Cavitation enhancement elevates the efficiency and consistency of as-

says for downstream applications aiding in drug discovery and pa-

tient diagnostics
Bryana M. Robinson1, Marjan Mehrab Mohseni1,2, Melodie N. Noel1, and Samantha G. Pattenden1,4
1
Division of Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery,
Eshelman School of Pharmacy, The University of North Carolina, Chapel Hill, North Carolina 27599, United States; 2Joint
Department of Biomedical Engineering, The University of North Carolina, Chapel Hill, North Carolina 27599, United States;
3
Lineberger Comprehensive Cancer Center.
Supporting Information Placeholder

ABSTRACT: Sonicating is specifically taking place in

DNA fragmentation is often necessary prior to
some downstream assays such as Formaldehyde
Assisted Isolation of Regulatory Ele-
ments(FAIRE), Chromatin Immunoprecipitation
(ChIP and next generation sequencing[1].
Acoustic sonication uses ultrasound to mechani-
cally shear DNA by cavitation. This method typi-
cally produces inconsistent results and is time
consuming since DNA extracted from cells or
tissue must be optimized each time to ensure that
fragmentation occurs to the desired size rang
[1].
Our study suggested that perfluorocarbon
nanodroplets enhance cavitation and results in
more efficient and consistent DNA fragmentation
with higher throughput.
Personalized medicine is a heavily
sought after commodity. Indispensable contextu-
ally the traditional approaches to diagnostics and
patient treatment bears a burden financially to the
institution and the family of the patient. We have
established a patented cavitation reagent that en-
hances sonication to increase the efficiency and
consistency of assays to aid in drug discovery
and patient diagnostics. Upon cavitation of the
perfluorocarbon containing Nanodroplets the
pressure released from the activation via ultra-
sound is fundamental for creating more efficient
and consistent fragmentation.[2]
the Q-Sonica and the Covaris. These machines
shear the genomic deoxyribonucleic acid (gDNA)
and withhold the conversion capability of acous-
tic energy to mechanical energy. A process that
fundamentally takes hours and is successive, es-
sentially now takes minutes and multitudinous.
Therefore making the concept of personalized
medicine more viable[3].
Production of consistent fragmentation in
a 96 well plate, would quantitatively exceed tra-
ditional methods of sonication. We postulate
more samples of gDNA can be sonicated with the
improved efficiency and consistency of
nanodroplet-mediated fragmenation. It would be
premised more suitable and efficient method for
gDNA and chromatin fragmentation for high
throughput applications in personalized ge-
nomics. 4]
Further analysis will reinforce this reju-
venated approach to diagnostics for a plethora of
samples, making it more efficient and affordable
method, aiding in the enhanced accessibility and
treatment of the patients due to being able to
conduct the necessary testing. Furthermore,
pharmaceuticals will attain the capability of soni-
cating a multitude of personalize samples in
minutes.
INTRODUCTION:
 Chromatin is a complex of DNA and proteins
that form chromosomes in the nucleus of cells
that are eukaryotic. Chromatin is DNA tightly
packed around histones. Euchromatin have ex-
posed regions that would be susceptible to muta-
tions. Histones in heterochromatin essentially
blocks regulatory gene elements from being ac-
cessible to transcription factors, which are pro-
teins that controls the rate of transcription of ge-
netic information that binds to a specific DNA
sequence. Formaldehyde-Assisted Isolation of
