Nano Letters

DNA nanotechnology relies on programmable anchoring of regions of single-stranded DNA through base pair hybridization to create nanoscale objects such as polyhedra, tubes, sheets, and other desired shapes. Recent work from our lab measured the energetics of base-stacking interactions and suggested that terminal stacking interactions between two adjacent strands could be an additional design parameter for DNA nanotechnology. Here, we explore that idea by creating DNA tetrahedra held together with sticky ends that contain identical base pairing interactions but different terminal stacking interactions. Testing all 16 possible combinations, we found that the melting temperature of DNA tetrahedra varied by up to 10 {degrees}C from altering a single base stack in the design. These results can inform stacking design to control DNA tetrahedra stability in a substantial and predictable way. To that end, we show that a 4 bp sticky end with weak terminal stacking does not form stable tetrahedra, while strengthening the stacks confers high stability with a 46.8 {+/-} 1.2 {degrees}C melting temperature, comparable to a 6 bp sticky end with weak stacking (49.7 {+/-} 2.9 {degrees}C). The results likely apply to other types of DNA nanostructures and suggest that terminal stacking interactions play an integral role in formation and stability of DNA nanostructures.

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

Coherent Raman scattering imaging has provided inherent chemical information of biomolecules without the need for any external labels.1-3 However, its working depth in deep-tissue imaging is extremely shallow because both the intrinsic scattering cross-section and image contrast are so small that even weak perturbation of the pump and Stokes beam focusing by the complex tissue causes the loss of the resolving power.4,5 Here, we propose a deep-tissue coherent Raman scattering (CRS) microscopy equipped with an advanced adaptive optics (AO) system measuring complex tissue aberration from elastic backscattering. Using this label-free AO-CRS microscopy, we demonstrate the vibrational imaging of lipid-rich substances such as myelin inside the mouse brain even through the thick and opaque cranial bones.

Optical nanoscale technologies often implement covalent or noncovalent strategies for the modification of nanoparticles, whereby both functionalizations are leveraged for multimodal applications but can affect the intrinsic fluorescence of nanoparticles. Specifically, single-walled carbon nanotubes (SWCNTs) can enable real-time imaging and cellular delivery; however, the introduction of covalent SWCNT sidewall functionalizations often attenuates SWCNT fluorescence. Herein, we leverage recent advances in SWCNT covalent functionalization chemistries that preserve the SWCNTs pristine graphitic lattice and intrinsic fluorescence and demonstrate that such covalently functionalized SWCNTs maintain fluorescence-based molecular recognition of neurotransmitter and protein analytes. We show that the covalently modified SWCNT nanosensor fluorescence response towards its analyte is preserved for certain nanosensors, presumably dependent on the steric hindrance introduced by the covalent functionalization that hinders noncovalent interactions with the SWCNT surface. We further demonstrate that these SWCNT nanosensors can be functionalized via their covalent handles to self-assemble on passivated microscopy slides, and discuss future use of these dual-functionalized SWCNT materials for multiplexed applications.

In the last decade, optical imaging methods have significantly improved our understanding of the information processing principles in the brain. Although many promising tools have been designed, sensors of membrane potential are lagging behind the rest. Semiconductor nanoparticles are an attractive alternative to classical voltage indicators, such as voltage-sensitive dyes and proteins. Such nanoparticles exhibit high sensitivity to external electric fields via the quantum-confined Stark effect. Here we report the development of lipid-coated semiconductor voltage-sensitive nanorods (vsNRs) that self-insert into the neuronal membrane. We describe a workflow to detect and process the photoluminescent signal of vsNRs after wide-field time-lapse recordings. We also present data indicating that vsNRs are feasible for sensing membrane potential in neurons at a single-particle level. This shows the potential of vsNRs for detection of neuronal activity with unprecedentedly high spatial and temporal resolution.

