Temperature-sensitive hydrogel loaded with DNase I alleviates epidural fibrosis in a mouse model of laminectomy

Excessive epidural fibrosis attached to the dura mater is the major cause of recurrent failed back surgery syndrome after spine surgery. Neutrophil extracellular traps (NETs) promote epidural fibrosis, raising the possibility that the DNA backbone of NETs may be a potential target in the therapy of epidural fibrosis. Human body temperature-sensitive hydroxypropyl chitin hydrogel solutions were prepared to encapsulate DNase I, which gradually decomposed in vivo. DNase I, which was released from temperature-sensitive hydrogels, destroyed the DNA backbone of NETs and dispersed the clustering of myeloperoxidase (MPO) in NETs. Evidence from MRI, H&E and Masson staining supported that hydroxypropyl chitin hydrogels loaded with DNase I were nontoxic and reduced epidural fibrosis. As expected, fibronectin in the wound was significantly abridged in the mice treated with hydrogels loaded with DNase I. Compared with the gelatin sponge absorbing DNase I, temperature-sensitive hydroxypropyl chitin hydrogels loaded with DNase I were more powerful in the therapy of epidural fibrosis. These results indicate that temperature-sensitive hydroxypropyl chitin hydrogels were effective in DNase I encapsulation and alleviation of epidural fibrosis in a mouse model of laminectomy.

The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, off-target mutagenesis by A3A has been readily detected in both cellular DNA and RNA, which has been shown to promote oncogenesis. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA, but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G) which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.

Amidine- and Amidoxime-Substituted Heterocycles: Synthesis, Antiproliferative Evaluations and DNA Binding

The novel 1,2,3-triazolyl-appended N- and O-heterocycles containing amidine 4–11 and amidoxime 12–22 moiety were prepared and evaluated for their antiproliferative activities in vitro. Among the series of amidine-substituted heterocycles, aromatic diamidine 5 and coumarine amidine 11 had the most potent growth-inhibitory effect on cervical carcinoma (HeLa), hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (SW620), with IC50 values in the nM range. Although compound 5 was toxic to non-tumor HFF cells, compound 11 showed certain selectivity. From the amidoxime series, quinoline amidoximes 18 and 20 showed antiproliferative effects on lung adenocarcinoma (A549), HeLa and SW620 cells emphasizing compound 20 that exhibited no cytostatic effect on normal HFF fibroblasts. Results of CD titrations and thermal melting experiments indicated that compounds 5 and 10 most likely bind inside the minor groove of AT-DNA and intercalate into AU-RNA. Compounds 6, 9 and 11 bind to AT-DNA with mixed binding mode, most probably minor groove binding accompanied with aggregate binding along the DNA backbone.

DNA bridges: A novel platform for single-molecule sequencing and other DNA-protein interaction applications

DNA molecular combing is a technique that stretches thousands of long individual DNA molecules (up to 10 Mbp) into a parallel configuration on surface. It has previously been proposed to sequence these molecules by synthesis. However, this approach poses two critical challenges: 1-Combed DNA molecules are overstretched and therefore a nonoptimal substrate for polymerase extension. 2-The combing surface sterically impedes full enzymatic access to the DNA backbone. Here, we introduce a novel approach that attaches thousands of molecules to a removable surface, with a tunable stretching factor. Next, we dissolve portions of the surface, leaving the DNA molecules suspended as ‘bridges’. We demonstrate that the suspended molecules are enzymatically accessible, and we have used an enzyme to incorporate labeled nucleotides, as predicted by the specific molecular sequence. Our results suggest that this novel platform is a promising candidate to achieve high-throughput sequencing of Mbp-long molecules, which could have additional genomic applications, such as the study of other protein-DNA interactions.

