48 A. Gabibov et al.
Fig. 4.2.Principle of the flow linear dichroism (FLD) method. Experimental condi-
tions: DNA polymers are oriented in reversible flow of reaction mixture. Combined
vector of orientation of chromophoric groups of DNA is then different from zero, its
value and sign depending on topological state of molecules and their orientation abil-
ity. Measured value is the ratio between the difference of the optical densities of the
oriented sample for two perpendicularly polarised rays and the optical density for
non-polarised light (∆A/A). Absolute values of reduced linear dichroism (ρ)were
measured by using a JASCO J500C spectropolarimeter equipped with an achro-
matic quarter-wavelength prism at 260 nm. Plasmid DNA was oriented by the flow
gradient provided by pumping the solution through the flow cell using the reciprocal
pump, designed and constructed by V.L. Makarov (Engelhardt Institute of Molecu-
lar Biology, Russian Academy of Sciences) [24]. The cell volume was 200μl; optical
path length was 2 mm; the frequency of reciprocal pump was 100 rpm; an average
flow gradient in the cell was 3,000 s−^1
An scDNA can be relatively adequate rendered as a rigid rod formed by a
double helix winded into the superhelix, which could further form superhelices
of higher order. For such moleculesρis defined as
ρ=1/2(3〈cos^2 Θ〉−1)(1/2(3〈cos^2 βi〉−1)), (4.13)whereΘis the angle between the main axis of DNA molecule and axis of
orientation of the molecules. The first coefficient is thus an orientation factor,
which under the condition of shear flow is determined by hydrodynamic prop-
erties of the molecule, and is dependent on the form of DNA molecule and
its rigidity. The rest of coefficients describes the intrinsic optical anisotropy
of scDNA. Parameterβi=tg−^1 (2πRi/Pi), whereRiandPiare radius and
pitch of theith order superhelix [21].
It seems to be clear that all variables (Θ,RiandPi) in this equation
are strongly dependent on〈σ〉. As an example, sc circular DNA can be
better oriented than the relaxed circles of the same counter length. However,
formation of the supercoiled DNA is accompanied by shortening the radius