Sp1-mediated miR-193b suppresses atopic dermatitis by regulating HMGB1.

Atopic dermatitis (AD) is a chronic and recurrent inflammatory skin disease. Keratinocyte dysfunction plays a central role in AD development. MicroRNA is a novel player in many inflammatory and immune skin diseases. In this study, we investigated the potential function and regulatory mechanism of miR-193b in AD. Inflamed human keratinocytes (HaCaT) were established by tumor necrosis factor (TNF)-α/interferon (IFN)-γ stimulation. Cell viability was measured using MTT assay, while the cell cycle was analyzed using flow cytometry. The cytokine levels were examined by enzyme-linked immunosorbent assay. The interaction between Sp1, miR-193b, and HMGB1 was analyzed using dual luciferase reporter and/or chromatin immunoprecipitation (ChIP) assays. Our results revealed that miR-193b upregulation enhanced the proliferation of TNF-α/IFN-γ-treated keratinocytes and repressed inflammatory injury. miR-193b negatively regulated high mobility group box 1 (HMGB1) expression by directly targeting HMGB1. Furthermore, HMGB1 knockdown promoted keratinocyte proliferation and inhibited inflammatory injury by repressing nuclear factor kappa-B (NF-κB) activation. During AD progression, HMGB1 overexpression abrogated increase of keratinocyte proliferation and repression of inflammatory injury caused by miR-193b overexpression. Moreover, transcription factor Sp1 was identified as the biological partner of the miR-193b promoter in promoting miR-193b expression. Therefore, Sp1 upregulation promotes keratinocyte proliferation and represses inflammatory injury during AD development via promoting miR-193b expression and repressing HMGB1/NF-κB activation.

Solution structure of a thrombin binding aptamer complex with a non-planar platinum(ii) compound.

Thrombin Binding Aptamer (TBA) is a monomolecular well-defined two G-tetrad antiparallel G-quadruplex DNA that inhibits the activity of human α-thrombin. In this report, we synthesized a quasi-cross-shaped platinum(ii) compound (L'2LPt) with one cyclometalated and two carbene ligands. We found L'2LPt has selective affinity to bind the TBA G-quadruplex. A fibrinogen clotting assay revealed that L'2LPt can abrogate the inhibitory activity of TBA against thrombin. We solved the 1 : 1 L'2LPt-TBA complex structure by NMR, which revealed a unique self-adaptive property of L'2LPt upon binding to TBA. In the complex, a carbene ligand of L'2LPt rotates to pair with the cyclometalated ligand to form a plane stacking over half of the TBA G-tetrad and covered by lateral TT loops. It is notable that the heavy atom Pt stays out of the G-tetrad. Meanwhile, the other carbene ligand remains relatively perpendicular and forms a hydrogen bond with a guanine to anchor the L'2LPt position. This structure exhibits a quasi-cross-shaped Pt(ii) compound bound to the G-quadruplex with an unusual "wall-mounted" binding mode. Our structures provide insights into the specific recognition of antiparallel G-quadruplex DNA by a self-adaptive Pt(ii) compound and useful information for the design of selective G-quadruplex targeting non-planar molecules.

G-quadruplex structural transition driven by a platinum compound.

G-quadruplex (G4) transitions play integral roles in regulating biological functions and can be modified by ligands. However, little is known about G4 transitions. Herein, we reveal distinct pathways of a platinum(II) compound Pt-phen converting parallel-stranded MYC G4 to a hybrid-type structure. Three NMR structures, 1:1 5'-end binding, 1:1 3'-end binding and 2:1 Pt-phen-MYC G4 complexes, were determined by NMR. We find that Pt-phen drives G4 transition at a low ratio. Under physiological 100 mM K+ conditions, a significant stable hydrogen-bonded T:T:A triad is formed at 3'-end of hybrid-type Myc1234, and consequently, Pt-phen first binds the 5'-end to form a 1:1 5'-end binding complex and then disrupts the 3' T:T:A triad and binds 3'-end to form a 2:1 complex with more Pt-phen. Remarkably, the G4 transition pathway is different in 5 mM K+ with Pt-phen first binding the 3'-end and then the 5'-end. 'Edgewise-loop and flanking/ligand/G-tetrad' sandwich structure formation and terminal T:T:A triad stabilization play decisive roles in advancing and altering transition pathways. Our work is the first to elucidate the molecular structures of G4 transitions driven by a small molecule. The ligand-driven G4 transition is a dynamic process that includes a quick G4 transition and multiple complexes formation.

Selectivity and Targeting of G-Quadruplex Binders Activated by Adaptive Binding and Controlled by Chemical Kinetics.

G-quadruplexes (G4s) are prevalent in oncogenes and are potential antitumor drug targets. However, binding selectivity of compounds to G4s still faces challenges. Herein, we report a platinum(II) complex (Pt1), whose affinity to G4-DNA is activated by adaptive binding and selectivity controlled by binding kinetics. The resolved structure of Pt1/VEGF-G4 (a promoter G4) shows that Pt1 matches 3'-G-tetrad of VEGF-G4 through Cl- -dissociation and loop rearrangement of VEGF-G4. Binding rate constants are determined by coordination bond breakage/formation, correlating fully with affinities. The selective rate-determining binding step, Cl- -dissociation upon G4-binding, is 2-3 orders of magnitude higher than dsDNA. Pt1 potently targets G4 in living cells, effectively represses VEGF expression, and inhibits vascular growth in zebrafish. We show adaptive G4-binding activation and controlled by kinetics, providing a complementary design principle for compounds targeting G4 or similar biomolecules.

Quantitative Detection of G-Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination.

G-quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G-quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE, which showed distinguishable fluorescence lifetime responses between G-quadruplexes and other DNA topologies, and fluorescence quantum yield (Φf ) enhancement upon G-quadruplex binding. We determined two NBTE-G-quadruplex complex structures with high Φf values by NMR spectroscopy. The structures indicated NBTE interacted with G-quadruplexes using three arms through π-π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φf values and lifetime response of NBTE upon G-quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G-quadruplex DNA in live cells with NBTE and found G-quadruplex DNA content in cancer cells is 4-fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.