Aptamers for Viral Detection and Inhibition.

Recent times have experienced more than ever the impact of viral infections in humans. Viral infections are known to cause diseases not only in humans but also in plants and animals. Here, we have compiled the literature review of aptamers selected and used for detection and inhibition of viral infections in all three categories: humans, animals, and plants. This review gives an in-depth introduction to aptamers, different types of aptamer selection (SELEX) methodologies, the benefits of using aptamers over commonly used antibody-based strategies, and the structural and functional mechanism of aptasensors for viral detection and therapy. The review is organized based on the different characterization and read-out tools used to detect virus-aptasensor interactions with a detailed index of existing virus-targeting aptamers. Along with addressing recent developments, we also discuss a way forward with aptamers for DNA nanotechnology-based detection and treatment of viral diseases. Overall, this review will serve as a comprehensive resource for aptamer-based strategies in viral diagnostics and treatment.

DNA modification and visualization on an origami-based enzyme nano-factory.

The past decade has seen enormous progress in DNA nanotechnology through the advent of DNA origami. Functionalizing the DNA origami for multiple applications is the recent focus of this field. Here we have constructed a novel DNA enzyme nano-factory, which modifies target DNA embedded on a DNA origami platform. The enzyme is programmed to reside in close proximity to the target DNA which enhances significantly the local concentration compared to solution-based DNA modification. To demonstrate this we have immobilized DNA methyltransferase M·TaqI next to the target DNA on the DNA origami and used this enzyme to sequence-specifically modify the target DNA with biotin using a cofactor analogue. Streptavidin binding to biotin is applied as a topographic marker to follow the machine cycle of this enzyme nano-factory using atomic force microscopy imaging. The nano-factory is demonstrated to be recyclable and holds the potential to be expanded to a multi-enzyme, multi-substrate operating system controlled by simple to complex molecules made of DNA, RNA or proteins.

DNA Translocation through Vertically Stacked 2D Layers of Graphene and Hexagonal Boron Nitride Heterostructure Nanopore.

Cost-effective, fast, and reliable DNA sequencing can be enabled by advances in nanopore-based methods, such as the use of atomically thin graphene membranes. However, strong interaction of DNA bases with graphene leads to undesirable effects such as sticking of DNA strands to the membrane surface. While surface functionalization is one way to counter this problem, here, we present another solution based on a heterostructure nanopore system, consisting of a monolayer of graphene and hexagonal boron nitride (hBN) each. Molecular dynamics studies of DNA translocation through this heterostructure nanopore revealed a surprising and crucial influence of the heterostructure layer order in controlling the base specific signal variability. Specifically, the heterostructure with graphene on top of hBN had nearly 3-10× lower signal variability than the one with hBN on top of graphene. Simulations point to the role of differential underside sticking of DNA bases as a possible reason for the observed influence of the layer order. Our studies can guide the development of experimental systems to study and exploit DNA translocation through two-dimensional heterostructure nanopores for single molecule sequencing and sensing applications.

Aptamer-Programmed DNA Nanodevices for Advanced, Targeted Cancer Theranostics.

DNA has been demonstrated to be a versatile material for construction at the nanoscale. DNA nanodevices are highly programmable and allow functionalization with multiple entities such as imaging modalities (fluorophores), targeting entities (aptamers), drug conjugation (chemical linkers), and triggered release (photoresponsive molecules). These features enhance the use of DNA nanodevices in biological applications, catalyzing the rapid growth of this domain of research. In this review, we focus on recent progress in the development and use of aptamer-functionalized DNA nanodevices as theranostic agents, their characterization, applications as delivery platforms, and advantages. We provide a brief background on the development of aptamers and DNA nanodevices in biomedical applications, and we present specific applications of these entities in cancer diagnosis and therapeutics. We conclude with a perspective on the challenges and possible solutions for the clinical translation of aptamer-functionalized DNA nanodevices in the domain of cancer therapeutics.

Effect of single nanoparticle-nanopore interaction strength on ionic current modulation.

