Local conformation of confined DNA studied using emission polarization anisotropy
Conference poster, 2010
In nanochannels with dimensions smaller than the DNA radius of gyration, DNA will extend
along the channel. We investigate long DNA confined in nanochannels using fluorescence
microscopy and intercalated dyes. Studies of the dynamics and statics of the DNA extension
or position in such nanoscale confinements as a function of e.g. DNA contour length, degree
and shape of confinement as well as ionic strength have yielded new insights in the physical
properties of DNA with relevance for applications in genomics as well as fundamental
understanding of DNA packaging in vivo. Our work extends the field by not only studying the
location of the emitting dyes along a confined DNA molecule but also monitoring the
polarization of the emitted light. We use intercalating dyes (YOYO-1) whose emission is
polarized perpendicular to the DNA extension axis, and by measuring the emission polarized
parallel and perpendicular to the extension axis of the stretched DNA, information on the
local spatial distribution of the DNA backbone can be obtained. The results obtained are
analogous to linear dichroism (LD) but on a single-molecule level, and obtained in a highly
parallel fashion. We will discuss results in shallow (60 nm) and deep (180 nm) channels and
describe an example of how the technique can be used to investigate non-uniform stretching
of DNA on the single molecule level.
Comparing polarizations in two directions for DNA confined in channels of effective
diameters of 85 nm and 170 nm reveals a striking difference. Whereas the
DNA in the larger channels shows an isotropic polarization of the emitted light, the light is to
a large extent polarized perpendicular to the elongation of the DNA in the smaller channels. The ratio of the polarization parallel and perpendicular to the elongation
direction, I|| / I⊥, is a measure of the relative local orientation of the DNA backbone.
We believe that this technique will have a large impact on the studies of changes in DNA
conformation induced by protein binding or during DNA compactation as well as in
fundamental polymer physics studies of DNA in confined environments, for example in
bacterial spores and viruses.
DNA conformation
polarization anisotropy
nanofluidics