Site-specific detection of circulating tumor DNA methylation in biological samples utilizing phosphorothioated primer-based loop-mediated isothermal amplification.

DNA methylation, a kind of epigenetic alteration, plays a vital role in tumorigenesis and offers a new class of targets for cancer treatment. DNA hypermethylation at the E-Box site (CACGTG, -288 bp) in the SLC22A2 promoter was related to multidrug resistance of renal cell carcinoma (RCC), which can provide the target for both treatment and monitoring. Herein, we developed a novel phosphorothioated primer based loop-mediated isothermal amplification (PS-LAMP) assay to detect circulating tumor DNA (ctDNA) methylation levels in E-Box sites in tumor tissue, urine, and plasma samples from patients with RCC. Bisulfite treatment converted methylated/unmethylated discrepancy to a single base discrepancy (C/U). PS-LAMP amplified the templates to a tremendous amount. One-step strand displacement (OSD) probe provided single base resolution in amplified products and finally realized the specific site methylation detection. Our proposed method provided a linear range from 0% to 100% for methylation levels and was available in samples at low concentrations (102 copies/µL). Visually observable colorimetric detection can be achieved by incorporating the OSD probe with gold nanoparticles (AuNP). Our assay performed better than traditional methods in biological samples with low ctDNA concentration. Further, we found a potential consistency of methylation levels between tumor tissue and plasma sample from the same patient (Spearman's ρ = 0.886, P = 0.019, n = 6). In general, this work provides a PS-LAMP assay combining OSD probes for site-specific methylation detection in various biological samples, offering a method for noninvasive detection.

Phosphorothioated and phosphate-terminal dumbbell (PP-TD) probe-based rapid detection of polynucleotide kinase activity.

Polynucleotide kinase (PNK), a bifunctional enzyme with 5'-kinase and 3'-phosphatase activities, plays an important role in DNA repair and is associated with various diseases. Here, we developed a primer-free, sensitive, and isothermal quantitative assay to detect PNK activity. In the presence of PNK, the 3'-phosphate group of the substrate was digested with 3'-OH, initiating the amplification reaction. Elongated dsDNA binds to the dsDNA-specific fluorescent dye EvaGreen, leading to a significant enhancement in fluorescence intensity. The limit of detection (LOD) of this method was 7.7 × 10-7 U µL-1, which is comparable or even superior to that of previously reported methods. This approach also showed good quantitative ability in complex cell lysates, indicating potential for biological sample analysis. Additionally, this facile and sensitive assay can be used to screen for PNK inhibitors. The proposed method provides a promising platform for sensitive PNK activity monitoring and inhibition screening for drug discovery and clinical treatment.

A self-assembly amplification strategy for ultra-sensitive detection of microRNA based on phosphorothioated probes.

Based on self-assembly amplification, we designed a novel microRNA (miRNA)-detection method with high specificity and sensitivity. Two unique DNA probes named Linker A and Linker B were modified with phosphorothioate (PS) at both ends. In the presence of the target miRNA, these two DNA probes were ligated together by T4 DNA ligase enzyme to form a dumbbell-shaped DNA. Then the dumbbell-shaped structure would be extended with Bst 2.0 DNA polymerase enzyme, triggering the strand displacement activity without using additional primers. These results revealed our method's ultralow detection limit (300 fM), excellent selectivity, simple operation, and capability to discriminate single-base mismatches. It is believed that this proposed approach would have great application potential in clinical diagnosis and other involved fields.

RT-LAMP assay combining multi-fluorescent probes for SARS-CoV-2 RNA detection and variant differentiation.

Simple and accurate testing tools for SARS-CoV-2 viral RNA detection are essential for the prevention of the spread of the virus and timely governmental actions. Herein, we present a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of ORF1ab and N gene fragments of SARS-CoV-2 in one pot. Using two primer sets and two molecular beacon (MB) probes respectively labelled with different fluorophore, positive results were obtained with a limit of detection of 20 and 2 copies/µL for ORF1ab and N gene fragments, respectively. Moreover, the RT-LAMP based assay was applied to detect single-site differences in S genes using two one-step displacement (OSD) probes targeting wild-type and mutant (P681R mutation was chosen as model) genes. Through that, the wild type strain and P681R mutant variant were well distinguished from each other, and a preliminary observation was also made on other mutations at this site such as P681H. The proposed method has high sensitivity for quantification and high specificity for mutation differentiation. In addition, it does not require accurate sophisticated thermal cycler instrumentation and can be used in clinical settings in resource-limited regions.

