Reconstructing the Free Energy Profiles Describing the Switching Mechanism of a pH-Dependent DNA Nanodevice from ABMD Simulations

The pH-responsive behavior of six triple-helix DNA nanoswitches, differing in the number of protonation centers (two or four) and in the length of the linker (5, 15 or 25 bases), connecting the double-helical region to the single-strand triplex-forming region, was characterized at the atomistic level through Adaptively Biased Molecular Dynamics simulations. The reconstruction of the free energy profiles of triplex-forming oligonucleotide unbinding from the double helix identified a different minimum energy path for the three diprotic nanoswitches, depending on the length of the connecting linker and leading to a different per-base unbinding profile. The same analyses carried out on the tetraprotic switches indicated that, in the presence of four protonation centers, the unbinding process occurs independently of the linker length. The simulation data provide an atomistic explanation for previously published experimental results showing, only in the diprotic switch, a two unit increase in the pKa switching mechanism decreasing the linker length from 25 to 5 bases, endorsing the validity of computational methods for the design and refinement of functional DNA nanodevices.

Antigene and Antiproliferative Effects of Triplex-Forming Oligonucleotide (TFO) Targeted on hmgb1 Gene in Human Hepatoma Cells

Background: The high mobility group box 1 (hmgb1) is one of the frequently over-expressed genes whose aberrant expression is reported in a number of human cancers. Various strategies are underway to inhibit hmgb1 expression in cancer cells having considerable therapeutic value. Objective: The present work involves selective transcriptional inhibition of the hmgb1 gene using selective DNA triplex structure-based gene technology. Here, the promoter region of the hmgb1 gene at position (-183 to -165) from the transcription start site as a target was selected using bioinformatic tools. Methods: The DNA triplex formation by the DNA of the target gene and TFO was confirmed using UV absorption spectroscopy, Circular Dichroism, and Isothermal Calorimetry. Results: Treatment of HepG2 cell with specific Triplex-forming Oligonucleotide significantly downregulated HMGB1 expression level at mRNA and protein levels by 50%, while the classical anticancer drugs, actinomycin/ adriamycin as positive controls showed 65% and the combination of TFO and drug decreased by 70%. The anti-proliferative effects of TFO correlated well with the fact of accumulation of cells in the Go phase and apoptotic cell death. Further, the binding of anti-cancer drugs to hmgb1 is stronger in DNA triplex state as compared to hmgb1 alone, suggesting the combination therapy as a better option. Conclusion: Therefore, the ability of hmgb1 targeted triplex-forming oligonucleotide in combination with triplex selective anticancer drug holds promise in the treatment of malignancies associated with hmgb1 overexpression. The result obtained may open up new vistas to provide a basis for the rational drug design and searching for high-affinity ligands with a high triplex selectivity.

Systematic Evaluation of Hybridization Property and Photo-Crosslinking Ability of Modified Oligonucleotides Containing Benzophenone-Bearing Glycol Nucleoside Analogs

Antigene technology is the one strategy for the artificial regulation of gene expression by the formation of triple structure on triplex forming oligonucleotide (TFO) with dsDNA. For the enhancement of the thermal stability of triplex structure, photo-crosslinking reaction is attractive by the covalent bond formation between TFO and dsDNA. In this paper, we designed the novel TFOs containing benzophenone moiety as a photo-crosslinkable agent. Several types of glycol nucleoside analogs having glycol scaffold and benzophenone residues with different linker length were prepared, and the these were incorporated into TFOs. The thermal stability of triplex and the photo-crosslinking reaction efficiency of TFOs toward dsDNA was systematically evaluated.

A triplex-forming linear probe for sequence-specific detection of duplex DNA with high sensitivity and affinity

We have developed a d-threoninol-based triplex-forming oligonucleotide (TFO) linear probe that can fluorescently detect target double-stranded DNA in a sequence-specific manner by forming stable triplex with high signal/background ratio.

Gold-DNA nanosunflowers for efficient gene silencing with controllable transformation

The development of an efficient delivery system for enhanced and controlled gene interference–based therapeutics is still facing great challenges. Fortunately, the flourishing field of nanotechnology provides more effective strategies for nucleic acid delivery. Here, the triplex-forming oligonucleotide sequence and its complementary strand were used to mediate self-assembly of ultrasmall gold nanoparticles. The obtained sunflower-like nanostructures exhibited strong near-infrared (NIR) absorption and photothermal conversion ability. Upon NIR irradiation, the large-sized nanostructure could disassemble and generate ultrasmall nanoparticles modified with c-myc oncogene silencing sequence, which could directly target the cell nucleus. Moreover, the controlled gene silencing effect could be realized by synergistically controlling the preincubation time with the self-assembled nanostructure (in vitro and in vivo) and NIR irradiation time point. This study provides a new approach for constructing more efficient and tailorable nanocarriers for gene interference applications.