Regulatory Elements [FAIRE] is an attractive
approach in identifying various regions of eu-
chromatin[1].
Fragmentation has essentially been considered
a bottleneck in the next generation sequencing
pipeline. A variety of limitations creates that bot-
tleneck including: biased fragmentation, poor ge-
nomic DNA quality, and lack of standardization
in size and reproducibility. We are specifically
focusing on the component of having a standard-
ized fragmentation size for downstream assays.
The ideal size for a quality strand is 300-500 base
pairs. Optimization and repetition of the method
is fundamental for being able to produce down-
stream assays. Initially, fragmentation was done
in various machines that convert mechanical en-
ergy to acoustic energy. The E110 machine has
been optimized and has produced consistent
fragmentation. However, this machine only pro-
cesses one sample at a time and is considered
lengthy compared to the LE220 which sonicates
8 samples at a time.[2]. The Q-sonica and the
LE220 is what we focused on specifically. Both
machines produced fragmentation and the Q-
Sonica showed significant cavitation. With our
cavitation reagent known as nanodroplets that are
perflourocarbon encapsulated and has a lipid
shell. It is a cavitation reagent it that enables-
denabled the increase in cavitation and when in-
corporated into the LE220 produced more con-
sistent fragmentation and exceeds the traditional
method by 8-fold. Essentially, high-throughput is
being established because in the LE220 8 sam-
ples are fragmented at a time, and that bottleneck
of creating consistent fragmentation in a efficient
amount of time is being debunked. Being able to
reproduce consistent fragmentation aids in the
overall understanding of various diseases due to
being able efficiently sonicate and analyze di-
verse molecular signatures multitudinously. This
is the very reason we are optimizing sonication in
the LE220. We want to make diagnostics more
accessible to the general population by reducing
the time that it takes to process genomic DNA for
determining mutations by high throughput se-
quencing. That means that “personalized medi-
cine” or understanding the genetic makeup of an
individual diseased cell would be less expensive
and therefore more generally accessible. Many
diseases thrive because of its manifestation and
proliferation[3]. This appealing approach makes
determining therapeutic options more accessible
for the patients with those rare or undiagnosed
diseases, being that more personalized samples
are sonicated quickly and proficiently
.understanding disease-specific genetic and epi-
genetic changes in individual patients aids in a
plethora of biological breakthroughs in medicine
for downstream assays[4].
Traditionally, diagnostics are costly and occur
successively.
We are supporting that the capability of
laboratories to fragment genomic DNA has been
made more viable, through our method of
sonication, whilst incorporating our biological
compound. It efficiently creates consistency and
increasing throughput.
METHODS AND RESULTS:
Microbubble formulation
Nanodroplets are essential because it serves as
our cavitation reagent. They are condensed
microbubbles. They are in a liquid form (the
microbubbles are in gas form) and are 250 um in
size. The highly compressible core of
perfluorocarbon gas enables the microbubble to
compress and expand in a process notarized as
cavitation. Our sonicators the Covaris and the Q-
Sonica produces high acoustic energy. With the
incorporation of our microbubble the cavitation
becomes very intense. The result is a plethora of
mechanical energy due to the collapsing of the
microbubble.

