(174ad) Functionalizing Cell Membrane with a Multifunctional DNA-Origami Platform for Biomulecular Detection | AIChE

(174ad) Functionalizing Cell Membrane with a Multifunctional DNA-Origami Platform for Biomulecular Detection


Shahhosseini, M. - Presenter, Ohio State Universoty
Castro, C. E., The Ohio State University
Akbari, E., The Ohio State University
Song, J. W., The Ohio State University
v\:* {behavior:url(#default#VML);} o\:* {behavior:url(#default#VML);} w\:* {behavior:url(#default#VML);} .shape {behavior:url(#default#VML);}

NBL User NBL User 2 84 2019-04-12T21:55:00Z 2019-04-12T21:55:00Z 2 1101 6277 52 14 7364 14.0

Clean Clean false false false false EN-US JA X-NONE

/* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin;}

none;text-autospace:none"> major-latin;mso-no-proof:yes">Functionalizing Cell Membrane with a
Multifunctional DNA-origami platform for Sensing the Cell Microenvironment

none;text-autospace:none"> major-latin;mso-no-proof:yes">

none;text-autospace:none"> major-latin;mso-no-proof:yes">Introduction Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin;
mso-bidi-font-family:Arial">: The plasma membrane is the primary communicating
interface between the cell and extracellular environment. Thus, the ability to
engineer new functions into the cell membrane to externally monitor cellular
interactions is of great significance. Previous studies have focused on
engineering cell surface proteins or incorporating synthetic protein constructs
into the cell membrane, which are often highly challenging. Therefore,
designing a robust approach to incorporate multifunctional nanodevices
with diverse structural and dynamic functions into the cell membrane can enable
engineering of the cell surface as a functional material. " times new roman>In recent
decades biomarkers have become essential in diagnosis and assessing patients'
responses to therapy of various diseases.  Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin;
mso-bidi-font-family:Arial"> There are currently multiple methods to detect biomarkers
for early disease diagnosis, often by performing liquid biopsies. However, these
methods have limitations such as demand for specialized equipment, low
sensitivity and insufficient specificity. Furthermore, none of the available
techniques provide in-situ monitoring
of cell-biomarker interaction directly in the cell microenvironment, which may
provide the additional benefit of elucidating mechanisms of disease progression
mediated by those interactions. To address these limitations, we aim to incorporate
DNA origami sensing platforms into the cell membrane to detect biomarkers in
the microenvironment. Our initial focus is to detect circulating tumor DNA (ctDNA), mutated genes that are released by dead cancer
cells, and platelet-derived growth factor (PDGF), which can contribute to
angiogenesis in tumor microenvironment.  in-situ with
fluorescence based reporting. We further aim to combine ctDNA
and PDGF detection with other functions like sensing pH, other proteins, and
physical forces at the cell surface with the aim of spatially and temporally
profiling cellular interactions in tumor microenvironments. major-latin;mso-ansi-language:EN"> mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin;mso-bidi-font-family:

none;text-autospace:none"> major-latin;mso-no-proof:yes">Materials and Methods: major-latin;mso-ansi-language:EN"> DNA origami is the self-assembly of complex
nanostructures using DNA as a programmable building block [1]. We recently
reported an approach to engineer cell-membrane function by embedding
DNA-origami nanodevices onto the cell surface [2]. major-latin;mso-bidi-font-family:Arial">Our method implements a cholesterol
conjugated DNA strand serving as the membrane incorporated oligo
(MIO) to serve as an anchoring site for the binding of a DNA origami membrane
bound breadboard (MBB). First, we confirmed the functionalization of the cell
membrane with MIO by visualization of a complementary fluorescent oligo (Fig. 1A). Next, we utilized an intermediate oligo that binds to the MIO and extends a single-stranded
DNA binding site away from the surface for subsequent binding of the MBB (Fig.
1B). Using this functionalization scheme, we effectively coated the surface of
five cell types with MBB structures (Fig. 1C, image shown for B Cells). In
addition, confocal imaging techniques confirmed MBB presence around the cell
periphery (Fig. 1D). mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin;mso-ansi-language:
EN">Next, we modified the MBB  with major-latin;mso-bidi-font-family:Arial">overhangs on the outward facing side, to
serve as sensing ports. mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin;mso-ansi-language:
EN">We programmed two ports to detect presence of 2 different DNA sequences
(targets) on the cell surface by emitting fluorescence signal in two different
channels. We modified nanodevices (Cell Sensing Platform, CSP) with
flourophores and fluoroscense quenching molecules (Q) such that introduction of
DNA targets of a specific sequence results in displacement of quenching
molecules and hence an increase fluoroscense signal. We functionalized the
lipid membrane of CH12 and HL60 cells with nanodevices and demonstrated
detection of two distinct DNA targets on surface of cells via live cell
fluorescence imaging (Fig. 2A). Furthurmore, we programmed one of the ports for
detection of mso-hansi-theme-font:major-latin;mso-bidi-font-family:Arial">platelet-derived
growth factor (PDGF), using a previously reported Aptamer
[3]. Binding between PDGF and the aptamer induces a
conformational change in the aptamer resulting in
reduction in the fluorescence readout. Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin;

