Specific quantitative detection of N6-methyladenosine at single-base resolution by extension-based isothermal exponential amplification reaction (E-IEXPAR).

BACKGROUND: N6-methyladenosine (m6A) is a common modification in RNA, crucial for various cellular functions and associated with human diseases. Quantification of m6A at single-base resolution is of great significance for exploring its biological roles and related disease research. However, existing analysis techniques, such as polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP), face challenges like the requirement for thermal cycling or intricate primer design. Therefore, it is urgent to establish a simple, non-thermal cycling and highly sensitive assay for m6A. RESULTS: Leveraging the inhibitory effect of m6A on primer elongation and uncomplicated feature of the isothermal exponential amplification reaction (IEXPAR), we have developed an extension-based IEXPAR (E-IEXPAR). This approach requires just a single extension primer and one template, simplifying the design process in comparison to the more complex primer requirements of the LAMP methods. The reactions are conducted at constant temperatures, therby elimiating the use of thermal cycling that needed in PCR methods. By combining IEXPAR with an extension reaction, E-IEXPAR can identify m6A in RNA concentrations as low as 4 fM. We have also introduced a new analytical model to process E-IEXPAR results, which can aid to minimize the impact of unmodified adenine (A) on m6A measurements, enabling accurate m6A quantification in small mixed samples and cellular RNA specimens. SIGNIFICANCE AND NOVELTY: E-IEXPAR streamlines m6A detection by eliminating the need for intricate primer design and thermal cycling, which are common in current analytical methods. Its utilization of an extension reaction for the initial identification of m6A, coupled with a novel calculation model tailored to E-IEXPAR outcomes, ensures accurate m6A selectivity in mixed samples. As a result, E-IEXPAR offers a reliable, straightforward, and potentially economical approach for specifically assaying m6A in both biological function studies and clinical research.

Double blocking gap-filling-ligation coupled with cascade isothermal amplification for ultrasensitive quantification of N6-methyladenosine.

We developed a method for quantifying N6-methyladenosine at one-nucleotide resolution based on double blocking gap-filling-ligation and cascade isothermal amplification. This proposed method can detect as low as 1 fM target RNA, achieving selectivity up to approximately 100-fold between m6A and A, and has been successfully applied to the analysis of m6A at specific sites in cell samples.

Multiple thermocycles followed by LAMP with only two primers for ultrasensitive colorimetric viral RNA testing and tracking at single-base resolution.

Rapid, accurate and high throughput measurement of infectious viruses is an urgent need to prevent viral transmission. Loop-mediated isothermal amplification (LAMP) is an attractive isothermal amplification method for nucleic acid detection, especially for point-of-care (POC) testing, but it needs at least four primers and its sensitivity is also limited when integrating with visual detection methods. Herein, by designing only two primers to precisely recognize the four regions of the target, we developed a multiple thermocycles-based LAMP method (MTC-LAMP) for sensitive and specific testing and tracking of viral RNA. We also introduced a novel SYBR Green I (SG)-assisted stable colorimetric assay induced by the amplification products through the charge neutralization effect of positively charged SG toward gold nanoparticles (AuNPs). The ultralow nonspecific background of the double exponential amplification improved the detection sensitivity to near single-molecule level (1 aM, 3 copies in 5 µL solution), which was higher than RT-PCR and RT-LAMP. After adding AuNPs, a significant color difference between target and blank was immediately observed by naked eye. By introducing a peptide nucleic acid (PNA) clamp into our colorimetric MTC-LAMP assay, the specific distinguish of virus variants at single-base resolution was observed without the requirement of any equipment. This assay shows great potential for large-scale screening and tracking of the threatening viruses with ultrahigh sensitivity and pronounced colorimetric output, which is of great importance for pandemic control.

Specific multiplexed detection of mRNA splice variants based on size-coding DNA probes and universal PCR amplification.

Alternative messenger RNA (mRNA) splicing is a vital regulatory process during the gene expression of higher eukaryotes. The specific and sensitive quantification of disease-related mRNA splice variants in biological and clinical samples is becoming particularly important. Reverse transcription polymerase chain reaction (RT-PCR), the most classical strategy for the assay of mRNA splice variants, cannot avoid false positive signals, which poses a challenge to the specificity of mRNA splice variant detection. In this paper, by rationally designing two DNA probes with double recognition at the splice site and different lengths, different mRNA splice variants could generate amplification products of unique lengths. Combined with capillary electrophoresis (CE) separation, the product peak of the corresponding mRNA splice variant can be specifically detected, which can avoid false-positive signals caused by non-specific amplification of PCR, greatly improving the specificity of the mRNA splice variant assay. In addition, universal PCR amplification eliminates amplification bias caused by different primer sequences and improves quantitative accuracy. Furthermore, the proposed method can simultaneously detect multiple mRNA splice variants as low as 100 aM in a one-tube reaction and has been successfully applied to the assay of variants in cell samples, which will provide a new strategy for mRNA splice variant-based clinical diagnosis and research.

