Structural Study of DNA Condensation Induced by Novel Phosphorylcholine-Based Copolymers for Gene Delivery and Relevance to DNA Protection

Abstract

Poly[2-(dimethylamino)ethyl methacrylate-b-2-methacryloyloxyethyl phosphorylcholine] (DMA-MPC) is currently under investigation as a new vector candidate for gene therapy. The DMA block has been previously demonstrated to condense DNA effectively. The MPC block contains a phosphorylcholine (PC) headgroup, which can be found naturally in the outside of the cell membrane. This PC-based polymer is extremely hydrophilic and acts as a biocompatible steric stabilizer. In this study, we assess in detail the morphologies of DNA complexes obtained using the diblock copolymer series DMA(x)MPC30 (where the mean degree of polymerization of the MPC block was fixed at 30 and the DMA block length was systematically varied) using transmission electron microscopy (TEM) and liquid atomic force microscopy (AFM). Both techniques indicate more compact complex morphologies (more efficient condensation) as the length of the cationic DMA block increases. However, the detailed morphologies of the DMA(x)MPC30-DNA complexes observed by TEM in vacuo and by AFM in aqueous medium are different. This phenomena is believed to be related to the highly hydrophilic nature of the MPC block. TEM studies revealed that the morphology of the complexes changes from loosely condensed structures to highly condensed rods, toroids, and oval-shaped particles as the DMA moiety increases. In contrast, morphological changes from plectonemic loops to flower-like and rectangular block-like structures, with an increase in highly condensed central regions, are observed by in situ AFM studies. The relative population of each structure is clearly dependent on the polymer molecular composition. Enzymatic degradation assays revealed that only the DMA homopolymer provided effective DNA protection against DNase I degradation, while other highly condensed copolymer complexes, as judged from TEM and gel electrophoresis, only partially protected the DNA. However, AFM images indicated that the same highly condensed complexes have less condensed regions, which we believe to be the initiation sites for enzymatic attack. This indicates that the open structures observed by AFM of the DNA complexation by the DMA(x)MPC30 copolymer series are closer to in vivo morphology when compared to TEM.

Electrophoresis of a toroid along the axis of a cylindrical pore

“…This includes all bacterial DNA, all plasmid DNA from within eukaryotic cells, and most viral DNA. Within living cells, DNA is highly condensed in toroidal arrays as compared with free DNA in solution, especially in sperm cells and viruses where gene transcription is inactive [1][2][3], and DNA condensation in vitro has attracted much attention in gene therapy [4]. Kong et al [5] determined the structure of the beta subunit in Escherichia coli and found that it was also a toroid, which contained a hole big enough to encircle double-stranded DNA, and proposed that the beta subunit acts as a sliding clamp to hole the polymerase III.…”

Critical behavior of megabase-size DNA toward the transition into a compact state

“…Similar effects on the appearance of parallel ordering have been described in previous reports. [56][57][58] However, the degree of parallel ordering has not yet been fully analyzed quantitatively at the single-molecule level.…”

Polycationic graft copolymers of poly(N-vinylpyrrolidone) as non-viral vectors for gene transfection

“…Systems such as PEO-b-PDMAEMA [26], Pluronic-b-PDMAEMA [27], PEG-modified PDMAEMA [28] and PDMAEMA-b-PMPC (poly (methacryloyloxyethyl phosphorylcholine)) [29] have been widely studied as potential gene delivery systems.…”