44 research outputs found
Restriction Enzymes as a Target for DNA-Based Sensing and Structural Rearrangement
DNA
nanostructures have been shown viable for the creation of complex
logic-enabled sensing motifs. To date, most of these types of devices
have been limited to the interaction with strictly DNA-type inputs.
Restriction endonuclease represents a class of enzyme with endogenous
specificity to DNA, and we hypothesize that these can be integrated
with a DNA structure for use as inputs to trigger structural transformation
and structural rearrangement. In this work, we reconfigured a three-arm
DNA switch, which utilizes a cyclic FoĢrster resonance energy
transfer interaction between three dyes to produce complex output
for the detection of three separate input regions to respond to restriction
endonucleases, and investigated the efficacy of the enzyme targets.
We demonstrate the ability to use three enzymes in one switch with
no nonspecific interaction between cleavage sites. Further, we show
that the enzymatic digestion can be harnessed to expose an active
toehold into the DNA structure, allowing for single-pot addition of
a small oligo in solution
Time-Gated DNA Photonic Wires with FoĢrster Resonance Energy Transfer Cascades Initiated by a Luminescent Terbium Donor
Functional
DNA nanotechnology is a rapidly growing area of research
with many prospective photonic applications, including roles as wires
and switches, logic operators, and smart biological probes and delivery
vectors. Photonic wire constructs are one such example and comprise
a FoĢrster resonance energy transfer (FRET) cascade between
fluorescent dyes arranged periodically along a DNA scaffold. To date,
the majority of research on photonic wires has focused on setting
new benchmarks for efficient energy transfer over more steps and across
longer distances, using almost exclusively organic fluorescent dyes
and strictly DNA structures. Here, we expand the range of materials
utilized with DNA photonic wires by demonstrating the use of a luminescent
terbium complex (Tb) as an initial donor for a four-step FRET cascade
along a ā¼15 nm long DNA/locked nucleic acid (LNA) photonic
wire. The inclusion of LNA nucleotides increases the thermal stability
of the photonic wires while the Tb affords time-gated emission measurements
and other optical benefits. Time-gating minimizes unwanted background
emission, whether from direct excitation of fluorescent dyes along
the length of the photonic wire, from excess dye-labeled DNA strands
in the sample, or from a biological sample matrix. Observed efficiencies
for Tb-to-dye energy transfer are also closer to the predicted values
than those for dye-to-dye energy transfer, and the Tb can be used
as an initial FRET donor for a variety of next-in-line acceptors at
different spectral positions. We show that the key to using the Tb
as an effective initial donor is to optimally position the next-in-line
acceptor dye in a so-called āsweet spotā where the FRET
efficiency is sufficiently high for practicality, but not so high
as to suppress time-gated emission by shortening the Tb emission lifetime
to within the instrument lag or delay time necessary for measurements.
Overall, the initiation of a time-gated FRET cascade with a Tb donor
is a very promising strategy for the design, characterization, and
application of DNA-based photonic wires and other functional DNA nanostructures
Time-Gated FRET and DNA-Based Photonic Molecular Logic Gates: AND, OR, NAND, and NOR
Molecular logic devices (MLDs) constructed
from DNA are promising
for applications in bioanalysis, computing, and other applications
requiring Boolean logic. These MLDs accept oligonucleotide inputs
and generate fluorescence output through changes in structure. Although
fluorescent dyes are most common in MLD designs, nontraditional luminescent
materials with unique optical properties can potentially enhance MLD
capabilities. In this context, luminescent lanthanide complexes (LLCs)
have been largely overlooked. Here, we demonstrate a set of high-contrast
DNA photonic logic gates based on toehold-mediated strand displacement
and time-gated FRET. The gates include NAND, NOR, OR, and AND designs
that accept two unlabeled target oligonucleotide sequences as inputs.
Bright ātrueā output states utilize time-gated, FRET-sensitized
emission from an Alexa Fluor 546 (A546) dye acceptor paired with a
luminescent terbium cryptate (Tb) donor. Dark āfalseā
output states are generated through either displacement of the A546,
or through competitive and sequential quenching of the Tb or A546
by a dark quencher. Time-gated FRET and the long luminescence lifetime
and spectrally narrow emission lines of the Tb donor enable 4ā10-fold
contrast between Boolean outputs, ā¤10% signal variation for
a common output, multicolor implementation of two logic gates in parallel,
and effective performance in buffer and serum. These metrics exceed
those reported for many other logic gate designs with only fluorescent
dyes and with other non-LLC materials. Preliminary three-input AND
and NAND gates are also demonstrated. The powerful combination of
an LLC FRET donor with DNA-based logic gates is anticipated to have
many future applications in bioanalysis
Peptides for Specifically Targeting Nanoparticles to Cellular Organelles: <i>Quo Vadis</i>?