none;text-autospace:none"> major-latin;mso-hansi-theme-font:major-latin">Results and Discussion: major-latin"> major-latin;mso-hansi-theme-font:major-latin;mso-ansi-language:EN">We
functionalized the lipid membrane of CH12.LX and HL-60 cells with CSP and
demonstrated detection of two distinct DNA targets on surface of cells via live
cell fluorescence imaging (Fig. 2B). Preliminary results show that introduction
of each DNA target at 1µM, increases fluorescence signal by 50%. In addition, CSP
are able to simultaneously resolve the presence of the two target DNA sequences
over a broad range of concentrations (1nM to 1µM). Preliminary results also
suggest we can capture binding events on individual cells, and in some cases
with sub-cellular resolution, in real time. Furthurmore, PDGF detection
experiments show that we can detect PDGF as low as 1nM(Fig. 2A). Priliminarily
results also indicates that we can correlate spatial intensity of fluoroscence signal
around cells to PDGF concentration in the environment. We expect that
implementing DNA origami structures on cell membranes will enable us to profile
the spatiotemporal distribution of ctDNA, PDGF and other biomarkers or interstitial
properties in cellular microenvironment.

inter-ideograph"> Calibri;mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin;
EN">:  We have established the basic
methods for functionalizing cells with multi-functional DNA origami sensing
platforms and demonstrated the efficiency of nanodevices to detect multiple biomolecular
targets. For future steps, we plan to incorporate aptamers for multiple cancer
molecular biomarkers to enable multiplexed detection and profiling of the
cellular microenvironment. Specifically, we are interested in detection of
combinations of different cancer-related genes with other cancer biomarkers
(e.g. platelet-derived growth factor and pH) in cellular microenvironment. We
envision this technique can be implemented as a method for early detection of
cancer and a unique approach to study mechanisms of cancer progression at the
cellular level. major-latin;mso-hansi-theme-font:major-latin">

none;text-autospace:none"> major-latin;mso-hansi-theme-font:major-latin;mso-ansi-language:EN">


1. Arial;color:#222222;background:white">Castro, Carlos Ernesto, et al. Nature
 8.3 (2011): 221.

2. 10.0pt;font-family:Arial;color:#222222;background:white">Akbari, Ehsan, et al. Advanced Materials 29.46 (2017):

3 Arial;color:#222222;background:white">. mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin;mso-bidi-font-family:
Helvetica;color:black">  Arial;color:#222222;background:white">Zhao, Weian, et
al. Nature nanotechnology 6.8 (2011) 

none;text-autospace:none"> major-latin;mso-hansi-theme-font:major-latin;mso-ansi-language:EN">

none;text-autospace:none"> major-latin;mso-hansi-theme-font:major-latin;mso-ansi-language:EN">

none;text-autospace:none"> major-latin;mso-hansi-theme-font:major-latin;mso-no-proof:yes">


none;text-autospace:none"> major-latin;mso-hansi-theme-font:major-latin;mso-bidi-font-family:Arial">Figure
1 – Functionalization of Cell Membrane with DNA origami membrane bound
breadboard (MBB). A) The incorporation of cholesterol conjugated strand as the membrane incorporated oligo (MIO)
was confirmed using fluorophore conjugated
complementary oligo. B) The Functionalization steps
used to attach MBB to cell membrane coated with MIO. C) Successful attachment
of MBB to CH12.LX B cell membrane. The attachment was inhibited by using
a binding inhibitor oligo. D) Representative confocal
image of CH12.LX B cells coated with MBB. The scale bars are 10 µm.

none;text-autospace:none"> major-latin;mso-hansi-theme-font:major-latin;mso-bidi-font-family:Arial;

fig2.png mso-ascii-theme-font:major-latin;mso-hansi-theme-font:major-latin;mso-bidi-font-family:

none;text-autospace:none"> major-latin;mso-hansi-theme-font:major-latin;mso-bidi-font-family:Arial">

major-latin;mso-bidi-font-family:Arial">Figure 2 – Utilization of Cell
Sensing Platform to detect biomarkers. A) CSP was programmed to detect PDGF.
Blue signal shows incorporation of CSP into cell membrane and green signal
indicated detection of PDGF. Conformational change in PDGF Aptamer
causes its fluorophore and quencher to approximate
and consequently a signal reduction. B) CSP was utilized to detect the presence
of two different single stranded DNA targets. Two internal fluorophores
were incorporated into the MBB as readout for each target. Presence of target
DNA strands results in the displacement of the quencher oligoes
to increase the fluorescent signal recorded from each readout.

none;text-autospace:none"> major-latin;mso-hansi-theme-font:major-latin;mso-ansi-language:EN">