From Single DNA Molecules to an Entire Virus: an Investigation with Quantitative Fluorescence Microscopy and X-Ray Reflectivity

This PhD thesis presents a fluorescence microscopy study about
deoxyribose nucleic acid (DNA) in confined geometries. DNA serves
as an established model polymer well described by polymer physics.
In a first project, the dynamics of the DNA ejection through the
tight viral shaft from the highly packed phage capsid was
investigated. In a second project DNA was manipulated by linear
substrate structures. During phage assembly the phage DNA is very
tightly packaged into the phage capsid. The question arises if the
internal pressure resulting from the high DNA density inside the
capsid is responsible for the complete or at least the partial DNA
transfer into the host during infection. This was studied for the
first time on single T5 phages whose DNA release can be triggered
by the receptor protein FhuA in vitro. After T5 phages had adsorbed
onto a microfluidic chamber, the ejected DNA was fluorescently
stained and stretched in a hydrodynamic flow. The length could
thereby be measured continuously. With this setup, we succeeded for
the first time in visualizing the dynamics of the DNA ejection in
real time. The DNA release is not an all-or-none process but occurs
in a stepwise fashion and at a rate reaching 75 000 bp/s. The
pauses in between steps can last for over half an hour. The length
distribution of the ejected DNA was analyzed and characteristic
peaks were found at positions that coincided with the position of
single-stranded interruptions (nicks) of the phage genome. DNA
molecules adsorbed onto cationic fluid lipid membranes remain
laterally diffusive. During this thesis rectangular grooves of a
periodicity of 1000 – 1200nm and a depth of 40 – 120nm were
imprinted into the thermoplastic “cyclic olefin copolymer” (COC).
This substrate was coated with a lipid membrane. The influence of
such a support on the conformational behavior of DNA is
investigated here. The periodically structured membranes revealed
the ability to stretch long DNA molecules. During a collaboration,
L. Golubovic provided us with a theory explaining the observed
process. The DNA stretching phenomenon can be elucidated in terms
of a curvature dependent potential energy attained by the adsorbed
DNA molecules. The properties of lipid membranes supported by the
hydrophobic solid polymer COC are investigated. Lipid layers were
prepared by vesicle fusion and solvent exchange from varying
amounts of 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP,
cationic) and 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC,
neutral). The influence of the membrane charge and also of the
preparation method were investigated. All lipid mixtures coated the
COC surface homogeneously forming a fluid membrane as verified by
quantitative fluorescence microscopy, the method of Continuous
Bleaching and Fluorescence Recovery After Photobleaching (FRAP).
The average diffusion constant was found to be 0.8 µm2/s which is
higher by a factor of 5 than the typical membrane diffusion
constant for hydrophilic supports. The vertical electron density
profile of the supported membranes was determined by synchrotron
reflectivity experiments using a special microfluidic chamber
adapted to reflectivity experiments. The x-ray data can not be
fitted as expected with a model assuming a monolayer. They are
however in agreement with a compressed and less densely packed
bilayer. The head-to-head distance is 29˚A, the density of the
headgroups is ρh = 0.34 e−/°A3 and of the tails ρt = 0.26 e−/°A3.