Nuclease-induced stepwise photodropping (NISP) to precisely investigate single-stranded DNA degradation behaviors of exonucleases and endonucleases.

Here, we employed a fluorescence-based single molecule method called nuclease-induced stepwise photodropping (NISP) to measure in real time the DNA degradation mediated by mitochondrial genome maintenance exonuclease 1 (MGME1), a bidirectional single-stranded DNA (ssDNA)-specific exonuclease. The method detects a stepwise decrease in fluorescence signals from Cy3 fluorophores labeled on an immobilized DNA substrate. Using NISP, we successfully determined the DNA degradation rates of 6.3 ± 0.4 and 2.0 ± 0.1 nucleotides (nt) s-1 for MGME1 in the 5'-to-3' and 3'-to-5' directions, respectively. These results provide direct evidence of the stronger 5' directionality of MGME1, consistent with its established role in mitochondrial DNA maintenance. Importantly, when we employed NISP to investigate mung bean nuclease, an ss-specific endonuclease, we observed a markedly different NISP pattern, suggesting a distributive cleavage activity of the enzyme. Furthermore, we applied NISP to determine the ssDNA degradation behavior of the double-stranded-specific exonuclease, λ exonuclease. These findings underscore the capability of NISP to accurately and reliably measure the degradation of ssDNA by both exo- and endonucleases. Here, we demonstrate NISP as a powerful tool for investigating the ssDNA degradation behavior of nucleases at the single-molecule level.

Binding Behavior of Human Hepatoma-Derived Growth Factor on SMYD1.

The protein-induced fluorescence change technique was employed to investigate the interactions between proteins and their DNA substrates modified with the Cy3 fluorophore. It has been reported that the human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal proline-tryptophan-tryptophan-proline (PWWP) domain (the N-terminal 100 amino acids of HDGF) capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancer. This project investigated the specific binding behavior of HDGF, the PWWP domain, and the C140 domain (the C-terminal 140 amino acids of HDGF) sequentially using protein-induced fluorescence change. We found that the binding of HDGF and its related proteins on Cy3-labeled 15 bp SMYD1 dsDNA will cause a significant decrease in the recorded Cy3 fluorophore intensity, indicating the occurrence of protein-induced fluorescence quenching. The dissociation equilibrium constant was determined by fitting the bound fraction curve to a binding model. An approximate 10-time weaker SMYD1 binding affinity of the PWWP domain was found in comparison to HDGF. Moreover, the PWWP domain is required for DNA binding, and the C140 domain can enhance the DNA binding affinity. Furthermore, we found that the C140 domain can regulate the sequence-specific binding capability of HDGF on SMYD1.

Bioengineered Bacteriophage-Like Nanoparticles as RNAi Therapeutics to Enhance Radiotherapy against Glioblastomas.

Since glioblastomas (GBMs) are radioresistant malignancies and most GBM recurrences occur in radiotherapy, increasing the effectiveness of radiotherapy by gene-silencing has recently attracted attention. However, the difficulty in precisely tuning the composition and RNA loading in nanoparticles leads to batch-to-batch variations of the RNA therapeutics, thus significantly restricting their clinical translation. Here, we bioengineer bacteriophage Qß particles with a designed broccoli light-up three-way junction (b-3WJ) RNA scaffold (contains two siRNA/miRNA sequences and one light-up aptamer) packaging for the silencing of genes in radioresistant GBM cells. The in vitro results demonstrate that the cleavage of de novo designed b-3WJ RNA by Dicer enzyme can be easily monitored in real-time using fluorescence microscopy, and the TrQß@b-3WJLet-7gsiEGFR successfully knocks down EGFR and IKKα simultaneously and thereby inactivates NF-κB signaling to inhibit DNA repair. Delivery of TrQß@b-3WJLet-7gsiEGFR through convection-enhanced delivery (CED) infusion followed by 2Gy X-ray irradiation demonstrated that the median survival was prolonged to over 60 days compared with the 2Gy X-ray irradiated group (median survival: 31 days). Altogether, the results of this study could be critical for the design of RNAi-based genetic therapeutics, and CED infusion serves as a powerful delivery system for promoting radiotherapy against GBMs without evidence of systemic toxicity.