Variation of the relative fluorescence quantum yields, anisotropy and visocisty.

<p>(A) Variation of the relative fluorescence quantum yield of BC (▪), BC1 (•), and BC2 (▴) in the presence of poly(U)_•<b>.</b>poly(A)<b>_*</b>poly(U) as a function of excitation wavelength. (B) A plot of the variation of anisotropy values versus P/D ratio for the complexation of BC (▪), BC1 (•), BC2 (▴) with poly(U)_•poly(A)<b>_*</b>poly(U). (C) A plot of increase in helix contour length (L/Lo) versus r for the complexation of BC (▪), BC1 (•), BC2 (▴) with poly(U)_•poly(A)<b>_*</b>poly(U) at 25±0.5°C.</p

Thermal melting profiles of triplex with BC and BC analogs.

<p>Poly(U)_•poly(A)<b>_*</b>poly(U) (30.0 μM) treated with (A) BC (B) BC1 and (C) BC2. Curves 1–6 represent D/P in the range 0–0.6.</p

Chemical structures of BC, analogs and the base triplet of poly(U)_•poly(A)_*poly(U).

<p>(A) BC (B) BC analogs and (C) triplex.</p

Absorption spectral change of alkaloids in presence of triplex.

<p>(A) BC (B) BC1 (C) BC2 each of 5.0 μM treated with increasing concentrations of poly(U)_•poly(A)<b>_*</b>poly(U). P/D saturation for BC, BC1 and BC2 were 28.0, 20.0 and 15.0, respectively. Inset: respective non-cooperative Scatchard isotherms of the binding.</p

Fluorescence spectra of BC and BC analogs in presence of triplex.

<p>(A) BC (B) BC1 (C) BC2 (2.0 μM each) treated with increasing concentration of RNA triplex. P/D saturation for BC, BC1 and BC2 were 32.0, 24.0 and 18.0, respectively. Inset: respective non-cooperative Scatchard plots of binding.</p

ITC Derived Thermodynamic parameters for the binding of BC, BC1 and BC2 with poly(U)_•poly(A)_*poly(U) triplex at 25°C.^a

a<p>All the data in this table are derived from ITC experiments and are average of four determinations. <i>K_a</i> and Δ<i>H</i>^o values were determined from ITC profiles fitting to Origin 7 software as described in the text. n is the site size which is the reciprocal of the stoichiometry N. The values of Δ<i>G</i>^o and TΔ<i>S</i>^o were determined using the equations Δ<i>G</i>^o  =  − RTln <i>K_a</i> and Δ<i>G</i>^o<i> = </i>Δ<i>H</i>^o − TΔ<i>S</i>^o. All the ITC profiles were fit to a model of single binding site.</p

Binding parameters for poly(U)•poly(A)<b>_*</b>poly(U) triplex-alkaloid complexation obtained from McGhee-von Hippel analysis of the Scatchard plot from the spectrophotometric and fluorimetric titration data at 25°C^a.

a<p>Average of four determinations in each case. <i>K_i</i> is the intrinsic binding constant to an isolated binding site and ‘n’ represents the number of excluded sites.</p

Circular dichroic spectral titration of triplex with BC and BC analogs.

<p>Titration data of poly(U)_•poly(A)<b>_*</b>poly(U) (30 µM) treated with (A) 0.0, 3.0, 6.0, 9.0, 12.0, 18.0 µM of BC (B) 0.0, 3.0, 6.0, 9.0, 12.0, 15.0, 18.0, 24.0 µM of BC1 and (C) 0.0, 3.0, 6.0, 9.0, 12.0, 15.0, 18.0, 24.0 µM of BC2. Inset: A magnified representation of the induced CD spectra in the region 300–400 nm.</p

Effect of alkaloids on the thermal stability of the poly(U)_•poly(A)<b>_*</b>poly(U) triplex^a.

a<p>Average from three experiments. Error limits for individual <i>T_m</i> measurements are estimated to be within ±0.5°C in. <i>T_m’</i> (3 → 2) and <i>T_m”</i> (2→1) are triplex to duplex and duplex to single stranded transitions respectively. Δ<i>T_m</i>  =  (<i>T_m</i> of triplex-alkaloid complex – <i>T_m</i> of triplex or duplex).</p

Biophysical Studies on the Effect of the 13 Position Substitution of the Anticancer Alkaloid Berberine on Its DNA Binding

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 (CH₂)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 (CH₂)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 (CH₂)₃. Structural and thermodynamic data on the DNA binding aspects of the substituted berberines are presented in comparison with berberine