The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells.

Actinomycin D (ActD) is a natural antibiotic that inhibits the transcription of genes by interacting with a GC-rich duplex, a single-stranded or hairpin form of DNA, and then interfering with the action of RNA polymerase. In this study, we identified a novel molecular mechanism of anticancer activity of ActD as an oncogenic c-Myc promoter G-quadruplex binder. ActD selectively inhibits the elongation of oligonucleotides containing c-Myc promoter G-quadruplex sequence in PCR-stop assays. UV-vis spectroscopic and circular dichroism studies suggest that ActD interacts with c-Myc promoter G-quadruplex via a surface end stacking interaction, inducing a mixed-type conformation of the G-quadruplex. ActD selectively inhibits the cellular growth and synthesis of c-Myc mRNA in Ramos cells having the NHEIII(1) region in the translocated c-Myc gene. In addition, the results of promoter assays using two kinds of NHEIII(1) region mutants and wild-type constructs strongly support the idea that binding of ActD with G-quadruplex formed in the promoter region results in the reporter gene being turned off. Our study reveals a novel mechanism underlying the anticancer activity of ActD, whereby ActD interacts with oncogenic promoter G-quadruplex DNA to repress gene expression.

Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120–180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules.

Ultrasonography-guided percutaneous A1 pulley release for treatment of trigger finger reduces postoperative pain and complications, such as incomplete release, compared with a blind procedure.

This study demonstrates that posttraumatic heterotopic ossification, particularly in the posteromedial aspect of the capsule, is closely associated with loss of elbow flexion. Satisfactory restoration of elbow flexion can be obtained in the majority of patients by surgical release of the posterior band of the medial collateral ligament and excision of heterotopic bone.

BackgroundTo analyze clinical outcomes after anatomical reconstruction of distal radioulnar ligaments in patients with chronic post-traumatic instability of the distal radioulnar joint.MethodsAnatomical reconstruction was performed in 16 patients with subluxation or dynamic instability of distal radioulnar joint following trauma. Osteotomy was performed simultaneously in 10 patients with radial malunion. The average follow-up period was 18.9 months. For clinical outcome assessment, we performed the anteroposterior stress test, measured the range of motion and grip strength, and performed radiological examination. For assessment of the pain and function, we used the Patient Rated Wrist Evaluation, the Disabilities of the Arm, Shoulder and Hand, and the Modified Mayo Wrist Score.ResultsAnteroposterior stress test performed at the last follow-up showed normal in 12 patients, mild laxity in 3, and residual subluxation in one. The average Patient Rated Wrist Evaluation was 9.1 for pain and 11.2 for function. The average Disabilities of the Arm, Shoulder and Hand score was 10.5. The average Modified Mayo Wrist Score was 92.8; there were 10 excellent, 5 good, and 1 poor case. The average grip strength improved from 69.7 1b to 80.9 1b. A revision osteotomy was performed on the patient with residual subluxation in order to obtain normal alignment of the joint.ConclusionsAnatomical reconstruction of the distal radioulnar ligaments is recommended to restore distal radioulnar joint stability. In addition to ligament reconstruction, realignment of the distal radioulnar joint seems critical when the instability is combined with malunion of the radius.

G-quadruplex DNA formed in NHEIII1 region of oncogene promoter inhibits transcription of the genes. In this study, virtual screening combining pharmacophore-based search and structure-based docking screening was conducted to discover ligands binding to G-quadruplex in promoter region of c-MYC. Several hit ligands showed the selective PCR-arresting effects for oligonucleotide containing c-MYC G-quadruplex forming sequence. Among them, three hits selectively inhibited cell proliferation and decreased c-MYC mRNA level in Ramos cells, where NHEIII1 is included in translocated c-MYC gene for overexpression. Promoter assay using two kinds of constructs with wild-type and mutant sequences showed that interaction of these ligands with the G-quadruplex resulted in turning-off of the reporter gene. In conclusion, combined virtual screening methods were successfully used for discovery of selective c-MYC promoter G-quadruplex binders with anticancer activity.

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