Solid-state nanopores are rapidly emerging as promising platforms for developing various single molecule sensing applications. The modulation of ionic current through the pore due to translocation of the target molecule has been the dominant measurement modality in nanopore sensors. Here, we focus on the dwell time, which is the duration taken by the target molecule or particle to traverse the pore and study its dependence on the strength of interaction of the target with the pore using single gold nanoparticles (NPs) as targets interacting with a silicon nitride (SiN) nanopore. The strength of interaction, which in our case is electrostatic in nature, can be controlled by coating the nanoparticles with charged polymers. We report on an operating regime of this nanopore sensor, characterized by attractive interactions between the nanoparticle and the pore, where the dwell time is exponentially sensitive to the target-pore interaction. We used negatively and positively charged gold nanoparticles to control the strength of their interaction with the Silicon Nitride pore which is negatively charged. Our experiments revealed how this modulation of the electrostatic force greatly affects the dwell time. Positively charged NPs with strong attractive interactions with the pore resulted in increase of dwell times by 2-3 orders of magnitude, from 0.4 ms to 75.3 ms. This extreme sensitivity of the dwell time on the strength of interaction between a target and nanopore can be exploited in emerging nanopore sensor applications.

DNA Translocation through Hybrid Bilayer Nanopores.

Pore functionalization has been explored by several groups as a strategy to control DNA translocation through solid-state nanopores. Here we present a hybrid nanopore system consisting of single-layer graphene and a DNA origami layer to achieve base-selective control of DNA translocation rate through aligned nanopores of the two layers. This is achieved by incorporating unpaired dangling bases called overhangs to the origami near the pore region. Molecular dynamics simulations were used to optimize the design of the origami nanopore and the overhangs. Specifically, we considered the influence of the number and spatial distribution of overhangs on translocation times. The simulations revealed that specific interactions between the overhangs and the translocating single-stranded DNA resulted in base-specific residence times.

Atomic structures of RNA nanotubes and their comparison with DNA nanotubes.

We present a computational framework to model RNA based nanostructures and study their microscopic structures. We model hexagonal nanotubes made of 6 dsRNA (RNTs) connected by double crossover (DX) at different positions. Using several hundred nano-second (ns) long all-atom molecular dynamics simulations, we study the atomic structure, conformational change and elastic properties of RNTs in the presence of explicit water and ions. Based on several structural quantities such as root mean square deviation (RMSD) and root mean square fluctuation (RMSF), we find that the RNTs are almost as stable as DNA nanotubes (DNTs). Although the central portion of the RNTs maintain its cylindrical shape, both the terminal regions open up to give rise to a gating like behavior which can play a crucial role in drug delivery. From the bending angle distribution, we observe that the RNTs are more flexible than DNTs. The calculated persistence length of the RNTs is in the micron range which is an order of magnitude higher than that of a single dsRNA. The stretch modulus of the RNTs from the contour length distribution is in the range of 4-7 nN depending on the sequence. The calculated persistence length and stretch modulus are in the same range of values as in the case of DNTs. To understand the structural properties of RNTs at the individual base-pair level we have also calculated all the helicoidal parameters and analyzed the relative flexibility and rigidity of RNTs having a different sequence. These findings emphasized the fascinating properties of RNTs which will expedite further theoretical and experimental studies in this field.

Smartphone-based kanamycin sensing with ratiometric FRET.

Smartphone-based fluorescence detection is a promising avenue for biosensing that can aid on-site analysis. However, quantitative detection with fluorescence in the field has been limited due to challenges with robust excitation and calibration requirements. Here, we show that ratiometric analysis with Förster resonance energy transfer (FRET) between dye pairs on DNA aptamers can enable rapid and sensitive kanamycin detection. Since our detection scheme relies on ligand binding-induced changes in the aptamer tertiary structure, it is limited only by the kinetics of ligand binding to the aptamer. Our FRET-based kanamycin binding aptamer (KBA) sensor displays two linear ranges of 0.05-5 nM (detection limit of 0.18 nM) and 50-900 nM of kanamycin. The aptamer displays high specificity even in the presence of the 'natural' background from milk. By immobilizing the aptamer in the flow cell, our KBA sensor design is also suitable for repeated kanamycin detection. Finally, we show that the ratiometric FRET-based analysis can be implemented on a cheap custom-built smartphone setup. This smartphone-based FRET aptamer scheme detects kanamycin in a linear range of 50-500 nM with a limit of detection (LOD) of 28 nM.

