To examine the effect of the torsional constraints imposed on DNA substrates on Cas9 cleavage, we prepared con-strained DNA substrates using a DNA origami frame. By fixing the dsDNA at the connectors of the DNA frame, we created four different dsDNA substrates containing torsion-ally constrained or relaxed strands. We quantified the cleav-age of constrained and relaxed substrates by Cas9 with quantitative PCR. Moreover, we observed the Cas9/sgRNA complex bound to the DNA substrates and characterized the dissociation of the complex with high-speed AFM. The results revealed that the constrained non-target strand re-duced the cleavage efficiency of Cas9 drastically, whereas torsional constraints on the target strand had little effect on the cleavage. The present study suggests that highly ordered and constrained DNA structures could be obstacles for Cas9 and additionally provides insights in Cas9 dissociation at a single molecule level.

DNAzymes (Dz) 8-17 and 10-23 are two widely studied and well-characterized RNA-cleaving DNA catalysts. In an effort to further improve the understanding of the fragile interactions and dynamics of the enzymatic mechanism, this study examines the catalytic efficiency of minimally modified DNAzymes. Five single mutants of Dz8-17 and Dz10-23 were prepared by replacing the adenine residues in the corresponding catalytic cores with 3-deazaadenine units. Kinetic assays were used to assess the effect on the catalytic activity and thereby identify the importance of hydrogen bonding that arises from the N3 atoms. The results suggest that modifications at A15 and A15.0 of Dz8-17 have a significant influence and show a reduction in catalytic activity. Modification at each location in Dz10-23 results in a decrease of the observed rate constants, with A12 appearing to be the most affected with a reduction of ∼80% of kobs and ∼25% of the maximal cleavage rate compared to the wild-type DNAzyme. On the other hand, modification of A12 in Dz8-17 showed an ∼130% increase in kobs, thus unraveling a new potential site for the introduction of chemical modifications. A pH-profile analysis showed that the chemical cleavage step is rate-determining, regardless of the presence and/or location of the mutation. These findings point towards the importance of the N3-nitrogens of certain adenine nucleotides located within the catalytic cores of the DNAzymes for efficient catalytic activity and further suggest that they might directly partake in maintaining the appropriate tertiary structure. Therefore, it appears that minor groove interactions constitute an important feature of DNAzymes as well as ribozymes.

ABSTRACT:The generation of chemical alkylating agents from nitrosation of glycine and bile acid conjugates in the gastrointestinal tract is hypothesized to initiate carcinogenesis. O 6 -carboxymethylguanine (O 6 -CMG) is a product of DNA alkylation derived from nitrosated glycine. Although the tendency of the structurally related adduct O 6 -methylguanine to code for the misincoporation of TTP during DNA replication is well-established, the impact of the presence of the O 6 -CMG adduct in a DNA template on the efficiency and fidelity of translesion DNA synthesis (TLS) by human DNA polymerases (Pols) have hitherto not been described. Herein, we characterize the ability of the four human TLS Pols η, ι, κ and ζ and the replicative Pol δ to bypass O 6 -CMG in a prevalent mutational hot-spot for cancer. The results indicate that Pol η replicates past O 6 -CMG, incorporating dCMP or dAMP, whereas Pol κ exclusively performs error-free insertion and Pol ι displays the lowest fidelity. Additionally, we found that the subsequent extension step was carried out with high efficiency by TLS Pols η, κ and ζ, while Pol ι was unable to extend from a terminal mismatch. These results provide a first basis of O 6 -CMG-promoted base misincorporation by Y-and B-family polymerases potentially leading to mutational signatures associated with cancer.

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology. Video LinkThe video component of this article can be found at

Repair of airway epithelium after viral infection involves migration of epithelial cells to cover injured, denuded areas. We determined whether viral infection reduces the capability of bronchial epithelial cells to migrate and to attach to extracellular matrix proteins. Inoculation of bovine bronchial epithelial cells in vitro with bovine herpesvirus-1 reduced their ability to migrate in two different assays of cell migration. When attachment assays were performed, fewer cells attached to both control wells and matrix protein-precoated wells, suggesting that general mechanisms of adherence to substrates were altered by viral infection. Focal contact points of epithelial cells with the underlying matrix were evaluated with epifluorescence microscopy and monoclonal antibodies to vinculin and alpha v, an integrin chain. Disruption of focal contact points was seen early after infection and was prevented by an inhibitor of viral DNA polymerase, phosphonoacetic acid. Cycloheximide did not cause similar disruptions of focal contacts at early time points. Viral infection thus has marked effects on the interactions of bronchial epithelial cells with extracellular matrix and the organization of matrix to cytoskeleton links. The effects appear to be dependent in part on viral replication in the cells and are not simply due to reductions in host cell protein synthesis.