Polyvalent Nanobody Structure Designed for Boosting SARS-CoV-2 Inhibition.

Coronavirus transmission and mutations have brought intensive challenges on pandemic control and disease treatment. Developing robust and versatile antiviral drugs for viral neutralization is highly desired. Here, we created a new polyvalent nanobody (Nb) structure that shows the effective inhibition of SARS-CoV-2 infections. Our polyvalent Nb structure, called "PNS", is achieved by first conjugating single-stranded DNA (ssDNA) and the receptor-binding domain (RBD)-targeting Nb with retained binding ability to SARS-CoV-2 spike protein and then coalescing the ssDNA-Nb conjugates around a gold nanoparticle (AuNP) via DNA hybridization with a desired Nb density that offers spatial pattern-matching with that of the Nb binding sites on the trimeric spike. The surface plasmon resonance (SPR) assays show that the PNS binds the SARS-CoV-2 trimeric spike proteins with a ∼1000-fold improvement in affinity than that of monomeric Nbs. Furthermore, our viral entry inhibition assays using the PNS against SARS-CoV-2 WA/2020 and two recent variants of interest (BQ1.1 and XBB) show an over 400-fold enhancement in viral inhibition compared to free Nbs. Our PNS strategy built on a new DNA-protein conjugation chemistry provides a facile approach to developing robust virus inhibitors by using a corresponding virus-targeting Nb with a desired Nb density.

Self-Assembly of DNA Nanostructures in Different Cations.

The programmable nature of DNA allows the construction of custom-designed static and dynamic nanostructures, and assembly conditions typically require high concentrations of magnesium ions that restricts their applications. In other solution conditions tested for DNA nanostructure assembly, only a limited set of divalent and monovalent ions are used so far (typically Mg2+ and Na+ ). Here, we investigate the assembly of DNA nanostructures in a wide variety of ions using nanostructures of different sizes: a double-crossover motif (76 bp), a three-point-star motif (~134 bp), a DNA tetrahedron (534 bp) and a DNA origami triangle (7221 bp). We show successful assembly of a majority of these structures in Ca2+ , Ba2+ , Na+ , K+ and Li+ and provide quantified assembly yields using gel electrophoresis and visual confirmation of a DNA origami triangle using atomic force microscopy. We further show that structures assembled in monovalent ions (Na+ , K+ and Li+ ) exhibit up to a 10-fold higher nuclease resistance compared to those assembled in divalent ions (Mg2+ , Ca2+ and Ba2+ ). Our work presents new assembly conditions for a wide range of DNA nanostructures with enhanced biostability.

Designer DNA NanoGripper.

DNA has shown great biocompatibility, programmable mechanical properties, and structural addressability at the nanometer scale, making it a versatile material for building high precision nanorobotics for biomedical applications. Herein, we present design principle, synthesis, and characterization of a DNA nanorobotic hand, called the "NanoGripper", that contains a palm and four bendable fingers as inspired by human hands, bird claws, and bacteriophages evolved in nature. Each NanoGripper finger has three phalanges connected by two flexible and rotatable joints that are bendable in response to binding to other entities. Functions of the NanoGripper have been enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We showcase that the NanoGripper can be engineered to interact with and capture various objects with different dimensions, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. When carrying multiple DNA aptamer nanoswitches programmed to generate fluorescent signal enhanced on a photonic crystal platform, the NanoGripper functions as a sensitive viral biosensor that detects intact SARS-CoV-2 virions in human saliva with a limit of detection of ~ 100 copies/mL, providing RT-PCR equivalent sensitivity. Additionally, we use confocal microscopy to visualize how the NanoGripper-aptamer complex can effectively block viral entry into the host cells, indicating the viral inhibition. In summary, we report the design, synthesis, and characterization of a complex nanomachine that can be readily tailored for specific applications. The study highlights a path toward novel, feasible, and efficient solutions for the diagnosis and therapy of other diseases such as HIV and influenza.

Self-assembly of DNA nanostructures in different cations.

