Binding of protoberberine alkaloid coralyne with double stranded poly(A): a biophysical study.
Recognition of double stranded ribonucleic acid is a critical event in many biological pathways such as trafficking, editing and maturation of mRNA, interferon antiviral response and RNA interference. In the context of probing double stranded RNA binding small molecules, the interaction of the antitumor protoberberine alkaloid coralyne with double stranded poly(A) has been studied by various biophysical techniques. Typical hypochromic and bathochromic shifts in the absorption spectrum and appreciable quenching of the intrinsic fluorescence of coralyne indicated the strong affinity of coralyne to poly(A). The corresponding intrinsic binding constant evaluated from Scatchard analysis was in the order of 10(5) M(-1). The strong binding was further characterized by significant polarization of the alkaloid fluorescence and stabilization of poly(A) helix against thermal strand separation. The binding process was manifested by remarkable perturbation of the intrinsic circular dichroic spectrum of poly(A) with concomitant generation of optical activity in the bound alkaloid molecules that are otherwise achiral. Job plot analysis showed the binding stoichiometry of the interaction process to be two base pairs per alkaloid molecule. The energetics of the strong interaction was studied by isothermal titration and differential scanning calorimetric techniques that suggested the binding to be exothermic and favoured by both negative enthalpy and positive entropy changes. All these results, together with the Stern-Volmer quenching experiment in fluorescence, revealed the molecular details of the intercalation of coralyne into poly(A) duplex leading to its potential use as an agent in gene regulation in eukaryotic cells.
Giri
Prabal
P
Biophysical Chemistry Laboratory, Indian Institute of Chemical Biology, Kolkata, India. prabal_rs@iicb.res.in
Kumar
Gopinatha Suresh
GS
D001600:Berberine Alkaloids / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D002942:Circular Dichroism; D015394:Molecular Structure; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D001600:Berberine Alkaloids; D012333:RNA, Messenger; C000666:coralyne