7 research outputs found
Tension Sensing Nanoparticles for Mechano-Imaging at the Living/Nonliving Interface
Studying
chemomechanical coupling at interfaces is important for
fields ranging from lubrication and tribology to microfluidics and
cell biology. Several polymeric macro- and microscopic systems and
cantilevers have been developed to image forces at interfaces, but
few materials are amenable for molecular tension sensing. To address
this issue, we have developed a gold nanoparticle sensor for molecular
tension-based fluorescence microÂscopy. As a proof of concept,
we imaged the tension exerted by integrin receptors at the interface
between living cells and a substrate with high spatial (<1 ÎŒm)
resolution, at 100 ms acquisition times and with molecular specificity.
We report integrin tension values ranging from 1 to 15 pN and a mean
of âŒ1 pN within focal adhesions. Through the use of a conventional
fluorescence microscope, this method demonstrates a force sensitivity
that is 3 orders of magnitude greater than is achievable by traction
force microscopy or polydimethylsiloxane micropost arrays, which are the standard in cellular biomechanics
Tension Sensing Nanoparticles for Mechano-Imaging at the Living/Nonliving Interface
Studying
chemomechanical coupling at interfaces is important for
fields ranging from lubrication and tribology to microfluidics and
cell biology. Several polymeric macro- and microscopic systems and
cantilevers have been developed to image forces at interfaces, but
few materials are amenable for molecular tension sensing. To address
this issue, we have developed a gold nanoparticle sensor for molecular
tension-based fluorescence microÂscopy. As a proof of concept,
we imaged the tension exerted by integrin receptors at the interface
between living cells and a substrate with high spatial (<1 ÎŒm)
resolution, at 100 ms acquisition times and with molecular specificity.
We report integrin tension values ranging from 1 to 15 pN and a mean
of âŒ1 pN within focal adhesions. Through the use of a conventional
fluorescence microscope, this method demonstrates a force sensitivity
that is 3 orders of magnitude greater than is achievable by traction
force microscopy or polydimethylsiloxane micropost arrays, which are the standard in cellular biomechanics
Titin-Based Nanoparticle Tension Sensors Map High-Magnitude Integrin Forces within Focal Adhesions
Mechanical
forces transmitted through integrin transmembrane receptors play important
roles in a variety of cellular processes ranging from cell development
to tumorigenesis. Despite the importance of mechanics in integrin
function, the magnitude of integrin forces within adhesions remains
unclear. Literature suggests a range from 1 to 50 pN, but the upper
limit of integrin forces remains unknown. Herein we challenge integrins
with the most mechanically stable molecular tension probe, which is
comprised of the immunoglobulin 27th (I27) domain of cardiac titin
flanked with a fluorophore and gold nanoparticle. Cell experiments
show that integrin forces unfold the I27 domain, suggesting that integrin
forces exceed âŒ30â40 pN. The addition of a disulfide
bridge within I27 âclampsâ the probe and resists mechanical
unfolding. Importantly, incubation with a reducing agent initiates
SH exchange, thus unclamping I27 at a rate that is dependent on the
applied force. By recording the rate of SâS reduction in clamped
I27, we infer that integrins apply 110 ± 9 pN within focal adhesions
of rat embryonic fibroblasts. The rates of SâS exchange are
heterogeneous and integrin subtype-dependent. Nanoparticle titin tension
sensors along with kinetic analysis of unfolding demonstrate that
a subset of integrins apply tension many fold greater than previously
reported
Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle
Modifying RNA through
either splicing or editing is a fundamental
biological process for creating protein diversity from the same genetic
code. Developing novel chemical biology tools for RNA editing has
potential to transiently edit genes and to provide a better understanding
of RNA biochemistry. Current techniques used to modify RNA include
the use of ribozymes, adenosine deaminase, and tRNA endonucleases.
