Cell-Free Biosensing Genetic Circuit Coupled with Ribozyme Cleavage Reaction for Rapid and Sensitive Detection of Small Molecules

Synthetic biological systems have been utilized to develop a wide range of genetic circuits and components that enhance the performance of biosensing systems. Among them, cell-free systems are emerging as important platforms for synthetic biology applications. Genetic circuits play an essential role in cell-free systems, mainly consisting of sensing modules, regulation modules, and signal output modules. Currently, fluorescent proteins and aptamers are commonly used as signal outputs. However, these signal output modes cannot simultaneously achieve faster signal output, more accurate and reliable performance, and signal amplification. Ribozyme is a highly structured and catalytic RNA molecule that can specifically recognize and cut specific substrate sequences. Here, by adopting ribozyme as the signal output, we developed a cell-free biosensing genetic circuit coupled with the ribozyme cleavage reaction, enabling rapid and sensitive detection of small molecules. More importantly, we have also successfully constructed a 3D-printed sensor array and thereby achieved high-throughput analysis of an inhibitory drug. Furthermore, our method will help expand the application range of ribozyme in the field of synthetic biology and also optimize the signal output system of cell-free biosensing, thus promoting the development of cell-free synthetic biology in biomedical research, clinical diagnosis, environmental monitoring, and food inspection

Correction to A Dual-Enzyme-Assisted Three-Dimensional DNA Walking Machine Using T4 Polynucleotide Kinase as Activators and Application in Polynucleotide Kinase Assays

Correction to A Dual-Enzyme-Assisted Three-Dimensional DNA Walking Machine Using T4 Polynucleotide Kinase as Activators and Application in Polynucleotide Kinase Assay

DataSheet2_Management of BMI Is a Potential New Approach for the Prevention of Idiopathic Pulmonary Fibrosis.ZIP

Aims: Current idiopathic pulmonary fibrosis (IPF) therapies usually show a poor outcome or treatment efficacy. The search for new risk factors has significant implications in preventing, delaying, and treating IPF. The association between obesity and the risk of IPF is not clear. This study aimed to investigate the role of different obesity types in IPF risk, which provides the possibility of weight loss as a new approach for IPF prevention.Methods: We conducted a two-sample Mendelian randomization (MR) analysis to assess the causal effect of obesity on IPF risk. We collected summary data of genetically determined obesity-related traits, including body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) from large-scale consortia (the sample size ranging from 232,101 to 681,275), and genetic association with IPF from one of the largest meta-analyses including 2,668 cases. A total of 35–469 single nucleotide polymorphisms were selected as instrumental variables for obesity-related traits. We further performed multivariable MR to estimate the independent effect of BMI and WC on the risk of IPF.Results: Increased BMI and WC were associated with higher risk of IPF [odds ratio (OR) = 1.51, 95% confidence interval (CI) (1.22–1.87), p = 1.27 × 10–4, and OR = 1.71, 95% CI (1.08–2.72), p = 2.33 × 10–2, respectively]. Similar results for the BMI and WC were obtained in the replicated analysis. Subsequently, only the result for BMI survived following the multiple testing correction and showed good consistency with the weighted median estimator. Sensitivity analyses indicated that there was no heterogeneity or horizontal pleiotropy for MR estimations. Further multivariable MR suggested that the BMI showed the same direction and similar magnitude with that in the univariable MR analysis. There was little evidence to support the causal role of WHR on the risk of IPF in this study.Conclusion: Genetically determined BMI demonstrates a causal risk for IPF, which offers a novel insight into probing potential mechanisms. Meanwhile, these results also suggest that weight loss may be beneficial to IPF prevention.</p

DataSheet1_Management of BMI Is a Potential New Approach for the Prevention of Idiopathic Pulmonary Fibrosis.ZIP

