Comb-Type Copolymer: Stabilization of Triplex DNA and Possible Application in Antigene Strategy

Abstract

By employing a reductive amination reaction between the epsilon-amino groups of poly(L-lysine) (PLL) and the reductive ends of the hydrophilic dextran (Dex) side chain, we have prepared different comb-type copolymers which varied in the degree of grafting and the length of the hydrophilic Dex chains. The resulting copolymers, poly(L-lysine)-graft-dextran (PLL-g-Dex), were tested for their ability to stabilize triplex DNA in vitro under physiologically relevant conditions. Thermal denaturation (UV-Tm) and circular dichroism experiments revealed that the graft copolymer with the higher degree of grafting of long Dex chains significantly increased the thermal stability of triplex structure of poly(dA). 2poly(dT) by more than 50 degreesC without affecting the transition between triplex and single-stranded DNA or the native structure of DNA. Of importance is that when triplex formation involving a 30-mer target duplex from rat alpha1 (I) collagen promoter was analyzed by an in vitro electrophoretic mobility shift assay, the graft copolymer also remarkably diminished potassium inhibition of the purine motif triplex formation up to 200 mM as well as pH-dependence of the pyrimidine motif triplex formation. Moreover the triplex-stabilizing efficiency of the copolymer was significantly higher than that of other oligocations like spermine and spermidine. We suggest that a molecular design of comb-type copolymers consisting of various types of polycation backbones (e.g., PLL) grafted with different hydrophilic side chains (e.g., Dex) is a novel strategy to create efficient triplex stabilizers that will certainly shed light on possible in vivo application of the antigene strategy.