SENSORS AND ACTUATORS B-CHEMICAL
·
2020
·
IF 7.1
Kaiwu Zhang,Xuan Wang,Caicheng Long,Jin Xu,Zixin Jiang,Bo Feng,Peng Zhang,Junjie Fei,Taiping Qing
DNA templated fluorescent copper nanoclusters (DNA-CuNCs) have demonstrated enormous potential for biochemical analysis. However, most of them are focused on DNA-related sensing. Here, we designed a hairpin DNA/RNA chimeric chain for the formation of fluorescent CuNCs and further applied it to detect reverse transcription-associated ribonuclease H (RNase H). The DNA/RNA chimera templated copper nanoclusters (DNA/RNA-CuNCs) exhibited similar spectral properties with DNA-CuNCs. In the presence of RNase H, the RNA segment in chimera is hydrolyzed and release short oligonucleotides, which fail to synthsize fluorescent CuNCs. The capabilities for RNase H detection from complex samples and screening of inhibitors were verified. This strategy exhibited high sensitivity for RNase H with a detectable minimum concentration of 5.5 × 10 −4 U mL -1 , which is lower than the developed fluorescent methods. This work will open up new thoughts to design new template for metal nanoclusters and broaden their application in RNA-related sensing.
研究领域:
生物化学
·
分子生物学
·
分析化学
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产品关联
DOI
高影响因子
Vaccines
·
2026
·Vol. 14
·期 7
·581
·
IF 3.5
Ren Yang,Xiaohong Wu,Xiaowei Zhang,Shengqing Fu,Kaiping Gu,Zhe Lv,Xiaoli Li,Qunying Mao
Background:The m7G cap structure, which mimics the natural cap of eukaryotic mRNA, is a critical determinant of mRNA vaccine efficacy, safety, and stability. However, its precise quantification remains challenging due to complex impurity profiles and the high physicochemical similarity between the target cap and related impurities. Although liquid chromatography mass spectrometry (LC-MS) is widely employed for this purpose, current methodologies still face significant limitations, including labor-intensive sample preparation, low analytical throughput, poor reproducibility in quantifying low-level impurities, and a lack of universally applicable strategies across diverse mRNA vaccine platforms.Methods: We systematically optimized sample preparation and LC-MS detection workflows. RNase H-mediated cleavage was compared with DNAzymes, guide DNA probes were rationally designed, and thermostable RNase H was introduced for one-step denaturation and cleavage. To establish an accurate, efficient, and universal sample preparation workflow. Chromatographic conditions were optimized using an ion-pairing reagent system to suppress ESI-MS metal adducts. Eliminating sample purification improves recovery, reduces manual handling errors, and boosts assay efficiency.Results: Through optimally designed guide DNA probes, RNase H cleavage specificity reached ≥98% with high cleavage efficiency, offering higher efficiency than DNAzyme. Furthermore, the incorporation of thermostable RNase H enabled a single-step workflow combining high-temperature denaturation and site-specific cleavage, substantially streamlining sample preparation. On the chromatographic side, optimization of the ion-pairing reagent system effectively suppressed metal adduct formation in electrospray ionization mass spectrometry (ESI-MS). This advancement enabled direct injection of the 5′ cap fragments without purification, achieving high-recovery quantification while demonstrating broad compatibility across mainstream LC-MS platforms. The optimized assay reduces the total analytical workflow from 4~6 h to under 1.5 h.Conclusions:Combining high accuracy, robustness, and broad platform compatibility, this method offers a universal, high-throughput analytical solution for mRNA vaccine quality control and continuous process development.
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DOI
高影响因子
ANALYST
·
2015
·Vol. 140
·期 13
·4566-4575
·
IF 4.107
Jacky F. C. Loo,S. S. Wang,F. Peng,J. A. He,L. He,Y. C. Guo,D. Y. Gu,H. C. Kwok,S. Y. Wu,H. P. Ho,W. D. Xie,Y. H. Shao,S. K. Kong
As in all RNA viruses, influenza viruses change and mutate constantly because their RNA polymerase has no proofreading ability. This poses a serious threat to public health nowadays. In addition, traditional pathogen-based detection methods may not be able to report an infection from an unknown type or a subtype of virus if its nucleotide sequence is not known. Because of these factors, targeting host microRNA signatures may be an alternative to classify infections and distinguish types of pathogens as microRNAs are produced in humans shortly after infection. Although this approach is in its infant stage, there is an urgent need to develop a rapid reporter assay for microRNA for disease control and prevention. As a proof of concept, we report herein for the first time a non-PCR MARS (MicroRNA-RNase-SPR) assay to detect the microRNA miR-29a-3p from human subjects infected with influenza virus H1N1 by surface plasmon resonance (SPR). In our MARS assay, RNase H is employed to specifically hydrolyze the RNA probes immobilized on the gold surface where they hybridize with its cognate target cDNAs miR-29a-3p, where it was formed from reverse transcription with mature miR-29a-3p specific stem-looped primers. After the digestion of the RNA probe by RNase H, the intact cDNA was released from the RNA–DNA hybrid and bound to a new RNA probe for another enzymatic reaction cycle to amplify signals. With assay optimization, the detection limit of our MARS assay for miR-29a-3p was found to be 1 nM, and this new assay could be completed within 1 hour without thermal cycling. This non-PCR assay with high selectivity for mature microRNA provides a new platform for rapid disease diagnosis, quarantine and disease control.
研究领域:
病毒学
·
分子生物学
·
诊断学
·
公共卫生
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）