An efficient, scarless, selection-free technology for phage engineering.

Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1-14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50-201 bases, for insertions of 20 bases, and for four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.[Figure: see text].

Inhibition of host cell division by T5 protein 008 (Hdi).

IMPORTANCE: We have identified a novel phage-encoded inhibitor of the major cytoskeletal protein in bacterial division, FtsZ. The inhibition is shown to confer T5 bacteriophage with a growth advantage in dividing hosts. Our studies demonstrate a strategy in bacteriophages to maximize their progeny number by inhibiting escape of one of the daughter cells of an infected bacterium. They further emphasize that FtsZ is a natural target for bacterial growth inhibition.

Highly active CRISPR-adaptation proteins revealed by a robust enrichment technology.

Natural prokaryotic defense via the CRISPR-Cas system requires spacer integration into the CRISPR array in a process called adaptation. To search for adaptation proteins with enhanced capabilities, we established a robust perpetual DNA packaging and transfer (PeDPaT) system that uses a strain of T7 phage to package plasmids and transfer them without killing the host, and then uses a different strain of T7 phage to repeat the cycle. We used PeDPaT to identify better adaptation proteins-Cas1 and Cas2-by enriching mutants that provide higher adaptation efficiency. We identified two mutant Cas1 proteins that show up to 10-fold enhanced adaptation in vivo. In vitro, one mutant has higher integration and DNA binding activities, and another has a higher disintegration activity compared to the wild-type Cas1. Lastly, we showed that their specificity for selecting a protospacer adjacent motif is decreased. The PeDPaT technology may be used for many robust screens requiring efficient and effortless DNA transduction.

Thou shalt not cleave DNA-only repress transcription: A compact Cas protein representing a new CRISPR-Cas subtype.

Wu et al.1 characterize Cas12m, a compact Cas protein that silences transcription without cleaving DNA and is a prototype protein of the novel CRISPR-Cas subtype V-M.

Cleavage of Abasic Sites in DNA by an Aminoquinoxaline Compound: Augmented Cytotoxicity and DNA Damage in Combination with an Anticancer Drug Chlorambucil in Human Colorectal Carcinoma Cells.

The elevated level of endogenous oxidative DNA damage and spontaneous deamination of DNA bases in cancer cells substantially increase the abasic sites in DNA via base excision repairs (BERs). Thus, the predominant BER pathway is a favorable target for cancer therapy. Interestingly, elevated levels of glutathione (GSH) in certain cancer cells, such as colon cancer, are associated with acquired resistance to several chemotherapeutic agents, which increase the difficulty for the treatment of cancer. Here, we have reported an ideal nitro group-containing monoquinoxaline DNA intercalator (1d), which is reduced into a fluorescent quinoxaline amine (1e) in the presence of GSH; concurrently, 1e (∼100 nM concentration) selectively causes the in vitro cleavage of abasic sites in DNA. 1e also binds to the tetrahydrofuran analogue of the abasic site in the nanomolar to low micromolar range depending on the nucleotide sequence opposite to the abasic site and also induces a structural change in abasic DNA. Furthermore, the amine compound (1e) augments the response of the specific bifunctional alkylating drug chlorambucil at a much lower concentration in the human colorectal carcinoma cell (HCT-116), and their combination shows a potential strategy for targeted therapy. Alone or in combination, 1d and 1e lead to a cascade of cellular events such as induction of DNA double-stranded breaks and cell arrest at G0/G1 and G2/M phases, eventually leading to apoptotic cell death in HCT-116 cells. Hence, the outcome of this study provides a definitive approach that will help optimize the therapeutic applications for targeting the abasic site in cancer cells.

A phage mechanism for selective nicking of dUMP-containing DNA.

Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.

Interaction of a Triantennary Quinoline Glycoconjugate with the Asialoglycoprotein Receptor.

Targeted intracellular delivery is an efficient strategy for developing therapeutics against cancer and other intracellular infections. Nonspecific drug delivery shows limited clinical applications owing to high dosage, cytotoxicity, nonspecific action, high cost, etc. Therefore, targeted delivery of less cytotoxic drug candidates to hepatocytes through ASGPR-mediated endocytosis could be an efficient strategy to surmount the prevailing shortcomings. In the present work, the gene encoding ASGPR-H1-CRD was amplified from Huh7 cells, cloned into pET 11a vector, and the ASGPR-H1-CRD protein was expressed and purified from E. coli. A novel triantennary galactose-conjugated quinoline derivative 4 was synthesized that demonstrates 17-fold higher binding affinity to isolated ASGPR-H1-CRD protein receptor (Kd ∼54 µM) in comparison to D-galactose (Kd ∼900 µM). Moreover, micro-calorimetric studies for the interaction of glycoconjugate 4 with ASGPR protein on live hepatocytes showed notable thermal response in case of ASGPR-containing Huh7 cells, in comparison to non-ASGPR Chang cells. These results might serve as an approach towards targeted delivery of small glycoconjugates to hepatocytes.

Quinoxaline derivatives disrupt the base stacking of hepatitis C virus-internal ribosome entry site RNA: reduce translation and replication.

RNA-biased small molecules with a monoquinoxaline core target the L-shaped structure of subdomain IIa of Hepatitis C virus internal ribosome entry site (IRES) RNA in proximity to the Mg2+ binding site. The binding event leads to the destacking of RNA bases, resulting in the inhibition of IRES-mediated translation and HCV RNA replication.

Intercalator-Induced DNA Superstructure Formation: Doxorubicin and a Synthetic Quinoxaline Derivative.

Small molecules that intercalate DNA have tremendous therapeutic potential. Typically, DNA intercalators do not alter the overall DNA double-helical structure, except locally at the intercalation sites. In a previous report, we showed that a quinoxaline-based intercalator with a mandatory benzyl substitution (1d) induced an unusually large circular dichroism signal upon DNA binding, suggesting the formation of intercalated DNA superstructures. However, no detailed structural studies have been reported. Using atomic force microscopy, we have probed the nature of the superstructure and report the formation of a plectonemically oversupercoiled structure of pBR322 plasmid DNA by 1d, where close association of distant DNA double-helical stretches is the predominant motif. Without the benzyl moiety (1a), no such DNA superstructure was observed. Similar superstructures were also observed with doxorubicin (dox), a therapeutically important DNA intercalator, suggesting that the superstructure is common to some intercalators. The superstructure formation, for both intercalators, was observed to be GC-specific. Interestingly, at higher concentrations (1d and dox), the DNA superstructure led to DNA condensation, a phenomenon typically associated with polyamines but not intercalators. The superstructure may have important biological relevance in connection to a recent study in which dox was shown to evict histone at micromolar concentrations.

The Benzyl Moiety in a Quinoxaline-Based Scaffold Acts as a DNA Intercalation Switch.

Quinoxaline antibiotics intercalate dsDNA and exhibit antitumor properties. However, they are difficult to synthesize and their structural complexity impedes a clear mechanistic understanding of DNA binding. Therefore design and synthesis of minimal-intercalators, using only part of the antibiotic scaffold so as to retain the key DNA-binding property, is extremely important. Reported is a unique example of a monomeric quinoxaline derivative of a 6-nitroquinoxaline-2,3-diamine scaffold which binds dsDNA by two different modes. While benzyl derivatives bound DNA in a sequential fashion, with intercalation as the second event, nonbenzyl derivatives showed only the first binding event. The benzyl intercalation switch provides important insights about molecular architecture which control specific DNA binding modes and would be useful in designing functionally important monomeric quinoxaline DNA binders and benchmarking molecular simulations.