CRISPR/Cas13a induced exponential amplification for highly sensitive and specific detection of circular RNA.

Rapid, sensitive and specific determination of circular RNA (circRNA) is of great significance for both biological research and clinical diagnosis. Specific recognition of target circRNA is now facing major challenges due to the fact that large amount of corresponding linear RNA is coexisting and possesses the same sequences except the junction sequence of circRNA. Herein, we firstly utilize CRISPR/Cas13a to specifically recognize the unique junction sequence of target circRNA and innovatively develop a CRISPR/Cas13a induced exponential amplification assay for sensitive and specific detection of circRNA. A pair of stem-loop DNA primers are elaborately designed with a pair of complementary single-strand DNA and five uracil ribonucleotides as the cantilever at their 3' terminus. Once Cas13a recognizes target circRNA, the trans-cleavage activity of Cas13a can be activated and the uracil ribonucleotides will be cleaved. Thus, the 3' terminus of the stem-loop primers can extend along each other to generate a lot of double stem-loop DNAs which can initiate multiple loop-mediated isothermal amplification (LAMP). Taking advantage of the incessant cleavage activity of Cas13a and the high amplification efficiency of multiple LAMP reaction, as low as 1 fM target circRNA can be sensitively detected within 30 min. Due to the high specificity of Cas13a, the proposed assay has been successfully applied to the detection of circRNA in real biological samples without separation of corresponding linear RNAs. Moreover, the proposed assay has offered a versatile platform for the detection of all sequence-specific RNA targets, indicating that our CRISPR/Cas13a induced exponential amplification assay has great potential for the detection of RNA biomarkers in both fundamental studies and clinical diagnostics.

Multiple stem-loop primers induced cascaded loop-mediated isothermal amplification for direct recognition and specific detection of circular RNAs.

Circular RNAs (circRNAs), as a novel class of endogenous noncoding RNAs, play important roles in many biological functions, such as acting as microRNA sponges and cancer biomarkers. Rapid and sensitive detection of circRNAs will be of great benefit to better understand their biological functions especially in clinical diagnosis using circRNAs as biomarkers. Herein, by using multiple stem-loop primers (SLPs) to specifically recognize the unique junction site of circRNAs, we demonstrate a multiple SLPs-induced cascaded loop-mediated isothermal amplification (LAMP) assay which has the ability to directly and specifically discriminate circRNAs from homologous linear RNAs without any linear RNA removal procedure. Taking advantage of the displacement activity of Bst DNA polymerase, the rationally designed SLPs can directly recognize the target circRNA and generate a large amount of double stem-loop DNAs which can initiate a cascaded LAMP reaction with improved efficiency. The use of additional SLPs can accelerate the reaction rate; as a result, the LAMP reaction can be finished within 35 min which is much quicker than traditional LAMP. The proposed method can detect as low as 1 fM circRNA and has been successfully applied to the detection of circRNAs in total RNA samples extracted from cell lines.

Ultrasensitive homogeneous detection of microRNAs in a single cell with specifically designed exponential amplification.

We firstly developed an ultrasensitive method based on specifically designed exponential amplification for miRNA detection with simple operation in homogeneous solutions. The proposed assay can detect miRNAs at a concentration as low as 1 aM and can be successfully applied for routine miRNA detection in a single cell.

Ultrasensitive detection of circular RNA by accurate recognition of the specific junction site using stem-loop primer induced double exponential amplification.

Circular RNAs (circRNAs), as a class of newly emerging biomarkers, have shown to be associated with many fundamental life processes and diseases, especially cancer. However, various limitations in currently available detection methods have seriously restricted the development of the studies associated with circRNA's biological functions and the diagnosis of diseases by using circRNA as the biomarker. By specifically designing a pair of stem-loop primers (SLPs) exactly recognize the junction sequence of circRNA, we firstly establish a SLP induced double exponential amplification method for sensitive and specific detection of circRNA with the ability to directly discriminate circRNA from linear RNA. Through the extension of SLPs during thermo cycles, one circRNA can generate a large amount of double stem-loop structure DNA which can initiate the subsequent isothermal amplification, leading to the sensitive detection of as low as 10 aM circRNA which is the most sensitive method for circRNA detection up to now. The proposed method has successfully applied to the detection of circRNA in many kinds of cancer cells in homogeneous solution without any separation step.

Direct recognition and sensitive detection of circular RNA with ligation-based PCR.

Circular RNAs (circRNAs), which function as a kind of newly discovered biomarker, have been shown to play an important role in gene expression, signaling pathway and disease diagnosis. Accurate recognition and sensitive detection of sequence-specific circRNA of interest can advance our understanding of its biological functions and the diagnosis of human diseases. Herein, we have newly developed a highly sensitive and specific method to quantitatively detect circRNA through accurate ligation of two ingeniously designed DNA probes using splintR ligase at the junction site of circRNA, which can directly discriminate circRNA from the corresponding linear RNA. With PCR amplification of the ligated DNA probes, circRNA as low as 1 fM can be detected with the dynamic range spanning over five orders of magnitude. This method has been successfully applied to the detection of circRNA in total RNA samples extracted from cell lines.