When a nanoparticle enters a biological environment, the surface is rapidly coated with proteins to form a "protein corona". Presence of the protein corona surrounding the nanoparticle has significant implications for applying nanotechnologies within biological systems, affecting outcomes such as biodistribution and toxicity. Herein, we measure protein corona formation on single-stranded DNA wrapped single-walled carbon nanotubes (ssDNA-SWCNTs), a high-aspect ratio nanoparticle ideal for sensing and delivery applications, and polystyrene nanoparticles, a model nanoparticle system. The protein corona of each nanoparticle is studied in human blood plasma and cerebrospinal fluid. We characterize corona composition by proteomic mass spectrometry to determine abundant and differentially enriched vs. depleted corona proteins. High-binding corona proteins on ssDNA-SWCNTs include proteins involved in lipid binding and transport (clusterin and apolipoprotein A-I), complement activation (complement C3), and blood coagulation (fibrinogen). Of note, albumin is the most common blood protein (55% w/v), yet exhibits low-binding affinity towards ssDNA-SWCNTs, displaying 1300-fold lower bound concentration relative to native plasma. We investigate the role of electrostatic and entropic interactions driving selective protein corona formation, and find that hydrophobic interactions drive inner corona formation, while shielding of electrostatic interactions allows for outer corona formation. Lastly, we study real-time binding of proteins on ssDNA-SWCNTs and find relative agreement between proteins that are enriched and bind strongly, such as fibrinogen, and proteins that are depleted and bind marginally, such as albumin. Interestingly, certain proteins express contrary behavior in single-protein experiments than within the whole biofluid, highlighting the importance of cooperative mechanisms driving selective corona adsorption on the SWCNT surface. Knowledge of the protein corona composition, dynamics, and structure informs translation of engineered nanoparticles from in vitro design to effective in vivo application.

Developing insect cyborgs by integrating external components (optical, electrical or mechanical) with biological counterparts has a potential to offer elegant solutions for complex engineering problems.1 A key limiting step in the development of such biorobots arises at the nano-bio interface, i.e. between the organism and the nano implant that offers remote controllability.1,2 Often, invasive procedures are necessary that tend to severely compromise the navigation capabilities as well as the longevity of such biorobots. Therefore, we sought to develop a non-invasive solution using plasmonic nanostructures that can be photoexcited to generate heat with spatial and temporal control. We designed a nanotattoo using silk that can interface the plasmonic nanostructures with a biological tissue. Our results reveal that both structural and functional integrity of the biological tissues such as insect antenna, compound eyes and wings were preserved after the attachment of the nanotattoo. Finally, we demonstrate that insects with the plasmonic nanotattoos can be remote controlled using light and integrated with functional recognition elements to detect the chemical environment in the region of interest. In sum, we believe that the proposed technology will play a crucial role in the emerging fields of biorobotics and other nano-bio applications.

Due to the unique spatial addressability of DNA origami, targeting ligands (e.g. aptamers or antibodies) can be specifically positioned onto the surface of the nanostructure, constituting an essential tool for studying ligand-receptor interactions at the cell surface. While the design and ligand incorporation into DNA origami nanostructures are well-established, the study of cell surface interaction dynamics is still in the explorative phase, where in depth fundamental understanding on the molecular interactions remains underexplored. This study uniquely captures real-time encounters between DNA origami and cells in-situ using single particle tracking (SPT). Here, we functionalized DNA nanorods (NRs) with antibodies or aptamers specific to the epidermal growth factor receptor (EGFR) and used them to target EGFR-overexpressing cancer cells. SPT data revealed that ligand coated NRs selectively bound to the receptors expressed in target cancer cells, while non-functionalized NRs only display negligible cell interactions. Furthermore, we explored the effect of ligand density on the DNA origami, which revealed that aptamer-decorated NRs exhibit non-linear binding characteristics, whereas this effect in antibody-decorated NRs was less pronounced. This study provides new mechanistic insights into the fundamental understanding of DNA origami behaviour at the cell interface, with unprecedented spatiotemporal resolution, aiding the rational design of ligand-targeted DNA origami for biomedical applications.

The ability to noninvasively detect bacteria at any depth inside opaque tissues has important applications ranging from infection diagnostics to tracking therapeutic microbes in their mammalian host. Current examples of probes for detecting bacteria with strain-type specificity are largely based on optical dyes, which cannot be used to examine bacteria in deep tissues due to the physical limitation of light scattering. Here, we describe a new biomolecular probe for visualizing bacteria in a cell-type specific fashion using magnetic resonance imaging (MRI). The probe is based on a peptide that selectively binds manganese and is attached in high numbers to the capsid of filamentous phage. By genetically engineering phage particles to display this peptide, we are able to bring manganese ions to specific bacterial cells targeted by the phage, thereby producing MRI contrast. We show that this approach allows MRI-based detection of targeted E. coli strains while discriminating against non-target bacteria as well as mammalian cells. By engineering the phage coat to display a protein that targets cell surface receptors in V. cholerae, we further show that this approach can be applied to image other bacterial targets with MRI. Finally, as a preliminary example of in vivo applicability, we demonstrate MR imaging of phage-labeled V. cholerae cells implanted subcutaneously in mice. The nanomaterial developed here thus represents a path towards noninvasive detection and tracking of bacteria by combining the programmability of phage architecture with the ability to produce three- dimensional images of biological structures at any arbitrary depth with MRI.

