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Monday, February 17, 2014
DNA Structure and Dynamics II
1398-Pos Board B128
The Effects of Base Stacking on DNA Flexibility
Lauren S. Mogil, Justin P. Peters, Jim Maher.
BMB, Mayo Clinic, Rochester, MN, USA.
DNA is the main genetic component of life and understanding its basic properties is crucial for advancement in applied sciences. DNA, a long macromolecule, must complex with proteins and other molecules for packaging and
condensing to fit in the nucleus of a cell. Persistence length is a physical
property that defines the stiffness of a polymer, ranging from rod-like to
string-like. The persistence length of double-stranded DNA is ~150bp or
~50nm making it a relatively inflexible molecule. It is hypothesized that
the two main contributors limiting DNA flexibility are mutual charge repulsions along the DNA backbones and attractive base stacking interactions.
However, the relative contributions to DNA stiffness of electrostatic repulsion and base stacking forces are unknown. We use experimental T4 DNA
ligase-mediated cyclization kinetics experiments to measure the physical
properties of DNA, specifically DNA longitudinal and torsional flexibilities
and helical repeat.
We measured the impact on DNA stiffness of enhancing or diminishing stacking energy involving modified adenine and guanine bases. Diaminopurine
(DAP) is hypothesized to increase DNA bending stiffness by changing the
base geometry and dipole moment so as to enhance stacking energy and inosine
(dITP) is hypothesized to decrease DNA bending stiffness. Cyclization assays
were performed with radiolabeled DNA probes varying in length from 211bp to
201bp. The free ends of the probes are complementary and, in the presence of
T4 DNA ligase, yield various linear and circular species in vitro. The J-factor
(related to persistence length through the worm-like chain polymer model) can
be estimated in such assays. We found that the bending persistence length
changed slightly relative to natural DNA but it is the twist persistence length
that has a more dramatic change.
1399-Pos Board B129
Conformational Transition of Nanoslit Confined DNA at Low Ionic
JinYong Lee1, Kyubong Jo2.
Department of Chemistry, Sogang UNIV, Seoul, Korea, Republic of,
Deparrtment of Chemistry and Interdisciplinary Program of Integrated
Biotechnology, Sogang UNIV, Seoul, Korea, Republic of.
We investigate experimentally the effect of ionic strength on l DNA molecule
in glass/PDMS nanoslit by using fluorescence microscope. The radius of gyration Rg was measured as a function of slit height (250 ~ 1049 nm), and ionic
strength (0.183 ~ 1.75 mM) as a result, we observed the Rg values increases
as channel heights and ionic strengths decrease. Furthermore, based on Odijk’s
and de Gennes polymer physics theory, we interpret our experimental findings
as a function of channel heights and ionic strengths. We also study the effect of
EDTA concentration, radius of gyration of l DNA change drastically in
response to the varying EDTA concentration at low ionic strength condition.
Our results are useful for understanding the manipulation of biomolecules in
nanofluidic devices.
1400-Pos Board B130
Probing Sequence and Topological Specificity in the Binding of
Tetra(Methylpyridyl)Porphines to DNAs
Stephen A. Winkle1, Jennifer Barretta1, Diane Edgar1, Raul Castillo1,
Olga Roman1, Roxana Roque1, Jessica Millar2, Maria Ballester2.
Chemistry and Biochemistry, Florida International University, Miami, FL,
USA, 2Math, Science and Technology, Nova Southeastern University, Fort
Lauderdale, FL, USA.
The binding of a family of tetra(methylpyridyl)porphines to relaxed and
supercoiled circular phiX197RF DNA has been probed using restriction
endonuclease activity assays employing nine restriction enzymes (selected
for different cleavage sequences and different flanking sequences), mung
bean nuclease assays and E.coli topoisomerase assays. The restriction
enzyme assays indicate that placement of the methyl substituent alters the
sequence and topological specificity of binding. For example, the the restriction enzyme Mlu I (cleavage site ACGCGT), the ortho isomer yields partial
inhibition of cleavage of supercoiled and relaxed DNA, the meta isomer
yields inhibition of the relaxed DNA and no effect with supercoiled DNA,
and the para isomer gives enhanced cleavage of supercoiled and relaxed
DNA. With Dra I (TTTAAA), the ortho isomer again gives partial inhibition
cleavage of supercoiled and relaxed DNA, the meta isomer gives inhibition
of cleavage of relaxed AND supercoiled DNA, and the para isomer has no
effect on cleavage of either form. Variations in flanking sequences affect
the selectivity in binding. Mung bean digestion assays indicate that all three
isomers alter the conformation of the supercoiled DNA, with the ortho isomer giving the greatest distortion. Topoisomerase studies suggest that these
molecules unwind the DNA. Molecular modeling suggests that the placement
of the methyl substituent on the pyridyl rings affects the planarity of the molecules, which may alter the binding to DNA and lead to the observations
1401-Pos Board B131
Molecular Dynamic Studies of Z[WC] DNA and the B to Z-DNA Transition
Michael G. Lerner, Jinhee Kim, Alexander K. Seewald.