Aims: Current idiopathic pulmonary fibrosis (IPF) therapies usually show a poor outcome or treatment efficacy. The search for new risk factors has significant implications in preventing, delaying, and treating IPF. The association between obesity and the risk of IPF is not clear. This study aimed to investigate the role of different obesity types in IPF risk, which provides the possibility of weight loss as a new approach for IPF prevention.Methods: We conducted a two-sample Mendelian randomization (MR) analysis to assess the causal effect of obesity on IPF risk. We collected summary data of genetically determined obesity-related traits, including body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) from large-scale consortia (the sample size ranging from 232,101 to 681,275), and genetic association with IPF from one of the largest meta-analyses including 2,668 cases. A total of 35–469 single nucleotide polymorphisms were selected as instrumental variables for obesity-related traits. We further performed multivariable MR to estimate the independent effect of BMI and WC on the risk of IPF.Results: Increased BMI and WC were associated with higher risk of IPF [odds ratio (OR) = 1.51, 95% confidence interval (CI) (1.22–1.87), p = 1.27 × 10–4, and OR = 1.71, 95% CI (1.08–2.72), p = 2.33 × 10–2, respectively]. Similar results for the BMI and WC were obtained in the replicated analysis. Subsequently, only the result for BMI survived following the multiple testing correction and showed good consistency with the weighted median estimator. Sensitivity analyses indicated that there was no heterogeneity or horizontal pleiotropy for MR estimations. Further multivariable MR suggested that the BMI showed the same direction and similar magnitude with that in the univariable MR analysis. There was little evidence to support the causal role of WHR on the risk of IPF in this study.Conclusion: Genetically determined BMI demonstrates a causal risk for IPF, which offers a novel insight into probing potential mechanisms. Meanwhile, these results also suggest that weight loss may be beneficial to IPF prevention.</p

Assembly of Self-Cleaning Electrode Surface for the Development of Refreshable Biosensors

Passivation of electrode surface and tedious reconstruction of biosensing architectures have long plagued researchers for the development of electrochemical biosensors. Here, we report a novel self-cleaning electrode by modifying the commonly used working electrode with superhydrophobic and conductive nanocomposite. Owing to the superhydrophobicity and the chemical stability, the electrode avoids passivation result from both adsorption of molecules and oxidation in air. The high conductivity and the high effective area also allow the achievement of enhanced electrochemical signals. On the basis of comprehensive studies on this novel electrode, we have applied it in the fabrication of refreshable electrochemical biosensors for both electro-active and electro-inactive targets. For both cases, detection of the targets can be well performed, and the self-cleaning electrode can be refreshed by simply washing and applied for successive measurements in a long period

Table1_MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2.DOCX

MicroRNAs (miRNAs) as small non-coding RNA transcripts bind their complementary sequences in the 3′-untranslated region (3′-UTR) of target messenger RNAs (mRNAs) to regulate their expression. It is known that miR-372 belongs to the miR-371–373 gene cluster and has been found to be abnormally expressed in a variety of cancers, but its precise mechanism in cancer remains to be discovered. In this study, miR-372-3p expression was assessed in 153 frozen tissue samples, including primary diagnosed colon cancer and matched normal and adjacent tissues, using real time quantitative polymerase chain reaction (qPCR). An analysis of qPCR data revealed a significant reduction in miR-372-3p expression (by >2-fold) in colon cancer tissues in 51.5% (34/66) of patients. Consistent with this, mimicking the increased miR-372-3p levels in SW480 colon cancer cells significantly suppressed cell growth and proliferation. Although no direct correlation was found between the low level of miR-372-3p and certain tumor-related factors, such as p53, HRE-2, PMS2, MLH1, MSH2, MSH6, HDAC4, p21, and Wee1, in colon cancer tissues, an inverse relationship between miR-372-3p and Ki67 (a marker of proliferation) or miR-372-3p and MAP3K2(MEKK2), which plays a critical role in the MAPK signaling pathways, was confirmed using tissue samples. The target relationship between miR-372-3p and MAP3K2 was verified using luciferase assays in SW480 colon cancer cells. As expected, miR-372-3p mimics significantly suppressed the luciferase activity of pMIR-luc/MAP3K2 3′-UTR in cells, suggesting that miR-372-3p modulates the expression of MAP3K2 by directly targeting its 3′-UTR. Overall, the results obtained herein suggest that miR-372-3p may function as a tumor-suppressor miRNA in colon cancer by targeting MAP3K2.</p

Enzyme Reaction-Assisted Programmable Transcriptional Switches for Bioactive Molecule Detection