A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction.

Sensitive and accurate detection of site-specific DNA methylation is of critical significance for early diagnosis of human diseases, especially cancers. Herein, for the first time we employ a novel methylation-dependent restriction endonuclease GlaI to detect site-specific DNA methylation in a highly specific and sensitive way by coupling with isothermal exponential amplification reaction (EXPAR). GlaI can only cut the methylated target site with excellent selectivity but leave the unmethylated DNA intact. Then the newly exposed end fragments of methylated DNA can trigger EXPAR for highly efficient signal amplification while the intact unmethylated DNA will not initiate EXPAR at all. As such, only the methylated DNA is quantitatively and faithfully reflected by the real-time fluorescence signal of the GlaI-EXPAR system, and the potential false positive interference from unmethylated DNA can be effectively eliminated. Therefore, by integrating the unique features of GlaI for highly specific methylation discrimination and EXPAR for rapid and powerful signal amplification, the elegant GlaI-EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy. The detection limit of methylated DNA target has been pushed down to the aM level and the whole detection process of GlaI-EXPAR can be accomplished within a short time of 2 h. More importantly, ultrahigh specificity is achieved and as low as 0.01% methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA. This GlaI-EXPAR is also demonstrated to be capable of determining site-specific DNA methylations in real genomic DNA samples. Sharing the distinct advantages of ultrahigh sensitivity, outstanding specificity and facile operation, this new GlaI-EXPAR strategy may provide a robust and reliable platform for the detection of site-specific DNA methylations with low abundances.

Genetic Association Between CDKN1B rs2066827 Polymorphism and Susceptibility to Cancer.

Much attention has been directed to the association between cancer risk and rs2066827 polymorphism of the CDKN1B gene. However, the results are indefinitive and inconclusive. This study was devised to evaluate the hypothesis that rs2066827 polymorphism is associated with the risk of cancer.Computer-based databases (EMBASE, PubMed, and CNKI) were used to seek all case-control studies evaluating rs2066827 polymorphism and susceptibility to cancer. The genetic risk was assessed by calculating pooled odds ratio (OR) with its corresponding 95% confidence interval (CI). Fixed-effects pooled ORs were calculated by the Mantel-Haenszel method (Ph > 0.05), and random-effects pooled ORs were estimated by the DerSimonian-Laird method (Ph < 0.05).Data on rs2066827 polymorphism and cancer risk were available for 9038 cancer cases and 11,596 controls participating in 17 studies. Carriage of a TG genotype was associated with a minor but significant decrease in the risk of cancer (pooled OR 0.92, 95% CI: 0.86-0.99; model, TG vs. TT). We observed a moderately decreased risk of ovarian cancer based on 1829 cases and 2868 controls (pooled OR 0.85, 95% CI: 0.74-0.97; model, TG vs. TT). A slightly deceased risk of cancer was also indicated in Caucasians consisting of 6707 cases and 8279 controls (pooled OR 0.91, 95% CI: 0.85-0.98; model, TG vs. TT).These data suggest that carriage of a TG genotype at rs2066827 polymorphism may be associated with decreased susceptibility to cancer, ovarian cancer in particular.

The role of O(6)-methylguanine-DNA methyltransferase polymorphisms in colorectal cancer susceptibility: a meta analysis.

OBJECTIVE: O(6)-methylguanine DNA methyl-transferase gene (MGMT) is a central DNA repair mechanism with a significant role in removing DNA damage caused by alkylating agents and inhibiting human oncogenesis. Two single polymorphisms in the MGMT gene, Leu84Phe and Ile143Val, have been reported to affect DNA repair capability and enzymic activity, thereby leading to formation of different cancers. In this work, we quantitatively assess the associations between MGMT polymorphisms and risk of colorectal cancer (CRC), as previous studies has implicated inconsistencies in their results. METHODS: Analysis was performed on all usable data collected from the eligible studies that were searched in multiple bibliographical databases (PubMed, SCOPUS, and Embase). RESULTS: We obtained studies on Leu84Phe and Ile143Val, providing 6,154 and 7,371 samples, respectively. In the analysis on Leu84Phe, the SNP presented no global association with CRC at both the genotypic and the allelic level, but a trend towards an increased or decreased risk was shown in the models examined. Stratification by ethnicity revealed a significant increase in risk of CRC related to the Phe/Phe genotype in Caucasian samples (homozygote genetic model: OR=1.70, 95% CI=1.06-2.72; recessive genetic model: OR=1.80, 95% CI=1.12-2.87). CONCLUSIONS: Based on the statistical data, our meta-analysis indicates that Leu84Phe polymorphism in the MGMT gene may predispose Caucasians to CRC.