ConspectusThe interfacing of nanomaterials and especially
nanoparticles within all aspects of biological research continues
to grow at a nearly unabated pace with projected applications focusing
on powerful new tools for cellular labeling, imaging, and sensing,
theranostic materials, and drug delivery. At the most fundamental
level, many of these nanoparticles are meant to target not only very
specific cell-types, regardless of whether they are in a culture,
tissue, an animal model, or ultimately a patient, but also in many
cases a specific subcellular organelle. During this process, these
materials will undergo a complex journey that must first find the
target cell of interest, then be taken up by those cells across the
extracellular membrane, and ultimately localize to a desired subcellular
organelle, which may include the nucleus, plasma membrane, endolysosomal
system, mitochondria, cytosol, or endoplasmic reticulum. To accomplish
these complex tasks in the correct sequence, researchers are increasingly
interested in selecting for and exploiting targeting peptides that
can impart the requisite capabilities to a given nanoparticle construct.
There are also a number of related criteria that need careful consideration
for this undertaking centering on the nature and properties of the
peptide vector itself, the peptideānanoparticle conjugate characteristics,
and the target cell.Here, we highlight some important issues
and key research areas related to this burgeoning field. We begin
by providing a brief overview of some criteria for optimal attachment
of peptides to nanoparticles, the predominant methods by which nanoparticles
enter cells, and some of the peptide sequences that have been utilized
to facilitate nanoparticle delivery to cells focusing on those that
engender the initial targeting and uptake. Because almost all materials
delivered to cells by peptides utilize the endosomal system of vesicular
transport and in many cases remain sequestered within the vesicles,
we critically evaluate the issue of endosomal escape in the context
of some recently reported successes in this regard. Following from
this, peptides that have been reported to deliver nanoparticles to
specific subcellular compartments are examined with a focus on what
they delivered and the putative mechanisms by which they were able
to accomplish this. The last section focuses on two areas that are
critical to realizing this overall approach in the long term. The
first is how to select for peptidyl sequences capable of improved
or more specific cellular or subcellular targeting based upon principles
commonly associated with drug discovery. The second looks at what
has been done to create modular peptides that incorporate multiple
desirable functionalities within a single, contiguous sequence. This
provides a viable alternative to either the almost insurmountable
challenge of finding one sequence capable of all functions or, alternatively,
attaching different peptides with different functionalities to the
same nanoparticle in different ratios when trying to orchestrate their
net effects. Finally, we conclude with a brief perspective on the
future evolution and broader impact of this growing area of bionanoscience
Time-Resolved Nucleic Acid Hybridization Beacons Utilizing Unimolecular and Toehold-Mediated Strand Displacement Designs
Nucleic acid hybridization probes
are sought after for numerous
assay and imaging applications. These probes are often limited by
the properties of fluorescent dyes, prompting the development of new
probes where dyes are paired with novel or nontraditional luminescent
materials. Luminescent terbium complexes are an example of such a
material, and these complexes offer several unique spectroscopic advantages.
Here, we demonstrate two nonstem-loop designs for light-up nucleic
acid hybridization beacons that utilize time-resolved FoĢrster
resonance energy transfer (TR-FRET) between a luminescent Lumi4-Tb
cryptate (Tb) donor and a fluorescent reporter dye, where time-resolved
emission from the dye provides an analytical signal. Both designs
are based on probe oligonucleotides that are labeled at their opposite
termini with Tb and a fluorescent reporter dye. In one design, a probe
is partially blocked with a quencher dye-labeled oligonucleotide,
and target hybridization is signaled through toehold-mediated strand
displacement and loss of a competitive FRET pathway. In the other
design, the intrinsic folding properties of an unblocked probe are
utilized in combination with a temporal mechanism for signaling target
hybridization. This temporal mechanism is based on a recently elucidated
āsweet spotā for TR-FRET measurements and exploits distance
control over FRET efficiencies to shift the Tb lifetime within or
outside the time-gated detection window for measurements. Both the
blocked and unblocked beacons offer nanomolar (femtomole) detection
limits, response times on the order of minutes, multiplexing through
the use of different reporter dyes, and detection in complex matrices
such as serum and blood. The blocked beacons offer better mismatch
selectivity, whereas the unblocked beacons are simpler in design.