The programmable nature of DNA allows the construction of custom-designed static and dynamic nanostructures, and assembly conditions typically require high concentrations of magnesium ions which restricts their applications. In other solution conditions tested for DNA nanostructure assembly, only a limited set of divalent and monovalent ions have been used so far (typically Mg 2+ and Na + ). Here, we investigate the assembly of DNA nanostructures in a wide variety of ions using nanostructures of different sizes: a double-crossover motif (76 bp), a three-point-star motif (∼134 bp), a DNA tetrahedron (534 bp) and a DNA origami triangle (7221 bp). We show successful assembly of a majority of these structures in Ca 2+ , Ba 2+ , Na + , K + and Li + and provide quantified assembly yields using gel electrophoresis and visual confirmation of a DNA origami triangle using atomic force microscopy. We further show that structures assembled in monovalent ions (Na + , K + and Li + ) exhibit up to a 10-fold higher nuclease resistance compared to those assembled in divalent ions (Mg 2+ , Ca 2+ and Ba 2+ ). Our work presents new assembly conditions for a wide range of DNA nanostructures with enhanced biostability.

A Target Recycling Amplification Process for the Digital Detection of Exosomal MicroRNAs through Photonic Resonator Absorption Microscopy.

Exosomal microRNAs (miRNAs) have considerable potential as pivotal biomarkers to monitor cancer development, dis-ease progression, treatment effects and prognosis. Here, we report an efficient target recycling amplification process (TRAP) for the digital detection of miRNAs using photonic resonator absorption microscopy. We achieve multiplex digital detection with sub-attomolar sensitivity in 20 minutes, robust selectivity for single nucleotide variants, and a broad dynamic range from 1 aM to 1 pM. Compared with traditional qRT-PCR, TRAP showed similar accuracy in profiling exosomal miRNAs derived from cancer cells, but also exhibited at least 31-fold and 61-fold enhancement in the limits of miRNA-375 and miRNA-21 detection, respectively. The TRAP approach is ideal for exosomal or circulating miRNA biomarker quantification, where the miRNAs are present in low concentrations or sample volume, with potentials for frequent, low-cost, and minimally invasive point-of-care testing.

Identifying and Differentiating Topological G-Quadruplex Structures with DNA-Encoded Plasmonic Gold Nanoparticles.

DNA G-quadruplexes (G4s) have been identified as critical elements in modulating genomic functions and many other biological processes. Their functions are highly dependent on the primary nucleotides and secondary folding structures. Therefore, to understand their functions, methods to identify and differentiate structures of G4 with speed and accuracy are required but limited. In this report, we have applied a synthetic G4 DNA-encoded nanoparticle approach to identify and differentiate G4 DNA molecules with different topologies and nucleotide residues. We found that the resulting plasmonic properties of the gold nanoparticles, monitored by UV/Vis spectroscopy, are quite sensitive to different G4 structures, including stacking layers, loop sequences, capping bases on G4s, and topological structures. Through these systematic investigations, we demonstrate that this G4-encoded gold nanoparticle approach can be used to profile the G4 structures and distinguish G4s from human telomeres. Such a method may have wide applications in G4 research.

Efficient delivery of a DNA aptamer-based biosensor into plant cells for glucose sensing through thiol-mediated uptake.

DNA aptamers have been widely used as biosensors for detecting a variety of targets. Despite decades of success, they have not been applied to monitor any targets in plants, even though plants are a major platform for providing oxygen, food, and sustainable products ranging from energy fuels to chemicals, and high-value products such as pharmaceuticals. A major barrier to progress is a lack of efficient methods to deliver DNA into plant cells. We herein report a thiol-mediated uptake method that more efficiently delivers DNA into Arabidopsis and tobacco leaf cells than another state-of-the-art method, DNA nanostructures. Such a method allowed efficient delivery of a glucose DNA aptamer sensor into Arabidopsis for sensing glucose. This demonstration opens a new avenue to apply DNA aptamer sensors for functional studies of various targets, including metabolites, plant hormones, metal ions, and proteins in plants for a better understanding of the biodistribution and regulation of these species and their functions.

Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In Vivo Using High-Intensity Focused Ultrasound.

Detecting metal ions in vivo with a high spatiotemporal resolution is critical to understanding the roles of the metal ions in both healthy and disease states. Although spatiotemporal controls of metal-ion sensors using light have been demonstrated, the lack of penetration depth in tissue and in vivo has limited their application. To overcome this limitation, we herein report the use of high-intensity focused ultrasound (HIFU) to remotely deliver on-demand, spatiotemporally resolved thermal energy to activate the DNAzyme sensors at the targeted region both in vitro and in vivo. A Zn2+-selective DNAzyme probe is inactivated by a protector strand to block the formation of catalytic enzyme structure, which can then be activated by an HIFU-induced increase in the local temperature. With this design, Zn2+-specific fluorescent resonance energy transfer (FRET) imaging has been demonstrated by the new DNAzyme-HIFU probes in both HeLa cells and mice. The current method can be applied to monitor many other metal ions for in vivo imaging and medical diagnosis using metal-specific DNAzymes that have either been obtained or can be selected using in vitro selection.

Unique sequence-dependent properties of trinucleotide repeat monolayers: electrochemical, electrical, and topographic characterization.

Trinucleotide repeat (TNR) sequences widely exist in nature and their overgrowth is associated with two dozen neurodegenerative diseases in humans. These sequences have a unique helical flexibility, which affects their biophysical properties. A number of biophysical properties of these sequences have been studied in the past except their surface-tethered monolayers. To address the effect of sequence context and the associated helical flexibility on TNR monolayers, disease-relevant TNRs from three flexibility groups were surface-assembled on gold surfaces. The properties of the TNR films were studied, including charge transfer resistance (Rct) by electrochemical impedance spectroscopy (EIS), surface density by chronocoulometry (CC), surface topography by atomic force microscopy (AFM), and electrical conductivity by conducting atomic force microscopy (C-AFM). We found that the TNR film properties are characteristically sequence dependent rather than being dependent on their flexibility rank reported in the literature. The characteristic properties of TNR films studied here may be used for engineering label-free biosensors to detect neurological disorders and build DNA bioelectronics.

Engineering chemical reaction modules via programming the assembly of DNA hairpins.

The architect of enzyme-free chemical reaction modules, working as building blocks in implementing complex computing tasks, was achieved by modulating the assembly of DNA hairpins, including non-catalytic and catalytic systems. The performance of heterogeneous outputted sequences, which were programmed on different hairpins for triggering the downstream reaction, was asymmetric in the non-catalytic system, whereas symmetric in the catalytic system. Furthermore, complicated DNA-only chemical modules possessing controllable species of inputs or outputs were constructed based on our strategy. The kinetic studies revealed that the performance of the chemical modules was toehold length correlated; on the basis of which, a DNA concentration monitor was constructed.

Integrating DNA-strand-displacement circuitry with self-assembly of spherical nucleic acids.

Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures.

The combination of gold nanorods and nanoparticles with DNA nanodevices for logic gates construction.

In this work, two DNA nanodevices were constructed utilizing a DNA strand displacement reaction. With the assistance of gold nanoparticles (AuNPs) and gold nanorods (AuNRs), the autonomous reactions can be reflected from the aggregation states of nanoparticles. By sequence design and the two non-overlapping double hump-like UV-vis spectral peaks of AuNPs and AuNRs, two logic gates with multiple inputs and outputs were successfully run with expected outcomes. This method not only shows how to achieve computing with multiple logic calculations but also has great potential for multiple targets detection.

DNA-Encoded Tuning of Geometric and Plasmonic Properties of Nanoparticles Growing from Gold Nanorod Seeds.