Small extracellular vesicles (sEVs), or exosomes, play important roles in physiological and pathological cellular communication. sEVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. Studying the intricacies of sEV function and RNA-based communication requires tools capable of labeling sEV RNA. Here we developed a novel genetically encodable reporter system for tracking sEV RNAs comprising an sEV-loading RNA sequence, termed the EXO-Code, fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of RNA puncta and colocalization with markers of multivesicular bodies; imaging RNA puncta within sEVs; and quantification of sEVs. This technology represents a useful and versatile tool to interrogate the role of sEVs in cellular communication via RNA trafficking to sEVs, cellular sorting decisions, and sEV RNA cargo transfer to recipient cells.

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000s of bases are folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent-protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein (RNP) binding sites on the DNA nanostructures to increase shuttling of structures into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as measuring biophysical properties, into cell nuclei. Teaser SentenceCRISPR-Cas9 mediates nuclear transport and integration of nanostructured genes in human primary cells

The aggregation state and endosomal trapping of engineered nanocarriers once internalized into cells remain poorly characterized. Here, we visualized the membrane penetrating dynamics of semiconductor quantum dots (QDs) into the cytosol of T cells on a single-cell and single-nanoparticle basis. We water solubilized CdSe/CdZnS QDs with polymer encapsulants functionalized with a cell-penetrating peptide composed of an Asp-Ser-Ser (DSS) repeat sequence. T cells tolerated the 24-h incubation with QDs at concentrations of 5 nM or lower. Single-particle imaging demonstrated that the number of internalized nanoparticles was dependent upon the concentration of the probes for both control (peptide-free) and DSS-QDs. DSS-QDs were mostly distributed as monomers, whereas the control QDs were aggregated into clusters. Single-particle tracking using total internal reflection and highly inclined illumination showed that DSS-QDs were stationary near the activating surface and mobile within the cytosol of the T cell. A correlation exhibited between the mobility and aggregation state of individual QD clusters, with monomeric DSS-QDs showing the highest mobility. In addition, monomeric DSS-QDs displayed much faster diffusion than the endosomes. A small-molecule endosome marker confirmed the absence of colocalization between endosomes and DSS-QDs, indicating their endosomal escape. The ability to deliver and track individual QDs in the cytosol of live T cells creates inroads for the optimization of drug delivery and gene therapy through the use of nanoparticles.

Raman spectroscopy provides excellent specificity for in vivo preclinical imaging through a readout of fingerprint-like spectra. To achieve sufficient sensitivity for in vivo Raman imaging, metallic gold nanoparticles larger than 10 nm were employed to amplify Raman signals via surface-enhanced Raman scattering (SERS). However, the inability to excrete such large gold nanoparticles has restricted the translation of Raman imaging. Here we present Raman-active metallic gold supraclusters that are biodegradable and excretable as nanoclusters. Although the small size of the gold nanocluster building blocks compromises the electromagnetic field enhancement effect, the supraclusters exhibit bright and prominent Raman scattering comparable to that of large gold nanoparticle-based SERS nanotags due to high loading of NIR-resonant Raman dyes and much suppressed fluorescence background by metallic supraclusters. The bright Raman scattering of the supraclusters was pH-responsive, and we successfully performed in vivo Raman imaging of acidic tumors in mice. Furthermore, in contrast to large gold nanoparticles that remain in the liver and spleen, the supraclusters dissociated into small nanoclusters, and 73% of the administered dose to mice was excreted over 4 months. The highly excretable Raman supraclusters demonstrated here offer great potential for clinical applications of in vivo Raman imaging by replacing non-excretable large gold nanoparticles.

Genetically encoded pH sensors based on fluorescent proteins enable dynamic optical imaging of cellular processes such as endocytosis and exocytosis. To date, light scattering in thick tissue as well as photobleaching of fluorescent proteins prevent deep cellular imaging over sustained periods of time. To visualize intracellular pH variations across opaque organs, we introduce a genetically encoded acoustic pH sensor dubbed pHonon. We modified the outer gas vesicle protein (GvpC) of echogenic protein nanostructures via histidine point mutations. At low pH, engineered gas vesicles exhibit an increased shell stiffness which switched their acoustic response from nonlinear to linear. By combining pHonons with nonlinear ultrasound imaging, we captured dynamic deep tissue images of lysosomal acidification by macrophages in murine liver. The combination of pHonon with nonlinear ultrasound creates the possibility for basic studies of endo- and exocytic activity in deep tissue of living opaque organisms.

Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics and couplings in complex systems. However, the sensitivity of SRS is fundamentally limited to milli-molar level due to the shot noise and the small modulation depth. To overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrationally excited states. The thereafter relaxation heats up the surrounding and induces refractive index changes. By probing the refractive index changes with a laser beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved. Versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. SRP microscopy opens a new way to perform vibrational spectroscopic imaging with ultrahigh sensitivity. One-Sentence SummaryWe demonstrate a new spectroscopic imaging method that improves the signal intensity by >500-fold.

The addition of both cell-targeting moieties and polyethylene glycol (PEG) to nanoparticle (NP) drug delivery systems is a standard approach to improve the biodistribution, specificity, and uptake of therapeutic cargo. The spatial presentation of these molecules affects avidity of the NP to target cells in part through an interplay between the local ligand concentration and the steric hindrance imposed by PEG molecules. Here, we show that lipid phase separation in nanoparticles can modulate liposome avidity by changing the proximity of PEG and targeting protein molecules on a nanoparticle surface. Using lipid-anchored nickelnitrilotriacetic acid (Ni-NTA) as a model ligand, we demonstrate that the attachment of lipid anchored Ni-NTA and PEG molecules to distinct lipid domains in nanoparticles can enhance liposome binding to cancer cells by increasing ligand clustering and reducing steric hindrance. We then use this technique to enhance the binding of RGD-modified liposomes, which can bind to integrins overexpressed on many cancer cells. These results demonstrate the potential of lipid phase separation to modulate the spatial presentation of targeting and shielding molecules on nanocarriers, offering a powerful tool to enhance the efficacy of NP drug delivery systems.

Interacting with living systems typically involves the ability to address lipid membranes of cellular systems. The first step of interaction of a nanorobot with a cell will thus be the detection of binding to a lipid membrane. Leveraging the programmable nature of DNA origami, we engineered a biosensor harnessing single-molecule Fluorescence Resonance Energy Transfer (smFRET) as transduction mechanism for precise lipid vesicle detection. The system hinges on a hydrophobic ATTO647N modified single-stranded DNA (ssDNA) leash, protruding from a rectangular DNA origami. In a vesicle-free environment, the ssDNA adopts a coiled stance, ensuring high FRET efficiency. However, upon lipid vesicle binding to cholesterol anchors on the DNA origami, the hydrophobic ATTO647N induces the ssDNA to stretch towards the lipid bilayer, leading to reduced FRET efficiency. The strategic placement of cholesterol anchors further modulates this interaction, affecting the observed FRET populations. Beyond its role as a vesicle sensor, we show targeted cargo transport of the acceptor dye unit to the vesicle. The cargo transport is initiated by vesicle bound DNA and a strand displacement reaction. Our interaction platform opens pathways for innovative interaction such as biosensing and molecular transport with complex biosystems.

Calcium imaging has enabled major biological discoveries. However, the scattering of light by tissue limits the use of standard fluorescent calcium indicators in living animals. To address this limitation, we introduce the first genetically encoded ultrasonic reporter of calcium (URoC). Based on a unique class of air-filled protein nanostructures called gas vesicles, we engineered URoC to produce elevated nonlinear ultrasound signal upon binding to calcium ions. With URoC expressed in mammalian cells, we demonstrate noninvasive ultrasound imaging of calcium signaling in vivo during drug-induced receptor activation. URoC brings the depth and resolution advantages of ultrasound to the in vivo imaging of dynamic cellular function and paves the way for acoustic biosensing of a broader variety of biological signals.

DNA origami is a technique that allows the creation of precise, modular, and programmable nanostructures using DNA. These nanostructures have found use in several fields like biophysics, molecular biology, nanoelectronics, and nanophotonic due to their programmable nature as well as ability to organize other nanomaterials with high accuracy. However, they are fragile and unstable when removed from their optimal aqueous conditions. In contrast, other commonly used bottom-up methods for creating inorganic nanoparticles do not have these issues, but it is difficult to control the shape or spatial organization of ligands on these nanoparticles. In this study, we present a simple, highly controlled method for templated growth of silica on top of DNA origami while preserving all the salient features of DNA origami. Using the polyplex micellization (PM) strategy, we create DNA nanostructures that can withstand salt-free, buffer-free, alcohol-water mixtures, enabling us to control the material growth conditions while maintaining the monodispersity and organization of nanoelements. We demonstrate the growth of silica shells of different thicknesses on brick and ring-shaped DNA origami structures using the standard Stober process. We also demonstrate the thermostability of the silica-coated nanostructures as well as accessibility of surface sites programmed into the DNA origami after the silica growth in the final inorganic nanostructure.

Synthetic DNA motifs form the basis of nucleic acid nanotechnology, and their biochemical and biophysical properties determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.