A Novel Helper qPCR Assay for the Detection of miRNA Using Target/Helper Template for Primer Formation.

A novel, simple, and sensitive quantitative polymerase chain reaction (qPCR) technology, which is termed as helper qPCR, was established to detect miRNA. In this assay, the target miRNA sequence was introduced as helper template for a reaction switch preforming two-step real-time qPCR strategy. Firstly, the reverse primer was reverse transcribed to form "mediator primer" after binding to the target miRNA. Then, the mediator primer was further extended to form "active template" with annealing to the mediator template. In the end, the active template was amplified and detected by the qPCR reaction system with the help of reverse and forward primers. The SYBR Green dye was used for fluorescence quantification, which is quicker and cheaper than the fluorescent probes, as the detection limit of this assay was 1 pM. This helper qPCR system can be used for different miRNAs detection by redesigning reverse primer for target, indicating this strategy could afford good performance in detecting multiple miRNAs and has a promising application prospect.

Recent advances in therapeutic nucleic acids and their analytical methods.

Therapeutic nucleic acids are various chemically modified RNA or DNA with different functions, which mainly play roles at the gene level. Owing to its accurately targeting at pathogenic genes, nucleic acid based therapeutics have a wide range of application prospects. Recently, the improvement on chemical synthesis and delivery materials accelerated the development of therapeutic nucleic acids rapidly. Up to now, 17 nucleic acid based therapeutics approved by Food and Drug Administration (FDA) or European Medicines Agency (EMA). The development of therapeutics raised higher requirements for analytical methods, both in quality control and in clinical research. The first part of this review introduces different classes of therapeutic nucleic acids, including antisense oligonucleotide (ASO), RNA interference (RNAi) therapy, mRNA, aptamer and other classes which are under research. The second part reviews the therapeutic nucleic acids commercialized from 2019 to now. The third part discusses the analytical methods for nucleic acid based therapeutics, including liquid chromatography-based methods, capillary gel electrophoresis (CGE), hybridization enzyme-linked immunosorbent assay (ELISA) and other infrequently used methods. Finally, the advantages and shortcomings of these methods are summarized, and the future development of analysis methods are prospected.

The characterisation of the in vitro metabolism and transport of 6-hydroxykynurenic acid, an important constituent of Ginkgo biloba extracts.

6-Hydroxykynurenic acid (6-HKA) is a nitrogen-containing phenolic acid compound in Ginkgo biloba leaves. The pharmacological activities of 6-HKA have been reported and shown that 6-HKA has the potential to become a therapeutic drug and may play an important role in the treatment of nervous system diseases. However, there are few studies on the drug metabolism and transport of 6-HKA. The aim of this study is to investigate the in vitro metabolism of 6-HKA and its interaction with multiple important drug transporters.The in vitro metabolism experiments in the present study demonstrate that 6-HKA might not undergo phase-I or phase-II metabolism in hepatic microsomes/S9 of rats. In addition, some drug transporters, including OAT1/3, OCT2, MDR1, OATP1B1, MATE1/2K and OCTN2, were investigated. The cellular uptake assays indicate that 6-HKA exhibits inhibition to the transport of classical substrates mediated by OAT3, OCT2, MATE2K and OCTN2 but has no significant effect on the transport of substrates mediated by MDR1, OAT1, OATP1B1 or MATE1. Further investigation of cellular accumulation assays shows that 6-HKA might be the substrate of OAT3, but not OCT2 or OCTN2. The bidirectional transport study suggests that 6-HKA is not a substrate of MDR1.The information about the in vitro metabolism of 6-HKA and the interaction between 6-HKA and some transporters will help us to better understand the pharmacokinetic properties of 6-HKA and provide reference for its pharmacodynamics, DDIs and drug-food interactions studies.

Reverse transcription-based loop-mediated isothermal amplification strategy for real-time miRNA detection with phosphorothioated probes.

A novel reverse transcription-based loop-mediated isothermal amplification (LAMP) strategy for miRNA detection has been developed. This method consists of two stem-loop probes inspired by the dumbbell-shaped amplicons and inner primers used in conventional LAMP reactions. Termed "terminal hairpin formation and self-priming" (THSP), this reaction incorporates phosphorothioated (PS) modifications to achieve DNA folding and extension without primers. The final signal is monitored by a sequence-specific detection probe, which minimizes the background noise. We suggest that our rapid, facile, and reliable LAMP method will be a promising candidate for detecting miRNA in biomedical applications.