The temporal mechanism of signaling utilized with the unblocked beacons
also plays a significant role with the blocked beacons and represents
a new and effective strategy for developing FRET probes for bioassays
Complex Logic Functions Implemented with Quantum Dot Bionanophotonic Circuits
We combine quantum dots (QDs) with
long-lifetime terbium complexes (Tb), a near-IR Alexa Fluor dye (A647),
and self-assembling peptides to demonstrate combinatorial and sequential
bionanophotonic logic devices that function by time-gated FoĢrster
resonance energy transfer (FRET). Upon excitation, the Tb-QD-A647
FRET-complex produces time-dependent photoluminescent signatures from
multi-FRET pathways enabled by the capacitor-like behavior of the
Tb. The unique photoluminescent signatures are manipulated by ratiometrically
varying dye/Tb inputs and collection time. Fluorescent output is converted
into Boolean logic states to create complex arithmetic circuits including
the half-adder/half-subtractor, 2:1 multiplexer/1:2 demultiplexer,
and a 3-digit, 16-combination keypad lock
Multifunctional Liquid Crystal Nanoparticles for Intracellular Fluorescent Imaging and Drug Delivery
A continuing goal of nanoparticle (NP)-mediated drug delivery (NMDD) is the simultaneous improvement of drug efficacy coupled with tracking of the intracellular fate of the nanoparticle delivery vehicle and its drug cargo. Here, we present a robust multifunctional liquid crystal NP (LCNP)-based delivery system that affords facile intracellular fate tracking coupled with the efficient delivery and modulation of the anticancer therapeutic doxorubicin (Dox), employed here as a model drug cargo. The LCNPs consist of (1) a liquid crystal cross-linking agent, (2) a homologue of the organic chromophore perylene, and (3) a polymerizable surfactant containing a carboxylate headgroup. The NP core provides an environment to both incorporate fluorescent dye for spectrally tuned particle tracking and encapsulation of amphiphilic and/or hydrophobic agents for intracellular delivery. The carboxylate head groups enable conjugation to biologicals to facilitate the cellular uptake of the particles. Upon functionalization of the NPs with transferrin, we show the ability to differentially label the recycling endocytic pathway in HEK 293T/17 cells in a time-resolved manner with minimal cytotoxicity and with superior dye photostability compared to traditional organic fluorophores. Further, when passively loaded with Dox, the NPs mediate the rapid uptake and subsequent sustained release of Dox from within endocytic vesicles. We demonstrate the ability of the LCNPs to simultaneously serve as both an efficient delivery vehicle for Dox as well as a modulator of the drugās cytotoxicity. Specifically, the delivery of Dox as a LCNP conjugate results in a ā¼40-fold improvement in its IC<sub>50</sub> compared to free Dox in solution. Cumulatively, our results demonstrate the utility of the LCNPs as an effective nanomaterial for simultaneous cellular imaging, tracking, and delivery of drug cargos
Assembly of a Concentric FoĢrster Resonance Energy Transfer Relay on a Quantum Dot Scaffold: Characterization and Application to Multiplexed Protease Sensing
Semiconductor nanocrystals, or quantum dots (QDs), are one of the most widely utilized nanomaterials for biological applications. Their cumulative physicochemical and optical properties are both unique among nanomaterials and highly advantageous. In particular, FoĢrster resonance energy transfer (FRET) has been widely utilized as a spectroscopic tool with QDs, whether for characterizing QD bioconjugates as a āmolecular rulerā or for modulating QD luminescence āonā and āoffā in biosensing configurations. Here, we investigate the assembly and utility of a new āconcentricā FRET relay that comprises a central QD conjugated with multiple copies of two different peptides, each labeled with one of two fluorescent dyes, Alexa Fluor 555 (A555) or Alexa Fluor 647 (A647). Energy transfer occurs from the QD to the A555 (FRET<sub>1</sub>) then to the A647 (FRET<sub>2</sub>) and, to a lesser extent, directly from the QD to the A647 (FRET<sub>3</sub>). We show that such an arrangement can provide insight into the interfacial distribution of peptides assembled to the QD and can further be utilized for sensing proteolytic activity. In the latter, progress curves for digestion of the assembled peptides by two prototypical proteases, trypsin and chymotrypsin, were measured from the relative QD, A555 and A647 PL contributions, and used to extract MichaelisāMenten kinetic parameters. We further show that the concentric FRET relay, as a single nanoparticle vector, can track the tryptic activation of a proenzyme, chymotrypsinogen, to active chymotrypsin. The concentric FRET relay is thus a potentially powerful tool for the characterization of QD bioconjugates and multiplexed sensing of coupled biological activity
Colloidal Stability of Gold Nanoparticles Coated with Multithiol-Poly(ethylene glycol) Ligands: Importance of Structural Constraints of the Sulfur Anchoring Groups
Gold nanoparticles (AuNPs) coated
with a series of polyĀ(ethylene
glycol) (PEG) ligands appended with four different sulfur-based terminal
anchoring groups (monothiol, flexible dithiol, constrained dithiol,
disulfide) were prepared to explore how the structures of the sulfur-based
anchoring groups affect the colloidal stability in aqueous media.