Paranemic Crossover DNA: There and Back Again.

Over the past 35 years, DNA has been used to produce various nanometer-scale constructs, nanomechanical devices, and walkers. Construction of complex DNA nanostructures relies on the creation of rigid DNA motifs. Paranemic crossover (PX) DNA is one such motif that has played many roles in DNA nanotechnology. Specifically, PX cohesion has been used to connect topologically closed molecules, to assemble a three-dimensional object, and to create two-dimensional DNA crystals. Additionally, a sequence-dependent nanodevice based on conformational change between PX and its topoisomer, JX2, has been used in robust nanoscale assembly lines, as a key component in a DNA transducer, and to dictate polymer assembly. Furthermore, the PX motif has recently found a new role directly in basic biology, by possibly serving as the molecular structure for double-stranded DNA homology recognition, a prominent feature of molecular biology and essential for many crucial biological processes. This review discusses the many attributes and usages of PX-DNA-its design, characteristics, applications, and potential biological relevance-and aims to accelerate the understanding of PX-DNA motif in its many roles and manifestations.

Covalent Linkage of One-Dimensional DNA Arrays Bonded by Paranemic Cohesion.

The construction of DNA nanostructures from branched DNA motifs, or tiles, typically relies on the use of sticky-ended cohesion, owing to the specificity and programmability of DNA sequences. The stability of such constructs when unligated is restricted to a specific range of temperatures, owing to the disruption of base pairing at elevated temperatures. Paranemic (PX) cohesion was developed as an alternative to sticky ends for the cohesion of large topologically closed species that could be purified reliably on denaturing gels. However, PX cohesion is also of limited stability. In this work, we added sticky-ended interactions to PX-cohesive complexes to create interlocked complexes by functionalizing the sticky ends with psoralen, which can form cross-links between the two strands of a double helix. We were able to reinforce the stability of the constructs by creating covalent linkages between the 3'-ends and 5'-ends of the sticky ends; the sticky ends were added to double crossover domains via 3'-3' and 5'-5' linkages. Catenated arrays were obtained either by enzymatic ligation or by UV cross-linking. We have constructed finite-length one-dimensional arrays linked by interlocking loops and have positioned streptavidin-gold particles on these constructs.

A Programmable Transducer Self-Assembled from DNA.

A transducer consists of an input/output alphabet, a finite set of states, and a transition function. From an input symbol applied to a given state, the transition function determines the next state, and an output symbol. Using DNA, we have constructed a transducer that divides a number by 3. The input consists of a series of individually addressable 2-state DNA nanomechanical devices that control the orientations of a group of flat 6-helix DNA motifs; these motifs have edge domains tailed in sticky ends corresponding to the numbers 0 and 1. Three-domain DNA molecules (TX tiles) act as computational tiles that correspond to the transitions that the transducer can undergo. The output domain of these TX tiles contains sticky ends that also correspond to 0 or 1. Two different DNA tiles can chelate these output domains: A 5 nm gold nanoparticle is attached to the chelating tile that binds to 0-domains and a 10 nm gold nanoparticle is attached to the chelating tile that binds to 1-domains. The answer to the division is represented by the series of gold nanoparticles, which can be interpreted as a binary number. The answers of the computation are read out by examination of the transducer complexes under a transmission electron microscope. The start or end points of the output sequence can be indicated by the presence of a 15 nm gold nanoparticle. This work demonstrates two previously unreported features integrated in a single framework: [1] a system that combines DNA algorithmic self-assembly with DNA nanomechanical devices that control that input, and [2] the arrangement of non-DNA species, here metallic nanoparticles, through DNA algorithmic self-assembly. The nanomechanical devices are controlled by single-stranded DNA strands, allowing multiple input sequences to be applied to the rest of the system, thus guiding the algorithmic self-assembly to a variety of outputs.

Analyte-driven switching of DNA charge transport: de novo creation of electronic sensors for an early lung cancer biomarker.