The origin and impeded dissemination of the DNA phosphorothioation system in prokaryotes

Phosphorothioate (PT) modification by the dnd gene cluster is the first identified DNA backbone modification and constitute an epigenetic system with multiple functions, including antioxidant ability, restriction modification, and virus resistance. Despite these advantages for hosting dnd systems, they are surprisingly distributed sporadically among contemporary prokaryotic genomes. To address this ecological paradox, we systematically investigate the occurrence and phylogeny of dnd systems, and they are suggested to have originated in ancient Cyanobacteria after the Great Oxygenation Event. Interestingly, the occurrence of dnd systems and prophages is significantly negatively correlated. Further, we experimentally confirm that PT modification activates the filamentous phage SW1 by altering the binding affinity of repressor and the transcription level of its encoding gene. Competition assays, concurrent epigenomic and transcriptomic sequencing subsequently show that PT modification affects the expression of a variety of metabolic genes, which reduces the competitive fitness of the marine bacterium Shewanella piezotolerans WP3. Our findings strongly suggest that a series of negative effects on microorganisms caused by dnd systems limit horizontal gene transfer, thus leading to their sporadic distribution. Overall, our study reveals putative evolutionary scenario of the dnd system and provides novel insights into the physiological and ecological influences of PT modification.

Signal-based optical map alignment

In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a laborious manual process, Bionano Genomics platforms now offer high-throughput, automated optical mapping based on chips packed with nanochannels through which unwound DNA is guided and the fluorescent DNA backbone and specific restriction sites are recorded. Although the raw image data obtained is of high quality, the processing and assembly software accompanying the platforms is closed source and does not seem to make full use of data, labeling approximately half of the measured signals as unusable. Here we introduce two new software tools, independent of Bionano Genomics software, to extract and process molecules from raw images (OptiScan) and to perform molecule-to-molecule and molecule-to-reference alignments using a novel signal-based approach (OptiMap). We demonstrate that the molecules detected by OptiScan can yield better assemblies, and that the approach taken by OptiMap results in higher use of molecules from the raw data. These tools lay the foundation for a suite of open-source methods to process and analyze high-throughput optical mapping data. The Python implementations of the OptiTools are publicly available through http://www.bif.wur.nl/.

Supercoiling and looping promote DNA base accessibility and coordination among distant sites

DNA in cells is supercoiled and constrained into loops and this supercoiling and looping influence every aspect of DNA activity. We show here that negative supercoiling transmits mechanical stress along the DNA backbone to disrupt base pairing at specific distant sites. Cooperativity among distant sites localizes certain sequences to superhelical apices. Base pair disruption allows sharp bending at superhelical apices, which facilitates DNA writhing to relieve torsional strain. The coupling of these processes may help prevent extensive denaturation associated with genomic instability. Our results provide a model for how DNA can form short loops, which are required for many essential processes, and how cells may use DNA loops to position nicks to facilitate repair. Furthermore, our results reveal a complex interplay between site-specific disruptions to base pairing and the 3-D conformation of DNA, which influences how genomes are stored, replicated, transcribed, repaired, and many other aspects of DNA activity.

Small-Molecule Inhibitors Overcome Epigenetic Reprogramming for Cancer Therapy

Cancer treatment is a significant challenge for the global health system, although various pharmacological and therapeutic discoveries have been made. It has been widely established that cancer is associated with epigenetic modification, which is reversible and becomes an attractive target for drug development. Adding chemical groups to the DNA backbone and modifying histone proteins impart distinct characteristics on chromatin architecture. This process is mediated by various enzymes modifying chromatin structures to achieve the diversity of epigenetic space and the intricacy in gene expression files. After decades of effort, epigenetic modification has represented the hallmarks of different cancer types, and the enzymes involved in this process have provided novel targets for antitumor therapy development. Epigenetic drugs show significant effects on both preclinical and clinical studies in which the target development and research offer a promising direction for cancer therapy. Here, we summarize the different types of epigenetic enzymes which target corresponding protein domains, emphasize DNA methylation, histone modifications, and microRNA-mediated cooperation with epigenetic modification, and highlight recent achievements in developing targets for epigenetic inhibitor therapy. This article reviews current anticancer small-molecule inhibitors targeting epigenetic modified enzymes and displays their performances in different stages of clinical trials. Future studies are further needed to address their off-target effects and cytotoxicity to improve their clinical translation.