Carter, Ashley R.

Lee, Ji Hoon

Schwab, Robert D.

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Investigating the DNA Folding Mechanics of Protamine 

276a Monday, February 17, 2014metabolites of cytoxan and ifosfamide. These simulations include all 8 DNA
central sequences that can support a 1-3 guanine-guanine crosslink (5’-GNC-
3’ and 5’-CNG-3’ with N=A, T, G, or C) with both GC and AT-rich flanking
sequences. These simulations show that the likelihood of forming an inter-
strand crosslink has a strong sequence preference for the 5’-GNC-3’ orienta-
tion, in agreement with previous experiments and simulations. Additionally
we find a slight dependence of the crosslinking efficiency on the intervening
base. We also find that the distribution of crosslinking distances does not
follow a simple normal distribution, but instead shows the presence of several
DNA structural microstates with lifetimes of 10-100 ns. These simulations
provide a basis for the design of new DNA-crosslinking drugs optimized
for interstrand crosslinking.
1404-Pos Board B134
Characterization of DNA-CTAB Aggregates
Kathleen Westervelt, Pamela M. St John.
Chemistry, SUNY New Paltz, New Paltz, NY, USA.
Oligonucleotides will aggregate in the presence of a surfactant, much like the
DNA aggregates that form during the transportation of genes through cell
membranes. Aggregates formed from oligonucleotides of 20 to 100 bases
in length in the presence of the surfactant, CTAB (cetyl trimethylammonium
bromide) have been characterized by UV spectroscopy and electrophoresis.
UV spectroscopy showed that the absorbance increased as a result of light
scattering from various sized aggregates that formed between oligonucleo-
tides and CTAB when the ratio of CTAB to oligonucleotide concentration
was about 0.5 or larger. Electrophoresis has been used to compare the mo-
bilities of both single and double stranded DNA in the presence of CTAB.
The results show that DNA - CTAB gives rise to smeared bands in the
gels at room temperature indicative of a range of aggregate sizes where
the aggregates may be falling apart as they migrate through the gel. Bands
from gel electrophoresis of DNA-CTAB aggregates at 4 C do not appear
smeared indicating that the presence of aggregates is temperature dependent.
To better characterize these aggregates, we are currently staining the oligo-
nucleotides with a fluorescent dye and visualizing them using microscopy
and spectrofluorometry.
1405-Pos Board B135
Single Molecule Studies of Sequence Dependence Elasticity in DNA
Julia T. Bourg1, Krishnan Raghunathan1, Alan Kandinov1,
Joshua N. Milstein2, Jens-Christian Meiners3.
1Biophysics, University of Michigan, Ann Arbor, MI, USA, 2Chemical and
Physical Sciences, University of Toronto Mississauga, Mississauga, ON,
Canada, 3Physics and Biophysics, University of Michigan, Ann Arbor, MI,
USA.
DNA looping is a vital regulatory process in the cell to maintain proper func-
tion and viability. In protein-mediated looping, the DNA’s sequence dictates
both protein binding sites and the local mechanical properties of the DNA.
These mechanical properties, notably its elasticity and intrinsic curvature,
govern the ease at which loops can form. To probe the effects of sequence
dependence on elasticity and the loop formation process, single molecule ex-
periments were performed on short segments (~150bp) of DNA with varying
amounts of AT and GC content between the protein-binding operators. Teth-
ered particle motion (TPM) microscopy was employed to observe protein-
mediated DNA loop formation in this system, and an analytical model of the
loop formation process was used to calculate the elasticity of the DNA from
the observed loop formation rates. For comparison, axial constant-force optical
tweezers were used to directly stretch the same DNA molecules mechanically
to determine their persistence length as a measure for their elasticity. Our re-
sults indicate that the intraoperator sequence has a larger effect on elasticity in
the loop formation experiments than in the stretching experiments, which we
attribute to different elasticity regimes when the DNA is strongly bent as in a
DNA loop, compared to the thermally induced small curvature fluctuations in
stretched DNA.
1406-Pos Board B136
Mechanical Properties of DNA-Like Polymers
Justin P. Peters, L. James Maher III.
Mayo Clinic, Rochester, MN, USA.
This work continues to probe the mystery of what balance of forces creates the
extraordinary mechanical stiffness of DNA to bending and twisting. In partic-
ular, we have been trying to understand the extent to which the charge of DNA
contributes to stiffness through electrostatic stretching forces. Remarkably,
neither theory nor experiment has resolved this issue over six decades. We
have approached the problem by making a family of double-helical DNA-
like polymers in which one of the four normal bases is replaced with an anionic,cationic, or neutral analog that alters canonical stacking, charge, or both. The
