Papers, or it didn't happen.
Detection of cellular microRNAs with programmable DNA nanoswitches.
MicroRNAs are short non-coding regulatory RNAs that are increasingly used as disease biomarkers. Detection of microRNAs can be arduous and expensive, and often requires amplification, labeling, or radioactive probes. Here we report a single-step, non- enzymatic detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNA activated conditional looping of engineered switches), our assay has sub-attomole sensitivity and single-nucleotide specificity using an agarose gel electrophoresis readout. We detect cellular microRNAs from nanogram-scale RNA extracts of differentiating muscle cells, and demonstrate multiplexed detection of several microRNAs from one biological sample. We demonstrate one-hour detection without expensive equipment or reagents, making this assay a compelling alternative to qPCR and Northern blotting.
A ‘smart’tube holder enables real-time sample monitoring in a standard lab centrifuge.
The centrifuge is among the oldest and most widely used pieces of laboratory equipment, with significant applications that include clinical diagnostics and biomedical research. A major limitation of laboratory centrifuges is their “black box” nature, limiting sample observation to before and after centrifugation. Thus, optimized protocols require significant trial and error, while unoptimized protocols waste time by centrifuging longer than necessary or material due to incomplete sedimentation. Here, we developed an instrumented centrifuge tube receptacle compatible with several commercial benchtop centrifuges that can provide real-time sample analysis during centrifugation. We demonstrated the system by monitoring cell separations during centrifugation for different spin speeds, concentrations, buffers, cell types, and temperatures. We verified an adaptation where complete sedimentation turned off the centrifuge and notified the user by a text message. Our system adds new functionality to existing laboratory centrifuges, saving users time and providing useful feedback.
Addressable configurations of DNA nanostructures for rewritable memory.
DNA serves as nature's information storage molecule, and has been the primary focus of engineered systems for biological computing and data storage. Here we combine recent efforts in DNA self-assembly and toehold-mediated strand displacement to develop a rewritable multi-bit DNA memory system. The system operates by encoding information in distinct and reversible conformations of a DNA nanoswitch and decoding by gel electrophoresis. We demonstrate a 5-bit system capable of writing, erasing, and rewriting binary representations of alphanumeric symbols, as well as compatibility with ‘OR’ and ‘AND’ logic operations. Our strategy is simple to implement, requiring only a single mixing step at room temperature for each operation and standard gel electrophoresis to read the data. We envision such systems could find use in covert product labeling and barcoding, as well as secure messaging and authentication when combined with previously developed encryption strategies. Ultimately, this type of memory has exciting potential in biomedical sciences as data storage can be coupled to sensing of biological molecules.
Shear dependent LC purification of an engineered DNA nanoswitch and implications for DNA origami.
As DNA nanotechnology matures, there is increasing need for fast, reliable, and automated purification methods. Here, we develop UHPLC methods to purify self-assembled DNA nanoswitches, which are formed using DNA origami approaches and are designed to change conformations in response to a binding partner. We found that shear degradation hindered LC purification of the DNA nanoswitches, removing oligonucleotides from the scaffold strand and causing loss of function. However, proper choice of column, flow rate, and buffers enabled robust and automated purification of DNA nanoswitches without loss of function in under a half hour. Applying our approach to DNA origami structures, we found that ∼400 nm long nanotubes degraded under the gentlest flow conditions while ∼40 nm diameter nanospheres remained intact even under aggressive conditions. These examples show how fluid stresses can affect different DNA nanostructures during LC purification and suggest that shear forces may be relevant for some applications of DNA nanotechnology. Further development of this approach could lead to fast and automated purification of DNA nanostructures of various shapes and sizes, which would be an important advance for the field.
Click-based functionalization of a 2'-O-propargyl-modified branched DNA nanostructure.