The PEG-coated AuNPs were prepared by ligand exchange of citrate-stabilized
AuNPs with each ligand. The colloidal stability of the AuNPs in different
harsh environmental conditions was monitored visually and spectroscopically.
The AuNPs coated with dithiol- or disulfide-PEG exhibited improved
stability under high salt concentration and against ligand replacement
competition with dithiothreitol compared with those coated with their
monothiol counterpart. Importantly, the ligands with structurally
constrained dithiol or disulfide showed better colloidal stability
and higher sulfur coverage on the Au surface compared to the ligands
with more flexible dithiol and monothiol. X-ray photoelectron spectroscopy
also revealed that the disulfide-PEG ligand had the highest S coverage
on Au surface on the Au surface among the ligands studied. This result
was supported by energy minimization modeling studies: the structurally
more constrained disulfide ligand has the shortest SāS distance
and could pack more densely on the Au surface. The experimental results
indicate that the colloidal stability of the AuNPs is systematically
enhanced in the following order: monothiol < flexible dithiol <
constrained dithiol < disulfide. The present study indicates that
the colloidal stability of thiolated ligand-functionalized AuNPs can
be enhanced by (i) a multidentate chelating effect and (ii) use of
the constrained and compact structure of the multidentate anchoring
groups
Multiplexed Tracking of Protease Activity Using a Single Color of Quantum Dot Vector and a Time-Gated FoĢrster Resonance Energy Transfer Relay
Semiconductor quantum dots (QDs) are attractive probes
for optical
sensing and imaging due to their unique photophysical attributes and
nanoscale size. In particular, the development of assays and biosensors
based on QDs and FoĢrster resonance energy transfer (FRET) continues
to be a prominent focus of research. Here, we demonstrate the application
of QDs as simultaneous donors and acceptors in a time-gated FRET relay
for the multiplexed detection of protease activity. In contrast to
the current state-of-the-art, which uses multiple colors of QDs, multiplexing
was achieved using only a single color of QD. The other constituents
of the FRET relay, a luminescent terbium complex and fluorescent dye,
were assembled to QDs via peptides that were selected as substrates
for the model proteases trypsin and chymotrypsin. Loss of prompt FRET
between the QD and dye signaled the activity of chymotrypsin; loss
of time-gated FRET between the terbium and QD signaled the activity
of trypsin. We applied the FRET relay in a series of quantitative,
real-time kinetic assays of increasing biochemical complexity, including
multiplexed sensing, measuring inhibition in a multiplexed format,
and tracking the proteolytic activation of an inactive pro-protease
to its active form in a coupled, multienzyme system. These capabilities
were derived from a ratiometric analysis of the two FRET pathways
in the relay and permitted extraction of initial reaction rates, enzyme
specificity constants, and apparent inhibition constants. This work
adds to the growing body of research on multifunctional nanoparticles
and introduces multiplexed sensing as a novel capability for a single
nanoparticle vector. Furthermore, the ability to track both enzymes
within a coupled biological system using one vector represents a significant
advancement for nanoparticle-based biosensing. Prospective applications
in biochemical research, applied diagnostics, and drug discovery are
discussed