Earlham College, Richmond, IN, USA.
Although DNA is most commonly found in the right-handed B-DNA structure,
it is known that biologically active systems also contain left-handed ZII-DNA.
We investigate the possibility that Z[WC]-DNA serves as an intermediate
structure in the B to ZII transition. Molecular dynamics simulations indicate
that Z[WC] nonamers are stable structures with the current AMBER nucleic
acid force field. Steered molecular dynamics simulations indicate that, for
collective transitions of the whole strand, the B-Z[WC]-ZII pathway may
have a lower free-energy barrier than the direct B-ZII pathway. We then
used both steered and targeted molecular dynamics in combination with
umbrella sampling to produce potentials of mean force for the B to ZII transition along both pathways.
1402-Pos Board B132
Nanoconfined Circular DNA
Mohammadreza Alizadehheidari1, Erik Werner2, Charleston Noble3,
Lena Nyberg1, Joachim Fritzsche4, Bernhard Mehlig2, Jonas Tegenfeldt5,
Tobias Ambjo¨rnsson3, Fredrik Persson6, Fredrik Westerlund1.
Department of Chemical and Biological Engineering, Chalmers University
of Technology, Go¨teborg, Sweden, 2Department of Physics, Gothenburg
University, Go¨teborg, Sweden, 3Department of Astronomy and Theoretical
Physics, Lund University, Lund, Sweden, 4Department of Applied Physics,
Chalmers University of Technology, Go¨teborg, Sweden, 5Department of
Solid State Physics, Lund University, Lund, Sweden, 6Department of Cell
and Molecular Biology, Uppsala University, Uppsala, Sweden.
Nanofluidic channels have become a versatile tool to manipulate single DNA
molecules. They allow investigation of confined single DNA molecules from
a fundamental polymer physics perspective as well as for example in DNA barcoding techniques.
Covalently closed circular DNA is found in many biologically relevant contexts, such as bacterial plasmids, viruses and eukaryotic mitochondrial DNA.
Circular DNA is also interesting from a fundamental perspective. The circular
topology forces two strands to be close to each other in the nanochannel, which
changes the polymer physics behavior compared to linear DNA. Circular DNA
is difficult to study with traditional single molecule techniques because it
generally requires the attachment of handles.
Circular DNA in its folded state has less entropy and higher conformational
free energy than the unfolded state. Unfolding of circular DNA is therefore
entropically favorable. As a double-strand break occurs, circular DNA opens
up and unfolds to its linear conformation inside the nanochannel. This
study deals with the statics and dynamics of this transition from circular to
linear DNA.
We observe that the opening of the circle has a strong preference to occur in the
ends and discuss the mechanism behind this peculiar observation. We also
investigate the difference in extension between the circular and linear state.
We observe that their ratio decreases with decreased stretching, in agreement
with theoretical calculations.
Finally we demonstrate how we can identify bacterial plasmids carrying antibiotic resistance genes using a recently developed mapping technique based
on nanofluidic channels.
1403-Pos Board B133
Simulations of Crosslinking Efficiency and Sequence Specificity of
Nitrogen Mustard Anticancer Drugs
Moon Joon Park, Michael E. Colvin.
University of California, Merced, CA, USA.
Cytoxan and ifosfamide are important anti-cancer drugs that function by
forming covalent crosslinks between the two strands of double helical
DNA. The cytotoxicity of these drugs depends on their efficiency in forming
interstrand crosslinks versus intrastrand crosslinks or non-crosslinking DNA
damage which can cause mutations in normal cells leading to future cancers.
To better understand the factors favoring interstrand crosslinks, we have
performed a series of near-microsecond timescale molecular dynamics simulations of different sequences of DNA 11-mers bound to the activated