DNA has emerged as a versatile building block for programmable self-assembly. DNA-based nanostructures have been widely applied in biosensing, bioimaging, drug delivery, molecular computation and macromolecular scaffolding. A variety of strategies have been developed to functionalize these nanostructures. In this study, we report a facile click-based strategy to incorporate a metal chelating ligand and a fluorescent tag into a three-point-star DNA tile containing 2′-O-propargyl groups. Such a strategy opens up the possibility of functionalizing pre-assembled DNA strands to construct platforms for metal or drug delivery.
A wireless centrifuge force microscope (CFM) enables multiplexed single-molecule experiments in a commercial centrifuge.
The centrifuge force microscope (CFM) was recently introduced as a platform for massively parallel single-molecule manipulation and analysis. Here we developed a low-cost and self-contained CFM module that works directly within a commercial centrifuge, greatly improving accessibility and ease of use. Our instrument incorporates research grade video microscopy, a power source, a computer, and wireless transmission capability to simultaneously monitor many individually tethered microspheres. We validated the instrument by performing single-molecule force shearing of short DNA duplexes. For a 7 bp duplex, we observed over 1000 dissociation events due to force dependent shearing from 2 pN to 12 pN with dissociation times in the range of 10-100 s. We extended the measurement to a 10 bp duplex, applying a 12 pN force clamp and directly observing single-molecule dissociation over an 85 min experiment. Our new CFM module facilitates simple and inexpensive experiments that dramatically improve access to single-molecule analysis.
Beyond the fold: Emerging biological applications of DNA origami.
The use of DNA as a material for nanoscale construction has blossomed in the past decade. This is largely attributable to the DNA origami technique, which has enabled construction of nanostructures ranging from simple two‐dimensional sheets to complex three‐dimensional objects with defined curves and edges. These structures are amenable to site‐specific functionalization with nanometer precision, and have been shown to exhibit cellular biocompatibility and permeability. The DNA origami technique has already found widespread use in a variety of emerging biological applications such as biosensing, enzyme cascades, biomolecular analysis, biomimetics, and drug delivery. We highlight a few of these applications and comments on the prospects for this rapidly expanding field of research.
Complex thermodynamic behavior of single-stranded nucleic acid adsorption to graphene surfaces.
Graphene oxide has shown promise as a biosensor due to its preferential absorption of single-stranded polynucleotides and fluorescence quenching properties. The rational design of these biosensors, however, requires an improved understanding of the binding thermodynamics and ultimately a predictive model of sequence-specific binding. Toward these goals, here we directly measured the binding of nucleosides and oligonucleotides to graphene oxide nanoparticles using isothermal titration calorimetry and used the results to develop molecular models of graphene–nucleic acid interactions. Experimental and computational results from this study set the platform for informed design of graphene-based biosensors, further increasing their potential and application.
Evolution of DNA origami scaffolds.
AR Chandrasekaran, M Pushpanathan, K Halvorsen. Mater. Lett. 170, 221-224 (2016) [PDF].
Nanoscale materials made using DNA have been increasingly used for applications ranging from biosensors to nanoelectronics. Specifically, DNA origami – where one long single-stranded DNA scaffold is folded into nanoscale shapes and structures using short ‘staple’ oligonucleotides – typically relies on a single-stranded DNA scaffold derived from a viral genome. The sizes of structures that are made rely on the length of the scaffold strand; the most frequently used DNA scaffold is the single-stranded 7249-nucleotide circular M13mp18 genome. Modern techniques used in genome tailoring are now widely exploited for the creation of DNA scaffolds of various lengths for use in DNA origami. DNA scaffolds of lengths ranging from ~700-nucleotides to ~51,000 nucleotides have been prepared using biotechniques such as polymerase chain reaction, a combination of site-directed mutagenesis and site- and ligation independent cloning, and using the molecular toolbox of restriction and ligation enzymes. Such tailor-made DNA sca ffolds allow the creation of origami nanostructures of desired sizes.
Multiplexed single-molecule force spectroscopy using a centrifuge.
We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.
Programmable DNA nanoswitches for detection of nucleic acid sequences.