Bioactive molecules are highly worthwhile to recognize and explore the latent pathogenic mechanism. Conventional methods for bioactive molecule detection, including mass spectrometry and fluorescent probe imaging, are limited due to the complex processing and signal interference. Here, we designed enzyme-reaction-assisted programmable transcriptional switches for the detection of bioactive molecules. The approach is based on the use of programmable enzyme site-specific cleavage-assisted DNA triplex-based conformational switches that, upon responding to bioactive molecules, can trigger the transcription of fluorescent light-up aptamers. Thanks to the programmable nature of the sensing platform, the method can be adapted to different bioactive molecules, and we demonstrated the enzyme-small molecule catalytic reaction combination of myeloperoxidase (MPO)–hydrogen peroxide (H2O2) as a model that transcriptional switches was capable of detecting H2O2 and possessed the specificity and anti-interference ability in vitro. Furthermore, we successfully applied the switches into cells to observe the detection feasibility in vivo, and dynamically monitored changes of H2O2 in cellular oxidative stress levels. Therefore, we attempt to amalgamate the advantages of enzyme reaction with the pluripotency of programmable transcriptional switches, which can take both fields a step further, which may promote the research of biostimuli and the construction of DNA molecular devices

Table2_MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2.DOCX

MicroRNAs (miRNAs) as small non-coding RNA transcripts bind their complementary sequences in the 3′-untranslated region (3′-UTR) of target messenger RNAs (mRNAs) to regulate their expression. It is known that miR-372 belongs to the miR-371–373 gene cluster and has been found to be abnormally expressed in a variety of cancers, but its precise mechanism in cancer remains to be discovered. In this study, miR-372-3p expression was assessed in 153 frozen tissue samples, including primary diagnosed colon cancer and matched normal and adjacent tissues, using real time quantitative polymerase chain reaction (qPCR). An analysis of qPCR data revealed a significant reduction in miR-372-3p expression (by >2-fold) in colon cancer tissues in 51.5% (34/66) of patients. Consistent with this, mimicking the increased miR-372-3p levels in SW480 colon cancer cells significantly suppressed cell growth and proliferation. Although no direct correlation was found between the low level of miR-372-3p and certain tumor-related factors, such as p53, HRE-2, PMS2, MLH1, MSH2, MSH6, HDAC4, p21, and Wee1, in colon cancer tissues, an inverse relationship between miR-372-3p and Ki67 (a marker of proliferation) or miR-372-3p and MAP3K2(MEKK2), which plays a critical role in the MAPK signaling pathways, was confirmed using tissue samples. The target relationship between miR-372-3p and MAP3K2 was verified using luciferase assays in SW480 colon cancer cells. As expected, miR-372-3p mimics significantly suppressed the luciferase activity of pMIR-luc/MAP3K2 3′-UTR in cells, suggesting that miR-372-3p modulates the expression of MAP3K2 by directly targeting its 3′-UTR. Overall, the results obtained herein suggest that miR-372-3p may function as a tumor-suppressor miRNA in colon cancer by targeting MAP3K2.</p

DataSheet1_MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2.PDF

MicroRNAs (miRNAs) as small non-coding RNA transcripts bind their complementary sequences in the 3′-untranslated region (3′-UTR) of target messenger RNAs (mRNAs) to regulate their expression. It is known that miR-372 belongs to the miR-371–373 gene cluster and has been found to be abnormally expressed in a variety of cancers, but its precise mechanism in cancer remains to be discovered. In this study, miR-372-3p expression was assessed in 153 frozen tissue samples, including primary diagnosed colon cancer and matched normal and adjacent tissues, using real time quantitative polymerase chain reaction (qPCR). An analysis of qPCR data revealed a significant reduction in miR-372-3p expression (by >2-fold) in colon cancer tissues in 51.5% (34/66) of patients. Consistent with this, mimicking the increased miR-372-3p levels in SW480 colon cancer cells significantly suppressed cell growth and proliferation. Although no direct correlation was found between the low level of miR-372-3p and certain tumor-related factors, such as p53, HRE-2, PMS2, MLH1, MSH2, MSH6, HDAC4, p21, and Wee1, in colon cancer tissues, an inverse relationship between miR-372-3p and Ki67 (a marker of proliferation) or miR-372-3p and MAP3K2(MEKK2), which plays a critical role in the MAPK signaling pathways, was confirmed using tissue samples. The target relationship between miR-372-3p and MAP3K2 was verified using luciferase assays in SW480 colon cancer cells. As expected, miR-372-3p mimics significantly suppressed the luciferase activity of pMIR-luc/MAP3K2 3′-UTR in cells, suggesting that miR-372-3p modulates the expression of MAP3K2 by directly targeting its 3′-UTR. Overall, the results obtained herein suggest that miR-372-3p may function as a tumor-suppressor miRNA in colon cancer by targeting MAP3K2.</p