Molecular Transition Behavior of DNA-Lipid Films

It is well-known that naturally derived polyanions such as nucleic acids (RNA and DNA) can self-assemble with cationic lipids via electrostatic complexation. This complexation is driven thermodynamically by the release of counterions.[1] The structure of these complexes dispersed in water have been studied extensively by groups such as Safinya et al. and have been recognized as potentially useful in the field of gene delivery.[2] The structure of films in water is dominated by the nature of the lipid (e.g. DNA complexes with DDAB or DOPC tend to form lamellar structures whereas DNA complexes with DOPE tend to form inverted hexagonal structures). Within lamellar structured complexes the lipid assumes a bilayer formation and the DNA a B-form double helix. However amphiphilic lipids like DOPC and DOPE and their nucleic acid complexes are water soluble while cationic lipids like DDAB tend to form water insoluble complexes, depending on the length of the carbon chain tail. The cationic complexes are soluble in organic solvents like ethanol, isopropanol or chloroform and can be cast on a solid material such as Teflon or glass to obtain solid films.^[3],[4] These self-standing films have been characterized macroscopically by tensile properties and intercalation experiments. It has been reported that the DNA strands within these films can be aligned as the film is stretched, which has led to their application as a new type of anisotropically conductive material.[5] The tensile properties of these films are adjustable by mixing different kinds of nucleic acid (DNA and RNA) for the complex with DDAB[6] and are considered to be potentially useful as implants[7]. It was expected that these films have the same characteristic structure as the complexes in water. Thin films of the complexes have been prepared by layer by layer as well as Langmuir-Blodgett techniques and characterized by X-ray scattering spectroscopy. The state of the DNA within these films is a subject of controversy, as few papers have been able to propose a consistent structure (e.g. A-, B-, or C-form DNA).[8] Although some papers have suggested the DNA in such films is single stranded, the evidence is very thin as inappropriate controls are used, and often no structure is given to satisfactorily explain the state of the lipid.[9]

We focused in our work on the structure of DNA-DDAB films and found an unexpected model that is able to describe the behavior of the DNA and the lipid in the film[10]. Our DNA-DDAB films have been characterized by AFM, WAXS, SAXS, CD, fluorescence, and FT-IR spectroscopy. Initially we were surprised to learn that our films had a repeat unit spacing of 2.8 nm according to both our WAXS and AFM studies, as it was too small to accommodate both double stranded DNA (~2 nm) and a bilayer of DDAB (~2.4 nm experimentally).[11] However we also discovered that when immersed in water the repeat unit increases to the expected spacing of 4.4 nm and decreases to 2.8 nm as the film dries. This structural change can be monitored with FT-IR. We observe that the peaks corresponding to the paired and unpaired carbonyl stretches of guanine, cytosine, and thymine[12] increase and decrease depending on the environment of the film, suggesting that the structure of DNA in the film “switches” from single to double stranded as the film is wetted and reverses as it dries. We also observe from the IR peaks corresponding to the methyl endgroups of the DDAB lipid tails that DDAB likewise switches from a monolayer to a bilayer and back as the film is wetted and dried. We have also been able to observe the switch using fluorescent nucleic acid-intercalating dyes such as ethidium bromide, which fluoresce when the DNA is wet in the film and decay in signal as the film dries.

Based on these studies we propose a lamellar structure of the film consisting of monolayers of lipid paired with single stranded DNA molecules. We wish to further study the kinetics and mechanism of this structural switch, as well as the biodegrability of the film for further use as an implantable device.