Detection of nucleic acid sequences is important for applications such as medicine and forensics, but many detection strategies involve multiple time-consuming steps or require expensive lab equipment. Here we report a programmable DNA nanoswitch that undergoes a predefined conformational change upon binding a target sequence, flipping the switch from a linear “off” state to a looped “on” state. The presence of the target sequence is determined without amplification using standard gel electrophoresis to separate the on and off states. We showed sensitivity into the low picomolar range, as well as detection of a single target sequence from both a randomized pool of high concentration oligonucleotides and from a solution of fetal bovine serum (FBS), with no false positive detection in either case. By leveraging the already ubiquitous technique of gel electrophoresis, our low cost approach will be especially accessible to researchers in the biomedical sciences.
DNA nanoswitches: a quantitative platform for gel-based biomolecular interaction analysis.
We introduce a nanoscale experimental platform that enables kinetic and equilibrium measurements of a wide range of molecular interactions using a gel electrophoresis readout. Programmable, self-assembled DNA nanoswitches serve both as templates for positioning molecules and as sensitive, quantitative reporters of molecular association and dissociation. We demonstrated this low-cost, versatile, 'lab-on-a-molecule' system by characterizing ten different interactions, including a complex four-body interaction with five discernible states.
Cross-platform comparison of nucleic acid hybridization: Toward quantitative reference standards.
Measuring interactions between biological molecules is vitally important to both basic and applied research as well as development of pharmaceuticals. Although a wide and growing range of techniques is available to measure various kinetic and thermodynamic properties of interacting biomolecules, it can be difficult to compare data across techniques of different laboratories and personnel or even across different instruments using the same technique. Here we evaluate relevant biological interactions based on complementary DNA and RNA oligonucleotides that could be used as reference standards for many experimental systems. We measured thermodynamics of duplex formation using isothermal titration calorimetry, differential scanning calorimetry, and ultraviolet-visible (UV-vis) monitored denaturation/renaturation. These standards can be used to validate results, compare data from disparate techniques, act as a teaching tool for laboratory classes, or potentially to calibrate instruments. The RNA and DNA standards have many attractive features, including low cost, high purity, easily measurable concentrations, and minimal handling concerns, making them ideal for use as a reference material
Bioinspired multivalent DNA network for capture and release of cells.
Capture and isolation of flowing cells and particulates from body fluids has enormous implications in diagnosis, monitoring, and drug testing, yet monovalent adhesion molecules used for this purpose result in inefficient cell capture and difficulty in retrieving the captured cells. Inspired by marine creatures that present long tentacles containing multiple adhesive domains to effectively capture flowing food particulates, we developed a platform approach to capture and isolate cells using a 3D DNA network comprising repeating adhesive aptamer domains that extend over tens of micrometers into the solution. The DNA network was synthesized from a microfluidic surface by rolling circle amplification where critical parameters, including DNA graft density, length, and sequence, could readily be tailored. Using an aptamer that binds to protein tyrosine kinase-7 (PTK7) that is overexpressed on many human cancer cells, we demonstrate that the 3D DNA network significantly enhances the capture efficiency of lymphoblast CCRF-CEM cells over monovalent aptamers and antibodies, yet maintains a high purity of the captured cells. When incorporated in a herringbone microfluidic device, the 3D DNA network not only possessed significantly higher capture efficiency than monovalent aptamers and antibodies, but also outperformed previously reported cell-capture microfluidic devices at high flow rates. This work suggests that 3D DNA networks may have broad implications for detection and isolation of cells and other bioparticles.
Binary DNA nanostructures for data encryption.
We present a simple and secure system for encrypting and decrypting information using DNA self-assembly. Binary data is encoded in the geometry of DNA nanostructures with two distinct conformations. Removing or leaving out a single component reduces these structures to an encrypted solution of ssDNA, whereas adding back this missing “decryption key” causes the spontaneous formation of the message through self-assembly, enabling rapid read out via gel electrophoresis. Applications include authentication, secure messaging, and barcoding.
