Self-Assembly Nanostructure Induced by Regulation of G-Quadruplex DNA Topology via a Reduction-Sensitive Azobenzene Ligand on Cells.

The control of DNA assembly systems on cells has increasingly shown great importance for precisely targeted therapies. Here, we report a controllable DNA self-assembly system based on the regulation of G-quadruplex DNA topology by a reduction-sensitive azobenzene ligand. Specifically, three azobenzene multiamines are developed, and AzoDiTren is identified as the best G4 binder, which displays high affinity and specificity for G4 DNA. Moreover, the reduction-sensitive nature of the azobenzene scaffold allows AzoDiTren to induce a complete change of the G4 topology in a tissue-specific manner, even at high metal cation concentrations. On this basis, the AzoDiTren-induced G4 conformational switch achieves control of the self-assembly of G4-functionalized DNAs on cells. This strategy enables the regulation of G4 and DNA self-assembly by the bioreductant-responsive ligand.

Effect of Mini-PEGs Modification on the Enzymatic Digestion of D-Amino Acid-Containing Peptides under the Action of PROK.

It was previously reported that D-amino acid-containing peptides exhibited the ability to resist enzymatic hydrolysis. This study investigated the influence of mini-PEGs modification on enzymatic hydrolysis ability of D-amino acid-containing peptides. The results showed that PEGylation promoted enzymatic hydrolysis of the D-amino acid-containing peptide, especially, the cleavage rate of the D-amino acid-containing peptide 6-w with PEG3 modification at the N-ends was up to 17 times higher in the presence of proteinase K (PROK) compared to those without PEG3 modification. Moreover, analysis of the enzymatic cleavage sites demonstrated a similar cleavage pattern of the PEGylated D-amino acid-containing peptide to that of the unmodified peptide. The computational simulations further showed that the enhanced enzymatic hydrolysis ability can be attributed to the strong interaction between PROK and the peptide after PEG3 modification and the resulting formation of a mature catalytic triad structure.

Replicative bypass studies of l-deoxyribonucleosides in Vitro and in E. coli cell.

L-nucleosides were the most important antiviral lead compounds because they can inhibit viral DNA polymerase and DNA synthesis of many viruses, whereas they may lead to mutations in DNA replication and cause genomic instability. In this study, we reported the replicative bypass of L-deoxynucleosides in recombinant DNA by restriction enzyme-mediated assays to examine their impact on DNA replication in vitro and in E. coli cells. The results showed that a template L-dC inhibited Taq DNA polymerase reaction, whereas it can be bypassed by Vent (exo-) DNA polymerase as well as in cell replication, inserting correct nucleotides opposite L-dC. L-dG can be bypassed by Taq DNA polymerase and in E. coli cells, maintaining insertion of correct incoming nucleotides, and L-dG induced mutagenic replication by Vent (exo-) DNA polymerase. In contrast, L-dA can induced mutagenic replication in vitro and in E. coli cells. MD simulations were performed to investigate how DNA polymerase affected replicative bypass and mutations when D-nucleosides replaced with L-nucleosides. This study will provide a basis for the ability to assess the cytotoxic and mutagenic properties of the L-nucleoside drugs.

Redox manipulation of enzyme activity through physiologically active molecule.

The effective utility of physiologically active molecules is crucial in numerous biological processes. However, the regulation of enzyme functions through active substances remains challenging at present. Here, glutathione (GSH), produced in cells, was used to modulate the catalytic activity of thrombin without external stimulus. It was found that high concentrations of GSH was more conducive to initiate the cleavage of compound AzoDiTAB in the range of concentration used to mimic the difference between cancer and normal cells, which has practical implications for targeting cancel cells since GSH is overexpressed in cancer cells. Importantly, GSH treatment caused the deformation of G4 structure by cleaving AzoDiTAB and thus triggered the transition of thrombin from being free to be inhibited in complex biological systems. This work would open up a new route for the specific manipulation of enzyme-catalyzed systems in cancer cells.

New insight into enzymatic hydrolysis of peptides with site-specific amino acid d-isomerization.

The isomerization of l-amino acids in peptides and proteins into d-configuration under physiological conditions would affect the physiological dysfunction and caused protein conformational diseases. The presence of d-amino acids might change the higher-order structure of proteins and triggered abnormal aggregation. In order to better understand this phenomenon and promote degradation, we systematically studied the enzymatic hydrolysis of a series of peptides obtained by replacing l-amino acids in different positions of template peptide KYNETWRSED with d-amino acids under the action of Protease K. The results showed that, compared with normal peptide, isomerization of different amino acids had different effects on the anti-enzymatic hydrolysis of the peptides, especially d-tryptophan at position 6, which significantly inhibited enzymatic hydrolysis. The analysis of the peptide cleavage site revealed that the efficiency of enzymatic hydrolysis mainly depended on the isomerization of the amino acids at a specific site of the peptide cleavage. Further studies showed that the enzymatic hydrolysis of substrates could be facilitated by optimized reaction conditions such as temperature, pH, addition of metal ions, and change of buffer. In this way the accumulation of disease-associated d-amino acid containing polypeptides/proteins could be prevented.

Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase.

Due to highly enzymatic d-stereoselectivity, l-nucleotides (l-2'-deoxynucleoside 5'-triphosphates [l-dNTPs]) are not natural targets of polymerases. In this study, we synthesized series of l-thymidine (l-T)-modified DNA strands and evaluated the processivity of nucleotide incorporation for transcription by T7 RNA polymerase (RNAP) with an l-T-containing template. When single l-T was introduced into the transcribed region, transcription proceeded to afford the full-length transcript with different efficiencies. However, introduction of l-T into the non-transcribed region did not exhibit a noticeable change in the transcription efficiency. Surprisingly, when two consecutive or internal l-Ts were introduced into the transcribed region, no transcripts were detected. Compared to natural template, significant lags in NTP incorporation into the template T+4/N and T+7/N (where the number corresponds to the site of l-T position, and + means downstream of the transcribed region) were detected by kinetic analysis. Furthermore, affinity of template T+4/N was almost the same with T/N, whereas affinity of T+7/N was apparently increased. Furthermore, no mismatch opposite to l-T in the template was detected in transcription reactions via gel fidelity analysis. These results demonstrate the effects of chiral l-T in DNA on the efficiency and fidelity of RNA transcription mediated by T7 RNAP, which provides important knowledge about how mirror-image thymidine perturbs the flow of genetic information during RNA transcription and development of diseases caused by gene mutation.

Influence of d-Amino Acids in Beer on Formation of Uric Acid.

Excessive intake of beer could increase serum uric acid levels, leading to high risk of gout, which was previously attributed to high purine content in beer. Recent reports that purine-rich vegetables and bean products do not cause higher uric acid levels do not support this theory. Why excessive intake of beer could increase a high risk of gout has been unclear. Other factors affecting the accumulation of uric acid in the blood have been explored. Beer contains relatively high levels of d-amino acids due to the racemization of l-amino acids induced by food processing. d-amino acid was catalyzed by d-amino acid oxidase to produce H2O2, which is further oxidized in the presence of Fe2+ to produce hydroxyl radicals, resulting in DNA damage and formation of a large amount of purine bases, which are oxidized to uric acid by a series of enzymes. Some food ingredients, such as vitamins and I-, prompt d-amino acids to form uric acid. d-amino acids in beer are one of the key factors responsible for the increase in uric acid levels. The biological response of d-amino acids could explain gout occurrence in beer drinkers.