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Technical Program TRACK 5

to ensure the desired therapeutic effect and avoid the emergence of side m6A) detection. m6A is a methylation modification abundant in prokaryotic
effects due to the uncontrolled, systemic release of the GFs. We developed genomes, and also found in lower eukaryotes and higher plants. So far
biocompatible nanoporous silicon multistage vectors (MSV) to allow the detection of m6A relies on methods such as TLC, HPLC, MS, and enzymatic
efficient loading of a variety of bioactive molecules. To prevent the burst reactions, which are often laborious, expensive, with low specificity and
release from the open nanopores and provide enhanced protection to the varying reactivity. Unlike 5-methylcytosine (m5C), there is no chemical treat-
payload we engineered a tunable poly(DL-lactide-co-glycolide) acid (PLGA) ment that can facilitate the m6A detection. Whereas high-resolution melting
coating of the MSV. By controlling the physical and chemical properties of (HRM) analysis is able to detect a single m6A modification within a target
the PLGA layer we provided a further control on the long-term release of DNA via the destabilizing effect of m6A, HRM cannot pinpoint the location
therapeutic molecules. To test the ability of this platform to achieve sus- of m6A in the sequence. A simple and cost-effective way to identify single
tained temporal release and localized spatial distribution, we integrated it in m6A at any specific sites is therefore highly desired. Here, we developed a
an electrospun gelatin based patch. The patch was inspired by the biologi- robust, simple, enzyme-free and hybridization-based method for m6A detec-
cal properties and composition of the extracellular matrix (ECM) to avoid the tion with “pinpoint specificity”, using a new type of silver cluster-based DNA
immune rejection of the graft. To increase the early vascularization of the probe which we term methyladenine-specific NanoCluster Beacon (maNCB).
implant we loaded the PLGA-MSV with VEGF and PDGF-BB. The controlled To date, there is no hybridization technique that has the potential to reach
release of the two GFs can improve the local formation of neo-vasculature these remarkable results. To date, there is no hybridization technique that
at the site of the implant by locally enhancing and supporting angiogenesis. has the potential to reach this remarkable result.
The development of this biomimetic electrospun patch, functionalized with
a tunable delivery platform consisting of MSV and PLGA (PLGA-MSV), allows References:
for the programmable delivery of GFs in space and time. By orchestrating [1] Y.-A. Chen and J.M. Obliosca et al. J. Am. Chem. Soc., 2015, 137, 10476
the release of signaling molecules, this patch could be used in support of [2] J.M. Obliosca et al. Nanoscale, 2015, 7, 8332
any tissue restoration process requiring early vascularization. We charac- [3] J.M. Obliosca et al. ACS Nano, 2014, 8, 10150
terized the patch using scanning electron microscopy, Fourier transform
infrared spectroscopy, and confocal microscopy. The release of VEGF and 4:20pm Entropically controlled nanomechanical sensing with
PDGF-BB from PLGA-MSV was performed in physiological like conditions DNA origami
(PBS, 37ºC, under mild agitation). Samples were collected up to 4 weeks
and GF’s release kinetics was determined by ELISA. The patch allowed to
prevent the burst release of the loaded GFs and to prolong their delivery up Technical Presentation. NEMB2016-6063
to 4 weeks. Furthermore the stability and bioactivity of the loaded molecule
was preserved during the duration of the release process. We tested the Michael Hudoba, Yi Luo, Michael Poirier, The Ohio State Universi-
biocompatibility of the electrospun gelatin patches using human mesenchy- ty, Columbus, OH, United States, Carlos Castro, Ohio State univer-
mal stem cells (hMSC). Early passage hMSC were seeded onto the patch sity, Columbus, OH, United States
to assess cell growth and differentiation up to 1 month. The results of the in
vitro tests indicated uncompromised cell viability and full cell adhesion to DNA nanotechnology has emerged as a promising technology for appli-
the patch. cations such as single molecule sensing, super-resolution imaging, and
manipulating molecular components. Major advances in the last decade
The presented strategy provides the controlled release of VEGF and have enabled the precise design and fabrication of DNA nanostructures
PDGF-BB in a controlled fashion to enhance patch’s vascularization. Further with unprecedented geometric complexity; however, relative to natural bio-
investigations are ongoing to verify the efficacy of this platform to induce in molecular machines, the functional scope of DNA nanotechnology is limited
vivo vascularization, the localized delivery of GF and we expect to inevitably by an inability to design dynamic mechanical behavior such as complex
boost the regenerative microenvironment by promoting early angiogenesis motion, conformational dynamics, or force generation. Taking inspiration
at the site of the implant. from methods used in macroscopic machine design, we have recently
developed DNA nanostructures with well-defined 1D, 2D, and 3D motion,
and we have demonstrated the ability to actuate that motion via binding or
5-5 competitive release of DNA strands on the timescale of ~1 minute. We have
recently developed dynamic nanostructures that exhibit multiple stable
ASSEMBLED MATERIALS states separated by tunable energy barriers designed to allow thermally
driven conformational changes at room temperature. In addition, we have
Harris 4:00pm - 5:40pm demonstrated the ability to tune the kinetics of transition between the stable
states through structure design parameters that regulate the conformational
entropy by constraining thermal fluctuations in specific states. In particular,
4:00pm NanoCluster Beacons Enable Enzyme-Free N6-Meth- we have designed a dynamic DNA Origami Two-state nanodevice (DOT)
yladenine Detection consisting of two stiff double-stranded DNA bundle components connected
by 6 flexible single-stranded DNA linkers arranged symmetrically around
Technical Presentation. NEMB2016-6000 the circumference of the bundle. One of the linkers can close into a loop via
binding of a single DNA base-pairing interaction yielding a dynamic equilib-
Judy Obliosca, Yu-An Chen, Yen-Liang Liu, Cong Liu, Mary rium between a closed state where the two bundle components are pinned
together and an open state where they can exhibit large relative motion.
Gwozdz, Tim Yeh, University of Texas at Austin, Austin, TX, United This closing interaction was designed to allow for transient binding into the
States closed state with short lifetimes on the timescale of ~1 second. Equilibrium
behavior of the DOT measured both by transmission electron microscopy
NanoCluster Beacons (NCBs) are a new type of activatable molecular and single molecule fluorescence resonance energy transfer experiments
probes that are low cost, easy to prepare and have high fluorescence en- confirm a distribution of closed and open states. We further demonstrated
hancement ratios. NCBs employ DNA-templated, few-atom silver nanoclus- the ability to tune the kinetics of conformational transitions between states
ters (DNA/Ag NCs, with about 2~20 silver atoms per cluster) as reporters through structural design parameters that regulate conformational entropy.
which can significantly “light up” through interactions with a nearby DNA se- A major goal of our laboratory’s work is to develop devices where dynamic
quence (called an enhancer). Taking advantage of this fluorescence tunabil- behavior can be exploited to probe nanoscale physical properties or inter-
ity by altering the surrounding ligands, a property that is not commonly seen actions (e.g. molecular forces). Here we demonstrated that both the kinetics
among existing reporters, NCB soon evolved to a multicolor probe, termed and equilibrium behavior of DOTs serve as a readout of depletion forces
66 chameleon NanoCluster Beacon (cNCB), for single-nucleotide polymorphism that result from molecular crowding using polyethylene glycol as a crowd-
(SNP) detection. Here we bring the NCB detection to the next level by de- ing reagent. Given the length scale of ~100nm, this device could ultimately
signing a new NCB specifically for N6-methyladenine (hereafter denoted as be implemented to measure depletion forces inside cells where molecular