Physical manipulation of the Escherichia coli chromosome reveals its soft nature.
Replicating bacterial chromosomes continuously demix from each other and segregate within a compact volume inside the cell called the nucleoid. Although many proteins involved in this process have been identified, the nature of the global forces that shape and segregate the chromosomes has remained unclear because of limited knowledge of the micromechanical properties of the chromosome. In this work, we demonstrate experimentally the fundamentally soft nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment. We developed a unique “micropiston” and measured the force-compression behavior of single Escherichia coli chromosomes in confinement. Our data show that forces on the order of 100 pN and free energies on the order of 105 kBT are sufficient to compress the chromosome to its in vivo size. For comparison, the pressure required to hold the chromosome at this size is a thousand-fold smaller than the surrounding turgor pressure inside the cell. Furthermore, by manipulation of molecular crowding conditions (entropic forces), we were able to observe in real time fast (approximately 10 s), abrupt, reversible, and repeatable compaction–decompaction cycles of individual chromosomes in confinement. In contrast, we observed much slower dissociation kinetics of a histone-like protein HU from the whole chromosome during its in vivo to in vitro transition. These results for the first time provide quantitative, experimental support for a physical model in which the bacterial chromosome behaves as a loaded entropic spring in vivo.
Nanoengineering a single-molecule mechanical switch using DNA self-assembly.
The ability to manipulate and observe single biological molecules has led to both fundamental scientific discoveries and new methods in nanoscale engineering. A common challenge in many single-molecule experiments is reliably linking molecules to surfaces, and identifying their interactions. We have met this challenge by nanoengineering a novel DNA-based linker that behaves as a force-activated switch, providing a molecular signature that can eliminate errant data arising from non-specific and multiple interactions. By integrating a receptor and ligand into a single piece of DNA using DNA self-assembly, a single tether can be positively identified by force–extension behavior, and receptor–ligand unbinding easily identified by a sudden increase in tether length. Additionally, under proper conditions the exact same pair of molecules can be repeatedly bound and unbound. Our approach is simple, versatile and modular, and can be easily implemented using standard commercial reagents and laboratory equipment. In addition to improving the reliability and accuracy of force measurements, this single-molecule mechanical switch paves the way for high-throughput serial measurements, single-molecule on-rate studies, and investigations of population heterogeneity.
Massively parallel single-molecule manipulation using centrifugal force.
Precise manipulation of single molecules has already led to remarkable insights in physics, chemistry, biology, and medicine. However, widespread adoption of single-molecule techniques has been impeded by equipment cost and the laborious nature of making measurements one molecule at a time. We have solved these issues by developing an approach that enables massively parallel single-molecule force measurements using centrifugal force. This approach is realized in an instrument that we call the centrifuge force microscope in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force-field while their micro-to-nanoscopic motions are observed. We demonstrate high-throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Additionally, we verify the force accuracy of the instrument by measuring the well-established DNA overstretching transition at 66 ± 3 pN. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.
High-precision microsphere sorting using velocity sedimentation.
Monodisperse populations of microspheres are desirable for a variety of research and industrial applications, but many desirable sizes and materials can be difficult to synthesize and have limited commercial availability. In this paper, we present an effective, straightforward, and low cost method for sorting polydisperse microspheres into many separate monodisperse samples. The basic approach is to use velocity sedimentation through a density gradient in a long vertical column, followed by carefully targeted extraction. We demonstrate this technique by reducing the coefficient of variation of melamine microspheres from 13% to 1%–4% and glass microspheres from 35% to 3%–8%. This simple and inexpensive method can be used to sort microspheres of many sizes and materials, and is easily scalable, opening the possibility of cheap, monodisperse microspheres.
Mechanoenzymatic cleavage of the ultralarge vascular protein von Willebrand factor.