Systematically controlling the morphology of nanoparticles, especially those growing from gold nanorod (AuNR) seeds, are underexplored; however, the AuNR and its related morphologies have shown promises in many applications. Herein we report the use of programmable DNA sequences to control AuNR overgrowth, resulting in gold nanoparticles varying from nanodumbbell to nanooctahedron, as well as shapes in between, with high yield and reproducibility. Kinetic studies revealed two representative pathways for the shape control evolving into distinct nanostructures. Furthermore, the geometric and plasmonic properties of the gold nanoparticles could be precisely controlled by adjusting the base compositions of DNA sequences or by introducing phosphorothioate modifications in the DNA. As a result, the surface plasmon resonance (SPR) peaks of the nanoparticles can be fine-tuned in a wide range, from visible to second near-infrared (NIR-II) region beyond 1000 nm.

What Controls the "Off/On Switch" in the Toehold-Mediated Strand Displacement Reaction on DNA Conjugated Gold Nanoparticles?

In DNA dynamic nanotechnology, a toehold-mediated DNA strand-displacement reaction has demonstrated its capability in building complex autonomous system. In most cases, the reaction is performed in pure DNA solution that is essentially a one-phase system. In the present work, we systematically investigated the reaction in a heterogeneous media, in which the strand that implements a displacing action is conjugated on gold nanoparticles. By monitoring the kinetics of spherical nucleic acid (SNA) assembly driven by toehold-mediated strand displacement reaction, we observed significant differences, i.e., the abrupt jump in behavior of an "off/on switch", in the reaction rate when the invading toehold was extended to eight bases from seven bases. These phenomena are attributed to the effect of steric hindrance arising from the high density of invading strand conjugated to AuNPs. Based on these studies, an INHIBIT logic gate presenting good selectivity was developed.

An efficient DNA-fueled molecular machine for the discrimination of single-base changes.

A new strategy for single-base polymorphism (SNP) detection based on the assembly of DNA-AuNPs (gold nanoparticles) driven by a DNA-fueled molecular machine, is established and optimized. It is highly efficient, works at room temperature, and is easy to handle. A single-base change on an oligonucleotide strand is unambiguously discriminated for either SNPs or insertions and deletions (indels). The strategy is demonstrated to detect a mutation in the breast cancer gene BRCA1 in homogeneous solution at room temperature.

A fluorescent switch for sequentially and selectively sensing copper(II) and L-histidine in vitro and in living cells.

Herein, we report the development of a new fluorescent switch for sequential and selective sensing of Cu(2+) and L-histidine (L-His) in vitro and in living cells for the first time. In the absence of metal ions, Ac-SAACQ-Gly-Gly-Gly-Lys (FITC) (1) exhibits comparable fluorescence to that of free FITC. In the presence of metal ions, 1 selectively coordinates to Cu(2+) , causing its fluorescence emission to be quenched via photoinduced electron transfer. Interestingly, the as-formed 1­Cu(2+) complex selectively responds to L-His among the 20 natural amino acids by turning its fluorescence on. This property of fluorescence switch of 1 was successfully applied for qualitatively and quantitatively sensing Cu(2+) and L-His in vitro. Using this dual functional probe, we also sequentially imaged Cu(2+) and L-His in living HepG2 cells. Our new probe 1 could be applied for not only environmental monitoring but also biomolecule detection in the near future.

Synchronized assembly of gold nanoparticles driven by a dynamic DNA-fueled molecular machine.

A strategy for gold nanoparticle (AuNP) assembly driven by a dynamic DNA-fueled molecular machine is revealed here. In this machine, the aggregation of DNA-functionalized AuNPs is regulated by a series of toehold-mediated strand displacement reactions of DNA. The aggregation rate of the AuNPs can be regulated by controlling the amount of oligonucleotide catalyst. The versatility of the dynamic DNA-fueled molecular machine in the construction of two-component "OR" and "AND" logic gates has been demonstrated. This newly established strategy may find broad potential applications in terms of building up an "interface" that allows the combination of the strand displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, ultimately leading to the fabrication of a wide range of complex multicomponent devices and architectures.