resulting DNA-like polymers were characterized in a variety of ways, including
determining bend stiffness using cyclization kinetics and atomic force micro-
scopy experiments. Recent theory predicts that the variants with different
charge densities should have dramatically altered bending rigidity. Remark-
ably, we show that these DNA-like polymers have bend stiffness within the
range of reported values for sequence-dependent variation of the natural
DNA bases. On the other hand, DNA twist flexibility was strikingly different
among the polymers. These results suggest that electrostatic stretching forces
do not contribute measurably to DNA bend stiffness, showing the inadequacy
of recent theory and the need for new concepts.
1407-Pos Board B137
Investigating the DNA Folding Mechanics of Protamine
Ji Hoon Lee, Robert D. Schwab, Ashley R. Carter.
Amherst College, Amherst, MA, USA.
In spermatogenesis, an arginine-rich protein called protamine ultimately re-
places ~85 percent of the histones that bind DNA. After replacement, the
protamine proteins cause folding of the DNA into a series of toroids, which
allow for a high level of DNA compaction. This replacement and compac-
tion 1) reduces the head size of the sperm to enhance their hydrodynamicity,
2) decreases the likelihood of DNA damage, and 3) removes the epigenetic
markers passed on through histone modifications. Yet, little is known about
the exact mechanism of protamine-induced DNA folding. Here, we measure
DNA compaction using a tethered particle motion assay which allows us to
measure folding without applying any external forces. In a tethered particle
motion assay, a micron-sized bead is tethered to the surface via a DNA
molecule. During DNA folding, the end-to-end length of the DNA de-
creases, decreasing the motion of the bead. When we measured the motion
of the bead in the presence of low protamine concentrations (1 mM) we
observed discrete changes in the DNA length that occurred in ~200 nm in-
crements. We interpret these length changes as sequential looping of the
DNA into a toroid. Interestingly, the first step in the looping appears to be
reversible while subsequent steps are not, possibly due to inter-loop prot-
amine interactions. We then calculate the energetics for the looping and
compare our findings to current models. Our results show how protamine
is able to fold DNA into a super-compacted state necessary for efficient
sperm delivery, which provide novel insights into such limiting case in
epigenetics.
1408-Pos Board B138
Measuring Energetics of Sharp DNA Bending from Breakage Kinetics of
Small DNA Loops
Tung T. Le, Harold D. Kim.
School of physics, Georgia Institute of Technology, Atlanta, GA, USA.
Double-stranded DNA (dsDNA) can undergo diverse modes of conformational
changes inside a cell. Among them, dsDNA bending is closely associated with
genome packaging and gene regulation, and therefore a consistent polymer
model of dsDNA bending is essential for quantitative description of genome-
related processes. The behavior of dsDNA at large length scales can be well
described as a worm-like chain whose bending energy depends quadratically
on curvature. However, many studies on dsDNA bending at short length scales
have produced results contradictory to the worm-like chain model. Here we
show that the free energy of a dsDNA loop as short as 60 bp in contour length
can be well described by the wormlike chain model. We measured the breakage
rates of small dsDNA loop stabilized by sticky ends using Fluorescence Reso-
nance Energy Transfer (FRET). We found that the breakage rate vs. loop length
relationship to be in good agreement with the worm-like chain model, but not
with an alternative model which predicts superflexible behavior at short length
scales. Furthermore, we found that energetics of dsDNA begins to deviate from
the worm-like chain model below 60 bp. Our result pushes the lower limit of
the worm-like chain regime of dsDNA down to 60 bp, and suggests that
dsDNA undergoes structural transitions below this length.
1409-Pos Board B139
Polyethyleneimine-DNA Interactions
T.J. Thomas, Sripriya Venkiteswaran.
Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, USA.
Polyethyleneimine (PEI) is a cationic polymer with primary, secondary and
tertiary amino groups separated by an ethylene bridge. PEI is available in
both linear and branched forms, and many molecular weight forms are avail-
able commercially. PEI is amongst the first and very versatile transfection
agents to deliver DNA to cells for gene targeted studies and therapeutic ap-
plications. By virtue of their cationic nature under physiologically compatible
ionic and pH conditions, PEI can bind with DNA. Using total intensity and


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Investigating the DNA Folding Mechanics of Protamine

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