ABSTRACT
Inducible CRISPR-dCas9 transcription system has become a powerful tool for transcription regulation and sensing. Here, we develop a new concept of small-molecule-mediated split-aptamer assembly for inducible CRISPR-dCas9 transcription activation, allowing quantitative detection and imaging of S-adenosyl methionine (SAM) in live cells. This inducible transcription system is designed by integrating one fragment of a split SAM aptamer to guide RNA (gRNA) and the other to MS2 arrays. SAM-mediated reassembly of the split fragments recruits an MCP-fused transcription activator to the gRNA-dCas9 complex, activating the expression of a near-infrared fluorescent protein for imaging. We demonstrate that this inducible transcription system achieves quantitative detection of SAM with high sensitivity in live cells. Our system shows that methionine adenosyltransferase 1A (MAT1A) and MAT2A can both catalyze SAM production in live cells and the SAM levels in cancer cells can be increased via upregulation of MAT1A mRNA by epigenetic inhibitors. This split-aptamer assembly strategy could afford a new approach for controlling the CRISPR-dCas9 system, enabling conditional transcription regulation in response to endogenous metabolites in live cells.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , RNA, Guide, Kinetoplastida , CRISPR-Cas Systems , Gene Expression Regulation , RNA, Guide, Kinetoplastida/genetics , S-Adenosylmethionine , Transcriptional ActivationABSTRACT
Croton fruit poisoning was successfully identified through a series of chromatographic technologies. A suspected compound named 2-methyl-butenoic acid was screened out using gas chromatography-mass spectrometry, and the hydrolysate of isoguanosine in soup sample was further detected by high performance liquid chromatography-tandem mass spectrometry. The identification of croton fruit poisoning was confirmed basing on these laboratory tests together with the clinical symptoms of the patients. Especially, the false-negative result and the interferences were successfully excluded through simulation test, spectral analysis and chromatographic separation technique. Such study is significant for croton poisoning identification and provides beneficial references for disposing untargeted food poisoning incident.
Subject(s)
Croton , Tandem Mass Spectrometry , Humans , Gas Chromatography-Mass Spectrometry/methods , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid , Fruit/chemistryABSTRACT
Single-nanoparticle inductively coupled plasma mass spectrometry (SP-ICP-MS) has demonstrated unique advantages for the detection of biological samples. However, methods for enzyme activity detection based on SP-ICP-MS technology have been rarely explored. Here we report the development of a novel SP-ICP-MS assay for uracil-DNA glycosylase (UDG) activity detection based on its ability to specifically recognize and remove uracil to induce the cleavage of the DNA probe. Our design allows the generation of single gold nanoparticles correlated to the specific enzymatic reaction for a highly sensitive SP-ICP-MS measurement. The developed assay enables sensitive UDG activity detection with a detection limit of 0.0003 U/mL. The cell lysate analysis by the developed assay reveals its applicability for the detection of UDG activity in real samples. It is envisioned that our design may provide a new paradigm for developing the SP-ICP-MS assay for enzyme activity detection in biological samples.
Subject(s)
Metal Nanoparticles , Uracil-DNA Glycosidase , DNA Probes , Gold , Limit of DetectionABSTRACT
The detection of nucleic acid is critical for clinic diagnostics. Single-nanoparticle inductively coupled plasma mass spectrometry (SP-ICPMS) has demonstrated unique advantages for nucleic acid detection. Here we report the development of a novel SP-ICPMS DNA assay based on a target-induced hybridization chain reaction to achieve controlled spherical nucleic acid assembly. The assembly process generated large gold nanoparticle aggregates, and the number of aggregates could be counted by SP-ICPMS, which was closely correlated to the concentration of the target DNA. This simple homogeneous assay could analyze DNA within the range of 5 fM to 10 pM with excellent selectivity and applicability for real sample analysis. It is envisioned that the developed approach might create a useful SP-ICPMS platform for biomolecule detection.
Subject(s)
DNA/analysis , Nanoparticles/chemistry , Nucleic Acid Hybridization , Mass Spectrometry , Particle SizeABSTRACT
Herein, a novel 16S rRNA detection platform was achieved by combining a sandwich hybridization reaction, a single-molecule magnetic capture, and single particle-inductively coupled plasma mass spectrometry amplification. The assay was developed for the direct detection of RNA from dangerous human pathogens and enabled absolute and high-precision quantification of a target with a detection limit of 10 fM.
Subject(s)
Biological Assay/methods , DNA Primers/genetics , Mass Spectrometry/methods , RNA, Ribosomal, 16S/analysis , RNA, Ribosomal, 16S/genetics , Animals , Base Sequence , DNA Primers/radiation effects , Escherichia coli O157/genetics , Food Contamination/analysis , Gold/chemistry , Light , Limit of Detection , Magnetic Phenomena , Metal Nanoparticles/chemistry , Milk/microbiology , Nucleic Acid Hybridization , Photochemistry/methodsABSTRACT
An efficient method was developed for the simultaneous determination of 10 industrial dyes (basic orange 2, basic orange 21, basic orange 22, acid orange II, auramine, basic rhodamine B and Sudan I-IV) in the foodstuffs using high-performance liquid chromatography coupled with diode array detector. Samples were extracted with acetonitrile and cleaned up on a solid phase extraction cartridge using HLB. The chromatographic separation was achieved on a C18 column using a mobile phase consisting of methanol and 10 mmol/L ammonium acetate with 0.1% formic acid by gradient elution. Good linearity (r ≥ 0.9993) was observed between 0.050 and 5.0 µg/mL. The limits of detection were in the range of 0.007-0.01 mg/kg, high recoveries (80.6-104%) and good reproducibility (1.1-5.7%) were obtained. Such method is simple, feasible and accurate, which can be applied to the quantification of 10 dyes in food samples.
Subject(s)
Chromatography, High Pressure Liquid/methods , Food Analysis/methods , Food Coloring Agents/analysis , Capsicum , Food Coloring Agents/chemistry , Food Coloring Agents/isolation & purification , Limit of Detection , Linear Models , Meat/analysis , Plant Preparations/analysis , Reproducibility of Results , Solid Phase Extraction/methodsABSTRACT
The present paper presents a novel method for simultaneous determination of the three pesticides, carbaryl, carbendazim and 1-naphthol in honey. The excitation-emission matrix fluorescence data from calibration and prediction samples composed a three-way data array and then were analyzed using the second-order calibration method based on the self-weighted alternating trilinear decomposition (SWATLD). The second-order advantage of the SWATLD-based second-order calibration method was exploited, which makes it possible that calibration can be performed even in the presence of unknown interferences. The method was applied to determine the pesticides in the complex matrix of the honey sample. After the calibration step the recoveries of carbendazim and 1-naphthol were satisfactory. The quantification of carbaryl using the second-order standard addition method (SOSAM) based on SWATLD and the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) were performed to validate the concentration of carbaryl in honey samples.
