化学分析课程思政具体案例设计及思考

如何在化学核心课中融入课程思政元素,形成协同育人效应,是当前课程建设的一项重要任务。本文介绍了湖南大学分析化学教学团队在化学分析课程中的课程思政案例设计经验与思考,围绕化学分析各部分的教学特点,构建相应的教学案例,体现课程设计的教学目标和思政育人目标,同时提出具有普适性的课程思政设计理念。

Sensitive detection of DNA methyltransferase activity based on supercharged fluorescent protein and template-free DNA polymerization

DNA methylation, catalyzed by DNA methyltransferases(MTases), is a key component of genetic regulation, and DNA MTases have been regarded as potential targets in anticancer therapy. Herein, based on our previously developed DNA-mediated supercharged green fluorescent protein(Sc GFP)/graphene oxide(GO) interaction, coupled with methylation-initiated template-free DNA polymerization, we propose a novel fluorescence assay strategy for sensitive detection of DNA MTase activity. A hairpin DNA with a methylation-sensitive site and an amino-modified 3′-terminal(DNA-1) was designed and worked as a starting molecule. In the presence of DNA MTase, methylation-sensitive restriction endonuclease, and terminal deoxynucleotidyl transferase(Td T), DNA-1 can be sequentially methylated, cleaved, and further elongated. The resulting long DNA fragments quickly bind with Sc GFP and form the Sc GFP/DNA nanocomplex. Such nanocomplex can effectively protect Sc GFP from being adsorbed and quenched by GO. Without the methylation-initiated DNA polymerization, the fluorescence of Sc GFP will be quenched by GO. Thus, the DNA MTase activity, which is proportional to the amount of DNA polymerization products, can be measured by reading the fluorescence of Sc GFP/GO. The method was successfully used to detect the activity of DNA adenine methylation(Dam) MTase with a wide linear range(0.1–100 U/m L) and a low detection limit of 0.1 U/m L. In addition, the method showed high selectivity and the potential to be applied in a complex sample. Furthermore, this study was successfully extended to evaluate the inhibition effect of 5-fluorouracil on Dam MTase activity and detect Td T activity.

Coupling of proteolysis-triggered transcription and CRISPR-Cas12a for ultrasensitive protease detection

The efficient signal amplification capacity of several class 2 CRISPR-Cas systems with trans-cleavage activity has exhibited great value in molecular diagnostics,but its potential application for non-nucleic-acid targets is yet underdeveloped.Here,we deploy CRISPR-Cas system for the ultrasensitive detection of protease biomarkers by the coupling of proteolysis-triggered transcription.In this strategy,a protease-activatable RNA polymerase is adopted for the conversion of each protease-catalyzed proteolysis event into the output of multiple programable RNA sequences by in vitro transcription,and the transcribed RNA subsequently serves as the guide RNA of Cas12a proteins with trans-cleavage activity.The rational design of the transcribed RNA efficiently couples the signal conversion and amplification of proteolysis-triggered transcription and the self-signal amplification of CRISPR-Cas12a,resulting in a two-stage amplified detection of target protease.The versatility of this strategy has been demonstrated in the detection of protease biomarkers including MMP-2 and thrombin with femtomolar sensitivity,which is 5–6 orders of magnitude lower than that of the standard peptide-based methods.Moreover,the proposed method has been further applied in the analysis of MMP-2 secreted by different cancer cell lines as well the assessment of MMP-2 activity in clinical serum samples,providing a generic method for the ultrasensitive detection of protease biomarkers in biochemical research and clinical diagnosis.

Responsive MXene nanovehicles deliver CRISPR/Cas12a for boolean logic-controlled gene editing

Programmable and precise regulation of genetic information is crucial in bioengineering and biomedicine;however, it remains challenging to implement this objective. Here we deployed DNA-functionalized MXenes as a smart delivery system for spatiotemporally controllable genome editing. The MXene nanovehicles rationally integrated photothermal effect with nucleic acid strand displacement reaction, thereby allowing for the binary logic gate-controlled release of Cas ribonucleoprotein complexes in response to different input patterns of NIR light and nucleic acids. This system was highly programmable and could be harnessed to construct 2-input(AND, OR, and N-IMPLY) and 3-input(AND/OR and N-IMPLY/OR) logic gates for precise gene editing in mammalian cells. Moreover, an AND logic gate-controlled delivery system achieved selective induction of tumor cell death in a xenograft mice model using tissue-penetrating NIR light and cancer-relevant microRNA as the inputting cues.Therefore, the MXene nanovehicles adopted both the external and endogenous signals as the stimuli to precisely control gene editing under logic computation, presenting a helpful strategy for therapeutic genome editing.