A general approach is described for the de novo design and construction of aptamer-based electrochemical biosensors, for potentially any analyte of interest (ranging from small ligands to biological macromolecules). As a demonstration of the approach, we report the rapid development of a made-to-order electronic sensor for a newly reported early biomarker for lung cancer (CTAP III/NAP2). The steps include the in vitro selection and characterization of DNA aptamer sequences, design and biochemical testing of wholly DNA sensor constructs, and translation to a functional electrode-bound sensor format. The working principle of this distinct class of electronic biosensors is the enhancement of DNA-mediated charge transport in response to analyte binding. We first verify such analyte-responsive charge transport switching in solution, using biochemical methods; successful sensor variants were then immobilized on gold electrodes. We show that using these sensor-modified electrodes, CTAP III/NAP2 can be detected with both high specificity and sensitivity (K(d) ~1 nM) through a direct electrochemical reading. To investigate the underlying basis of analyte binding-induced conductivity switching, we carried out Förster Resonance Energy Transfer (FRET) experiments. The FRET data establish that analyte binding-induced conductivity switching in these sensors results from very subtle structural/conformational changes, rather than large scale, global folding events. The implications of this finding are discussed with respect to possible charge transport switching mechanisms in electrode-bound sensors. Overall, the approach we describe here represents a unique design principle for aptamer-based electrochemical sensors; its application should enable rapid, on-demand access to a class of portable biosensors that offer robust, inexpensive, and operationally simplified alternatives to conventional antibody-based immunoassays.

Adenosine-triggered elimination of methylene blue noncovalently bound to immobilized functional dsDNA-aptamer constructs.

Immobilization and electrochemical characterization of specially designed functional DNA-aptamer constructs are of great importance for the development of versatile biosensors (not limited to gene analysis) and the investigation of molecular interactions between DNA and other molecules. We have constructed a "DNA conformational switch" by incorporating the antiadenosine aptamer sequence in the middle of an otherwise cDNA double helix, as its structural change responds to the presence of small molecule ligands (e.g., adenosine). In particular, methylene blue (MB) was used as a model system to probe the rather complex interaction modes between small redox molecules and the dsDNA-aptamer construct. Besides intercalating with the double-stranded DNA stem, MB can stack with a single guanine base in the relatively unstructured aptamer domain or electrostatically bind to the DNA backbone. The decreased surface density of MB after adenosine binding indicated that the ligand-gated structural change of the dsDNA-aptamer construct can eliminate MB molecules that were originally bound to the aptamer domain but not those in the complementary stem.

The label-free unambiguous detection and symbolic display of single nucleotide polymorphisms on DNA origami.

Single nucleotide polymorphisms (SNPs) are the most common genetic variation in the human genome. Kinetic methods based on branch migration have proved successful for detecting SNPs because a mispair inhibits the progress of branch migration in the direction of the mispair. We have combined the effectiveness of kinetic methods with atomic force microscopy of DNA origami patterns to produce a direct visual readout of the target nucleotide contained in the probe sequence. The origami contains graphical representations of the four nucleotide alphabetic characters, A, T, G and C, and the symbol containing the test nucleotide identity vanishes in the presence of the probe. The system also works with pairs of probes, corresponding to heterozygous diploid genomes.

A DNA-based nanomechanical device with three robust states.

DNA has been used to build a variety of devices, ranging from those that are controlled by DNA structural transitions to those that are controlled by the addition of specific DNA strands. These sequence-dependent devices fulfill the promise of DNA in nanotechnology because a variety of devices in the same physical environment can be controlled individually. Many such devices have been reported, but most of them contain one or two structurally robust end states, in addition to a floppy intermediate or even a floppy end state. We describe a system in which three different structurally robust end states can be obtained, all resulting from the addition of different set strands to a single floppy intermediate. This system is an extension of the PX-JX(2) DNA device. The three states are related to each other by three different motions, a twofold rotation, a translation of approximately 2.1-2.5 nm, and a twofold screw rotation, which combines these two motions. We demonstrate the transitions by gel electrophoresis, by fluorescence resonance energy transfer, and by atomic force microscopy. The control of this system by DNA strands opens the door to trinary logic and to systems containing N devices that are able to attain 3(N) structural states.