318. Cationic Lipids/Ligands Induce DNA Helix Denaturation and Alter Transcriptional Activity

Abstract

Top of pageAbstract Cationic lipid-based formulations are promising gene delivery agents owing to their simplicity and self organizing properties. Although the physical properties of cationic lipids have been extensively worked out, the resultant low transfection efficiency observed in gene therapy protocols is ambiguous. We have investigated the effect of several cationic liposomes, namely 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), on transcription by Escherichia coli RNA polymerase at DNA templates complexed with the cationic lipid. The observed stimulation in transcription at excess DNA negative charges over lipid positive charge prompted us to investigate in detail the basic nature of Cationic Lipid DNA Complexes (CLDC) by absorbance spectroscopy and circular dichroism. Cetyl tri-methyl ammonium bromide (CTAB), a single chain cationic amphiphile and polyethylenimine (PEI), a cationic polymer, and other cationic lipids also bring about similar changes albeit to a lesser extent. The observed stimulation in transcription motivated us to explore in detail the molecular nature of CLDC by KMnO4 probing. Interestingly, thymidines followed by a purine showed higher susceptibility to cationic lipid/ligand-mediated melting of DNA. Other water-soluble cationic ligands such as polylysine, protamine and polyethyleneimine also demonstrated melting of the DNA but with variations. Similarly small cations such as spermine, spermidine and CTAB also rendered the DNA susceptible to modification by KMnO4. We present direct proof for melting of DNA upon interaction with cationic lipids/ligands. Structural changes subsequent to binding of cationic lipids/ligands to DNA may lead to instability and formation of DNA bubbles in double-stranded DNA. These observations would help in describing the CLDC completely.