Herein, we report a nanozyme that is capable of splicing virtually
any RNA stemâloop. This nanozyme is comprised of a gold nanoparticle
functionalized with three enzymes: two catalytic DNA strands with
ribonuclease function and an RNA ligase. The nanozyme cleaves and
then ligates RNA targets, performing a splicing reaction that is akin
to the function of the spliceosome. Our results show that the three-enzyme
reaction can remove a 19 nt segment from a 67 nt RNA loop with up
to 66% efficiency. The complete nanozyme can perform the same splice
reaction at 10% efficiency. These splicing nanozymes represent a new
promising approach for gene manipulation that has potential for applications
in living cells
Catalytic Deoxyribozyme-Modified Nanoparticles for RNAi-Independent Gene Regulation
DNAzymes are catalytic oligonucleotides with important applications in gene regulation, DNA computing, responsive soft materials, and ultrasensitive metal-ion sensing. The most significant challenge for using DNAzymes <i>in vivo</i> pertains to nontoxic delivery and maintaining function inside cells. We synthesized multivalent deoxyribozyme â10-23â gold nanoparticle (DzNP) conjugates, varying DNA density, linker length, enzyme orientation, and linker composition in order to study the role of the steric environment and gold surface chemistry on catalysis. DNAzyme catalytic efficiency was modulated by steric packing and proximity of the active loop to the gold surface. Importantly, the 10-23 DNAzyme was asymmetrically sensitive to the gold surface and when anchored through the 5âČ terminus was inhibited 32-fold. This property was used to generate DNAzymes whose catalytic activity is triggered by thiol displacement reactions or by photoexcitation at λ = 532 nm. Importantly, cell studies revealed that DzNPs are less susceptible to nuclease degradation, readily enter mammalian cells, and catalytically down-regulate GDF15 gene expression levels in breast cancer cells, thus addressing some of the key limitations in the adoption of DNAzymes for <i>in vivo</i> work
Quantum Dots Encapsulated within Phospholipid Membranes: Phase-Dependent Structure, Photostability, and Site-Selective Functionalization
Lipid vesicle encapsulation is an
efficient approach to transfer
quantum dots (QDs) into aqueous solutions, which is important for
renewable energy applications and biological imaging. However, little
is known about the molecular organization at the interface between
a QD and lipid membrane. To address this issue, we investigated the
properties of 3.0 nm CdSe QDs encapsulated within phospholipid membranes
displaying a range of phase transition temperatures (<i>T</i><sub>m</sub>). Theoretical and experimental results indicate that
the QD locally alters membrane structure, and in turn, the physical
state (phase) of the membrane controls the optical and chemical properties
of the QDs. Using photoluminescence, ICP-MS, optical microscopy, and
ligand exchange studies, we found that the <i>T</i><sub>m</sub> of the membrane controls optical and chemical properties
of lipid vesicle-embedded QDs. Importantly, QDs encapsulated within
gel-phase membranes were ultrastable, providing the most photostable
non-core/shell QDs in aqueous solution reported to date. Atomistic
molecular dynamics simulations support these observations and indicate
that membranes are locally disordered displaying greater disordered
organization near the particleâsolution interface. Using this
asymmetry in membrane organization near the particle, we identify
a new approach for site-selective modification of QDs by specifically
functionalizing the QD surface facing the outer lipid leaflet to generate
gold nanoparticleâQD assemblies programmed by WatsonâCrick
base-pairing
Nanoparticle Tension Probes Patterned at the Nanoscale: Impact of Integrin Clustering on Force Transmission
Herein
we aimed to understand how nanoscale clustering of RGD ligands
alters the mechano-regulation of their integrin receptors. We combined
molecular tension fluorescence microscopy with block copolymer micelle
nanolithography to fabricate substrates with arrays of precisely spaced
probes that can generate a 10-fold fluorescence response to pN-forces.
We found that the mechanism of sensing ligand spacing is force-mediated.
This strategy is broadly applicable to investigating receptor clustering
and its role in mechanotransduction pathways