We describe a new method for the assay of sequence-specific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNA footprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNA-binding proteins. We named this method as exonuclease III (ExoIII) protection assay with FRET probe. The FRET probe used in this assay was a duplex DNA which was designed to contain one FRET pair in the center and two flanking protein-binding sites. During protein detection, if a target protein exists, it will bind to the two protein-binding sites of the FRET probe and thus protect the FRET pair from ExoIII digestion, resulting in high FRET. However, if the target protein does not exist, the FRET pair on the naked FRET probe will be degraded by ExoIII, resulting in low FRET. Three kinds of recombinant transcription factors including NF-κB, SP1 and p50, and the target protein of NF-κB in HeLa cell nuclear extracts, were successfully detected by the assay. This assay can be extensively used in biomedical research targeted at DNA-binding proteins.

In this article, we checked the effect of 2,3-dimercaptosuccinic acid-coated Fe(3)O(4) nanoparticles on gene expression of mouse macrophage RAW264.7 cells and found that the transcription of several important genes related to intracellular iron homeostasis were significantly changed. We thus speculated that the cellular iron homeostasis might be disturbed by this nanoparticle through releasing iron ion in cells. To verify this speculation, we first confirmed the transcriptional changes of several key iron homeostasis- related genes, such as Tfrc, Trf, and Lcn2, using quantitative PCR, and found that an iron ion chelator, desferrioxamine, could alleviate the transcriptional alterations of two typical genes, Tfrc and Lcn2. Then, we designed and validated a method based on centrifugation for assaying intracellular irons in ion and nanoparticle state. After extensive measures of intracellular iron in two forms and total iron, we found that the intracellular iron ion significantly increased with intracellular total iron and nanoparticle iron, demonstrating degradation of this nanoparticle into iron ion in cells. We next mimicked the intralysosomal environment in vitro and verified that the internalized iron nanoparticle could release iron ion in lysosome. We found that as another important compensatory response to intracellular overload of iron ion, cells significantly downregulated the expressions of genes belonging to solute carrier family which are responsible for transferring many organic solutes into cells, such as Slc5a3 and Slc44a1, in order to prevent more organic solutes into cells and thus lower the intracellular osmosis. Based on these findings, we profiled a map of gene effects after cells were treated with this iron nanoparticle and concluded that the iron nanoparticles might be more detrimental to cell than iron ion due to its intracellular internalization fashion, nonspecific endocytosis.

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A new kind of polysiloxane‐supported ionogel is successfully designed via locking ionic liquids (ILs), 1‐ethyl‐3‐methylimidazolium bis(trifluoromethylsulfonyl)imide ([EMIM][Tf2N]), into poly(aminopropyl‐methylsiloxane) (PAPMS) grafted with [2‐(methacryloyloxy)ethyl] trimethylammonium chloride (METAC) in the presence of tannic acid (TA). The novel ionogel exhibits good mechanical and recovery properties, as well as high ionic conductivity (1.19 mS cm−1) at 25 °C. In addition, the totally physical dual‐crosslinked network based on ionic aggregates among METAC and the hydrogen bonds between PAPMS and TA provides excellent self‐healing ability, which allows the damaged ionogel to almost completely heal (≈83%) in 12 h at room temperature. Interestingly, the obtained ionogel also shows satisfactory adhesive behavior to various solid materials. Moreover, this novel ionogel can maintain its high ionic conductivity and recovery property even at subzero temperatures. Therefore, this polysiloxane‐supported ionogel is anticipated to be advantageous in flexible electronic devices such as sensors and supercapacitors, even at low temperatures.

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This study develops a new method for detecting target DNA based on Cas9 nuclease, which was named as CARP, representing CRISPR- or Cas9/sgRNAs-associated reverse PCR. This technique detects target DNA in three steps: (1) cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA; (2) ligating the cleaved DNA with DNA ligase; (3) amplifying target DNA with PCR. In the ligation step, the Cas9-cut target DNA was ligated into intramolecular circular or intermolecular concatenated linear DNA. In the PCR step, the ligated DNA was amplified with a pair of reverse primers. The technique was verified by detecting HPV16 and HPV18 L1 genes in nine different human papillomavirus (HPV) subtypes. The technique also detected the L1 and E6-E7 genes of two high-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells (HeLa and SiHa), in which no L1 and E6-E7 genes were detected in the HPV-negative cervical carcinoma cell, C-33a. By performing these proof-of-concept experiments, this study provides a new CRISPR-based DNA detection and typing method. Especially, the CARP method developed by this study is ready for the clinical HPV detection, which was supported by the final clinical sample detection. Graphical abstract CRISPR-associated reverse PCR (CARP) can be used to detect and type target DNA in a simple three-step procedure, cutting, ligation, and amplification.

Although some effective therapies have been available for cancer, it still poses a great threat to human health and life due to its drug resistance and low response in patients. Here, we develop a ferroptosis-based therapy by combining iron nanoparticles and cancer-specific gene interference. The expression of two iron metabolic genes (FPN and LCN2) was selectively knocked down in cancer cells by Cas13a or microRNA controlled by a NF-κB-specific promoter. Cells were simultaneously treated by iron nanoparticles. As a result, a significant ferroptosis was induced in a wide variety of cancer cells. However, the same treatment had little effect on normal cells. By transferring genes with adeno-associated virus and iron nanoparticles, the significant tumor growth inhibition and durable cure were obtained in mice with the therapy. In this work, we thus show a cancer therapy based on gene interference-enhanced ferroptosis.