Von Willebrand factor (VWF) is secreted as ultralarge multimers that are cleaved in the A2 domain by the metalloprotease ADAMTS13 to give smaller multimers. Cleaved VWF is activated by hydrodynamic forces found in arteriolar bleeding to promote hemostasis, whereas uncleaved VWF is activated at lower, physiologic shear stresses and causes thrombosis. Single-molecule experiments demonstrate that elongational forces in the range experienced by VWF in the vasculature unfold the A2 domain, and only the unfolded A2 domain is cleaved by ADAMTS13. In shear flow, tensile force on a VWF multimer increases with the square of multimer length and is highest at the middle, providing an efficient mechanism for homeostatic regulation of VWF size distribution by force-induced A2 unfolding and cleavage by ADAMTS13, as well as providing a counterbalance for VWF-mediated platelet aggregation.
Beyond the frame rate: Measuring high-frequency fluctuations with light-intensity modulation.
Power spectral density measurements of any sampled signal are typically restricted by both acquisition rate and frequency response limitations of instruments, which can be particularly prohibitive for video-based measurements. We have developed a new method called Intensity Modulation Spectral Analysis (IMSA) that circumvents these limitations, dramatically extending the effective detection bandwidth. We demonstrate this by video-tracking an optically-trapped microsphere while oscillating an LED illumination source. This approach allows us to quantify fluctuations of the microsphere at frequencies over 10 times higher than the Nyquist frequency, mimicking a significantly higher frame rate.
A new approach to analysis of single-molecule force measurements.
E Evans, K Halvorsen, K Kinoshita, WP Wong. Handbook of Single-Molecule Biophysics, 571-589 (2009).
A common aim in probing single molecular bonds or the structural stability of proteins is to measure the kinetic rates at which a bond dissociates or a protein changes conformation under conditions of changing force. Using sample data taken from tests of ligand–receptor unbinding and protein unfolding/refolding, we show that populations of “single molecule” events, arranged into statistical arrays expressing the numbers of bonds or initial conformers remaining as a function of time and cumulated into histograms of transitions over fixed time increments, provide the bases for a model-independent assay of the kinetic rates of transition throughout the course of an experiment. Most important, this assay for kinetic rates can be employed with any deterministic mode of force spectroscopy, whether the pulling force increases or decreases with time.
Imaging biomolecular interactions by fast three-dimensional tracking of laser-confined carrier particles.
The quantitative study of the near-equilibrium structural behavior of individual biomolecules requires high-resolution experimental approaches with longtime stability. We present a new technique to explore the dynamics of weak intramolecular interactions. It is based on the analysis of the 3D Brownian fluctuations of a laser-confined glass bead that is tethered to a flat surface by the biomolecule of interest. A continuous autofocusing mechanism allows us to maintain or adjust the height of the optical trap with nanometer accuracy over long periods of time. The resulting remarkably stable trapping potential adds a well-defined femto-to-piconewton force bias to the energy landscape of molecular configurations. A combination of optical interferometry and advanced pattern-tracking algorithms provides the 3D bead positions with nanometer spatial and >120 Hz temporal resolution. The analysis of accumulated 3D positions has allowed us not only to identify small single biomolecules but also to characterize their nanomechanical behavior, for example, the force−extension relations of short oligonucleotides and the unfolding/refolding transitions of small protein tethers.
The effect of integration time on fluctuation measurements: calibrating an optical trap in the presence of motion blur.
WP Wong, K Halvorsen. Opt. Express 14, 12517-12531 (2006) [PDF].
Dynamical instrument limitations, such as finite detection bandwidth, do not simply add statistical errors to fluctuation measurements, but can create significant systematic biases that affect the measurement of steady-state properties. Such effects must be considered when calibrating ultra-sensitive force probes by analyzing the observed Brownian fluctuations. In this article, we present a novel method for extracting the true spring constant and diffusion coefficient of a harmonically confined Brownian particle that extends the standard equipartition and power spectrum techniques to account for video-image motion blur. These results are confirmed both numerically with a Brownian dynamics simulation, and experimentally with laser optical tweezers.