The structural effects and thermodynamics of the DNA binding of six berberine analogues with alkyl chains of varying length and a terminal phenyl group at the C-13 position were investigated. All the analogues bound DNA noncooperatively in contrast to the cooperative binding of berberine. The binding affinity was higher and the effect of the chain length was only up to (CH(2))(3), after which the binding affinity decreased slightly. Intercalative binding with strong stabilization of the DNA helix was revealed. Binding resulted in the weakening of the base stacking with moderate conformational changes within the B-form. The binding was entropy driven in each case, the entropy contribution to the free energy increasing with the chain length up to the threshold (CH(2))(3). The complexation was dominated by nonpolyelectrolytic forces in each case; polyelectrolytic forces contributed only a quarter to the total free energy at 50 mM [Na(+)]. Overall, the phenylalkyl substitution at the C-13 position considerably enhanced the DNA binding and was highest for the analogue with (CH(2))(3). Structural and thermodynamic data on the DNA binding aspects of the substituted berberines are presented in comparison with berberine.

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Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

BackgroundBinding of two 9-O-(ω-amino) alkyl ether berberine analogs BC1 and BC2 to the RNA triplex poly(U)•poly(A)*poly(U) was studied by various biophysical techniques.Methodology/Principal FindingsBerberine analogs bind to the RNA triplex non-cooperatively. The affinity of binding was remarkably high by about 5 and 15 times, respectively, for BC1 and BC2 compared to berberine. The site size for the binding was around 4.3 for all. Based on ferrocyanide quenching, fluorescence polarization, quantum yield values and viscosity results a strong intercalative binding of BC1 and BC2 to the RNA triplex has been demonstrated. BC1 and BC2 stabilized the Hoogsteen base paired third strand by about 18.1 and 20.5°C compared to a 17.5°C stabilization by berberine. The binding was entropy driven compared to the enthalpy driven binding of berbeine, most likely due to additional contacts within the grooves of the triplex and disruption of the water structure by the alkyl side chain.Conclusions/SignificanceRemarkably higher binding affinity and stabilization effect of the RNA triplex by the amino alkyl berberine analogs was achieved compared to berberine. The length of the alkyl side chain influence in the triplex stabilization phenomena.

Interactions of the anionic surfactant sodium dodecyl sulfate (SDS) with the transport proteins bovine serum albumin (BSA) and human serum albumin (HSA) have been divulged using an external photoinduced proton transfer probe, norharmane (NHM). Steady-state fluorometry, time-resolved measurements, micropolarity analysis, circular dichroism (CD), and isothermal titration calorimetry (ITC) have been exploited for the study. With the gradual addition of SDS to the probe-bound proteins, the fluorometric responses of the different prototropic species of NHM exhibit an opposite pattern as to that observed while NHM binds to the proteins. The study reveals a sequential unfolding of the serum proteins with the gradual addition of SDS. ITC measures the heat changes associated with each step of the unfolding. ITC experiments, carried out at two different pH's, elucidate the nature of interaction between SDS and the two serum proteins. At a very high concentration of SDS, the external probe (NHM) is found to be dislodged from the protein environments to bind to the SDS micellar medium.