Cavitation
The following machines for sonication are
being utilized: LE220, and Q-Sonica. With the
perfluorocarbon containing microbubble
incorporated, our high-intensity sonicators shear
exhibit pure cavitation capacity of the
microbubbles under certain conditions.
Cavitation is measured through an extensive
testing of Potassium Iodide with the Nano
droplet. The LE220 exhibited a more focused
dispersion of energy. The Q-Sonica did however
exhibit positive results, however the culmination
that gDNA and chromatin will be fragmented in
the LE220 was established. Moreover through
various test consistency was established in both
machines and enabled favorable results.
Figure 1.
Figure 2
Figure 1. The following Is the KI test showing cavitation capacity
of nanodroplets. Half of the 96 well plate has the addition of
nanodroplet. While the other half Is used as a control and does not
Incorporate nanodroplets.
Figure 2. This Is the quantitative KI test In the Q-Sonica and
LE220 respectfully and clearly exhuberates a signifigant differ-
ence in cavitation with both p values measuring < .0001.
Tissue Culture
HEK (Human Embryonic Kidney) 293T cells
were grown ~ 1 million cells plates . on 15cm
petridish. . Upon passaging the cells were washed
with 10ml PBS (Phosphate Buffer Saline) per
plate of cells. They were then trysinized with
.125% trypsin . The new plates contained 1%
gelatin coating. Each new plate contained a total
volume of 20ml. this includes the cells and the
Media (L+Glutamine and 15% FBS). Zyppy
Plasmid Kit from Zymo Research (city?,
state?)was used for gDNA exctraction from
293T cells.
Crosslinking
For optical sonication of the HEK 293T . 1%
formaldehyde was added to the media contaning
cells. and shaken for 5 minutes. The glycine was
added to a final volume of 125M. This step stops
the crosslinking. Then, it was incubated for an
additional 5 minutes. The nuclei was isolated
with a series of rinses. The CIA rinse 1 was
added at 10ml volume centrifuged at 1,200g at 4
degrees Celsius for 5 minutes. The same was
done for CIA rinse 2. The Covaris Shaering
Buffer was adminsistered to the samples to wash
the salt. At this step, it is important to be careful
not to disrupt the pellet. This was followed by
centrifugation at 1,200g at 4 degrees Celsius for 5
minutes
Sonication and Preperation of Cells
The pellet was then resuspended in the shearing
buffer at volumes equating to 500,000 cells per
tube. The various time points determined based
on a series serious of tests. Sonication of HEK
293T cells were set
at 30 minutes and 15
minutes with three
samples that each
contained
nanodroplets and
three controls.
Figure 3.
The Covaris LE220 (Figure 3.) was used for
sonication. After sonication, cells were
transferred to 1.7 microfuge tube andcentrifuged
at 4C and 1800g for 5 min. to separate the soup
and the pellet. Pellet was resuspended in 100 ul
shearing buffer. Both pellet and soup were
transferred to PCR tube and 2ul RNase
(manufactor) was added to each pellet and when used with the ultrasound equipment
supernatant , and incubated at 37 degrees Celsius produced viable results for downstream assays.
for 10 minutes.2ul proteinase K(manufactor) was High throughput was created by being able to
added to the same samples and incubated at 55 sonicate a multitude of gDNA and Chromatin in a
degree for 30 min followin by incubation at 65 matter of minutes. This allows a more efficient
degree overnight to revers the formaldehyde and consistent fragmentation. The amount gDNA
cross-link and Chromatin samples being fragmentated in the
After the sonication, the DNA was extracted LE220 was quantitavly 96 wells. The pure
and purified (Zymo Research chip DNA clean cavitation of the nanodroplets showed the
and concentaration kit). ChIP binding buffer that efficieny of the droplets. This impressive
is added at a volume of 5 and mixed briefly. The approach to fragmentation is a staple in the
samples are transferred to spin columns diagnostic process and allowed a multtude of
centrifuged at 10,000x g for 30 seconds. 200 samples to efficently and consistanly sonicate in
microliters of wash buffer was added the seconds.
centrifuged at 10,000x g for 30 seconds. Finally
the elution buffer is then added and centrifuged at
10,000x g for 30 seconds. The pure DNA is
quantified with a Qubit. The results exuberated
that the 30 minutes and 15minutes were the ideal
optimization for the HEK 293T cells.
Electrophrasis and Tape Station
Checking the amount of sonication was done
through electrophrasis on 1.5% agarose gel. A
more quantifiable base pairing analysis was done
termed tape station.

Figure 4 Figure 5

Figure 4 shows the results in the LE220. In figure 4 the control did
not contain nanodroplets and shearing was minimal In comparison
to the samples with nanodroplets. Figure 5 showed the sonication
of the cells with the nanodroplets.

Figure 5
Tape Station and graphs
CONCLUSION:

The cavitation and sonication of gDNA and cells

was demostrated with the presence of cavitation
reagent. We found a signifigant difference in the
time required to sonicate the samples . There was
a signifigant improvement in high throughput
sonication of the samples. The cavitation reagent
 e0133014. doi:10.1371/journal.pone.0133014

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