 healthy volunteers matched for age, presenting for a routine physical examination. We analyzed the difference in serum metabolism between GC patients and healthy people through a comparative study of the average Raman spectra of the two groups. Four machine learning methods, one-dimensional convolutional neural network, random forest, support vector machine, and K-nearest neighbor were used to explore identifying two sets of Raman spectral data. The classification model was established by using 70% of the data as a training set and 30% as a test set. Using unseen data to test the model, the RF model yielded an accuracy of 92.8%, and the sensitivity and specificity were 94.7% and 90.8%. The performance of the RF model was further confirmed by the receiver operating characteristic (ROC) curve, with an area under the curve (AUC) of 0.9199. This exploratory work shows that serum Raman spectroscopy combined with RF has great potential in the machine-assisted classification of GC, and is expected to provide a non-destructive and convenient technology for the screening of GC patients.

An enzyme-powered, three-dimensional lame DNA walker.

Molecular machines constructed by three-dimensional (3-D) DNA walker have emerged as a hot topic in applications such as novel biosensors, cargo delivery platforms and intracellular imaging. Herein, we first propose a lame DNA walker that can randomly and autonomously move on microsphere-based 3-D track. The stochastic lame walker has a long leg mainly responsible for persistent movement and a short leg cutting substrates rapidly. Its motion is propelled by a nicking endonuclease cleavage of hybridized DNA tracks. Kinetic and persistent study show that the lame DNA walker enables reaction equilibrium at 30 min, need a cleat domain of at least 14 nucleotides and can persistently move on 3-D tracks with an average rate of 6.467 × 10-11 M s-1. We also demonstrate that the lame walker can be used to detect target DNA in the detection range of 10 pM-5 nM with high specificity by toehold exchange mechanism. This work will further expand the performance of 3-D DNA walkers and substantially contributes to the improved understanding of DNA walking systems.

A novel quantification platform for point-of-care testing of circulating MicroRNAs based on allosteric spherical nanoprobe.

MiRNA-150, a gene regulator that has been revealed to be abnormal expression in non-small cell lung cancer (NSCLC), can be regarded as a serum indicator for diagnosis and monitoring of NSCLC. Herein, a new sort of nanoprobe, termed allosteric spherical nanoprobe, was first developed to sense miRNA-150. Compared with conventional hairpin, this new nanoprobe possesses more enrichment capacity and reaction cross section. Structurally, it consists of magnetic nanoparticles and dual-hairpin. In the absence of miRNA-150, the spherical nanoprobes form hairpin structure through DNA self-assembly, which could promote the Förster resonance energy transfer (FRET) of fluorophore (FAM) and quencher (BHQ1) nearby. However, in the presence of target, the target-probe hybridization can open the hairpin and form the active "Y" structure which separated fluorophore and quencher to yield "signal on" fluorescence. In the manner of multipoint fluorescence detection, the target-bound allosteric spherical nanoprobe could provide high detection sensitivity with a linear range of 100 fM to 10 nM and a detection limit of 38 fM. More importantly, the proposed method can distinguish the expression of serum miRNA-150 among NSCLC patients and healthy people. Finally, we hoped that the potential bioanalytical application of this nanoprobe strategy will pave the way for point-of-care testing (POCT).

Two-layer three-dimensional DNA walker with highly integrated entropy-driven and enzyme-powered reactions for HIV detection.

Here, we propose a new two-layer three-dimensional (3-D) DNA walker sensor with highly integrated entropy-driven and enzyme-powered reactions for the first time. The 3-D DNA walker sensor is constructed by assembling densely carboxyfluorescein-labeled single strand oligonucleotides (inner-layer tracks) and nucleic acid complex S (outer-layer tracks) on a microparticle. In the presence of the target, outer and inner tracks are activated in turn, thereby releasing a great deal of the signal reporters for signal reading. As a result, our 3-D DNA walker sensor can realize the target detection in the range from 2 pM to 5 nM within one hour. Besides, the specific walker sensor can clearly distinguish even one-base mismatched target analogue. More importantly, our walker sensor can also test the target in human serum samples in the concentrations as low as 0.1 nM, which provides a bridge between real sample detection and clinical application. Certainly, this smart strategy could also be generalized to any target of interest by proper design.

An electrochemical biosensor for microRNA-196a detection based on cyclic enzymatic signal amplification and template-free DNA extension reaction with the adsorption of methylene blue.

A simple and sensitive electrochemical biosensor was developed for microRNA-196a detection, which is of important diagnostic significance for pancreatic cancer. It was based on cyclic enzymatic signal amplification (CESA) and template-free DNA extension reaction. In the presence of microRNA-196a, duplex-specific nuclease (DSN) catalyzed the digestion of the 3'-PO4 terminated capture probe (CP), resulting in the target recycling amplification. Meanwhile, the 3'-OH terminal of CP was exposed. Then, template-free DNA extension reaction was triggered by terminal deoxynucleotidyl transferase (TdT), producing amounts of single-stranded DNA (ssDNA). After ssDNA absorbed numerous methylene blue (MB), an ultrasensitive electrochemical readout was obtained. Based on this dual amplification mechanism, the proposed biosensor exhibited a high sensitivity for detection of microRNA-196a down to 15 aM with a linear range from 0.05 fM to 50 pM. This biosensor displayed high specificity, which could discriminate target microRNAs from one base mismatched microRNAs. It also showed good reproducibility and stability. Furthermore, it was successfully applied to the determination of microRNA-196a in plasma samples. In conclusion, with the excellent analytical performance, this biosensor might have the potential for application in clinical diagnostics of pancreatic cancer.

A novel SHARPIN-PRMT5-H3R2me1 axis is essential for lung cancer cell invasion.

SHARPIN (Shank-associated RH domain interacting protein) is the main component of the linear ubiquitin chain activation complex (LUBAC). SHARPIN is involved in regulating inflammation and cancer progression. However, whether SHARPIN plays an important role in lung cancer metastasis and the potential underlying mechanism are still unknown. Here, for the first time, we reported that SHARPIN expression is closely related to lung cancer progression. Moreover, SHARPIN plays a central role in controlling lung cancer cell metastasis. Mechanistic studies further revealed that PRMT5 (Protein arginine methyltransferase 5), responsible for catalyzing arginine methylation on histones, is a novel cofactor of SHARPIN. This finding provides the basis for further study of the crosstalk between protein ubiquitination and histone methylation. We further found that SHARPIN-PRMT5 is essential for the monomethylation of histones of chromatins at key metastasis-related genes, defining a new mechanism regulating cancer invasion. A novel MLL complex (ASH2 and WDR5) was implied in the link between histone arginine2 monomethylation (H3R2me1) and histone lysine4 trimethylation (H3K4me3) for the activation of metastasis-related genes. These novel findings establish a new epigenetic paradigm in which SHARPIN-PRMT5 has distinct roles in orchestrating chromatin environments for cancer-related genes via integrating signaling between H3R2me1 and H3K4me3.