Ultrasensitive and multiplexed miRNA detection system with DNA-PAINT.

MiRNAs hold great potential as biomarkers for the early detection and monitoring of diseases based on their differential expression profiles. Therefore, the sensitive, specific and accurate detection of miRNAs represents an emerging new tool to improve diagnosis and treatment of several diseases, cancer in particular. DNA origami-based miRNA detection is particularly advantageous as it allows to incorporate multiple attachment sites to capture different target miRNAs at the nanoscale. In this work, we present a DNA origami nanoarray system providing distance-dependent recognition of miRNAs by applying super-resolution microscopy technique; DNA-PAINT (point accumulation for imaging in nanoscale topography). The sensor can detect up to 4 miRNAs either separately or in combination based on the relative distance to the boundary markers on the structure using a single imager strand. The detection is highly sensitive, with a limit of detection down to the low femtomolar range (11 fM - 388 fM) and has a large dynamic range up to 10 nM without need for amplification. Moreover, our detection system can discriminate single base mismatches with low false positive rates. Using our strategy, we demonstrate the detection of endogenous miRNAs from cell extracts of cancer cell lines and plasma from breast cancer patients. Overall, we developed an ultrasensitive and amplification-free, DNA-PAINT imaging-based miRNA detection method using DNA origami nanoarray system for the detection of breast-cancer associated miRNAs which potentially provides a sensitive and accurate alternative to the current multiplexed diagnostic technologies.

DNA origami book biosensor for multiplex detection of cancer-associated nucleic acids.

DNA nanotechnology provides a promising approach for the development of biomedical point-of-care diagnostic nanoscale devices that are easy to use and cost-effective, highly sensitive and thus constitute an alternative to expensive, complex diagnostic devices. Moreover, DNA nanotechnology-based devices are particularly advantageous for applications in oncology, owing to being ideally suited for the detection of cancer-associated nucleic acids, including circulating tumor-derived DNA fragments (ctDNAs), circulating microRNAs (miRNAs) and other RNA species. Here, we present a dynamic DNA origami book biosensor that is precisely decorated with arrays of fluorophores acting as donors and acceptors and also fluorescence quenchers that produce a strong optical readout upon exposure to external stimuli for the single or dual detection of target oligonucleotides and miRNAs. This biosensor allowed the detection of target molecules either through the decrease of Förster resonance energy transfer (FRET) or an increase in the fluorescence intensity profile owing to a rotation of the constituent top layer of the structure. Single-DNA origami experiments showed that detection of two targets can be achieved simultaneously within 10 min with a limit of detection in the range of 1-10 pM. Overall, our DNA origami book biosensor design showed sensitive and specific detection of synthetic target oligonucleotides and natural miRNAs extracted from cancer cells. Based on these results, we foresee that our DNA origami biosensor may be developed into a cost-effective point-of-care diagnostic strategy for the specific and sensitive detection of a variety of DNAs and RNAs, such as ctDNAs, miRNAs, mRNAs, and viral DNA/RNAs in human samples.

Membrane-Interacting DNA Nanotubes Induce Cancer Cell Death.

DNA nanotechnology offers to build nanoscale structures with defined chemistries to precisely position biomolecules or drugs for selective cell targeting and drug delivery. Owing to the negatively charged nature of DNA, for delivery purposes, DNA is frequently conjugated with hydrophobic moieties, positively charged polymers/peptides and cell surface receptor-recognizing molecules or antibodies. Here, we designed and assembled cholesterol-modified DNA nanotubes to interact with cancer cells and conjugated them with cytochrome c to induce cancer cell apoptosis. By flow cytometry and confocal microscopy, we observed that DNA nanotubes efficiently bound to the plasma membrane as a function of the number of conjugated cholesterol moieties. The complex was taken up by the cells and localized to the endosomal compartment. Cholesterol-modified DNA nanotubes, but not unmodified ones, increased membrane permeability, caspase activation and cell death. Irreversible inhibition of caspase activity with a caspase inhibitor, however, only partially prevented cell death. Cytochrome c-conjugated DNA nanotubes were also efficiently taken up but did not increase the rate of cell death. These results demonstrate that cholesterol-modified DNA nanotubes induce cancer cell death associated with increased cell membrane permeability and are only partially dependent on caspase activity, consistent with a combined form of apoptotic and necrotic cell death. DNA nanotubes may be further developed as primary cytotoxic agents, or drug delivery vehicles, through cholesterol-mediated cellular membrane interactions and uptake.

Detection of HER2+ Breast Cancer Cells using Bioinspired DNA-Based Signal Amplification.

Circulating tumor cells (CTC) are promising biomarkers for metastatic cancer detection and monitoring progression. However, detection of CTCs remains challenging due to their low frequency and heterogeneity. Herein, we report a bioinspired approach to detect individual cancer cells, based on a signal amplification cascade using a programmable DNA hybridization chain reaction (HCR) circuit. We applied this approach to detect HER2+ cancer cells using the anti-HER2 antibody (trastuzumab) coupled to initiator DNA eliciting a HCR cascade that leads to a fluorescent signal at the cell surface. At 4 °C, this HCR detection scheme resulted in highly efficient, specific and sensitive signal amplification of the DNA hairpins specifically on the membrane of the HER2+ cells in a background of HER2- cells and peripheral blood leukocytes, which remained almost non-fluorescent. The results indicate that this system offers a new strategy that may be further developed toward an in vitro diagnostic platform for the sensitive and efficient detection of CTC.

Dexamethasone-conjugated DNA nanotubes as anti-inflammatory agents in vivo.

The biopolymer DNA allows to create nanoscale, biocompatible structures, which can be designed in a target-specific and stimuli-responsive manner. DNA carrier systems with these characteristics hold a great potential for nanomedical applications, such as for the treatment of inflammatory diseases. Here we used a DNA-based drug carrier system for the pH-dependent delivery of the glucocorticoid dexamethasone into macrophages, a cell type with a key role in the regulation of inflammation. Dexamethasone (Dex) nanotubes were internalized within minutes by MH-S macrophages in vitro and by tissue resident macrophages in the mouse cremaster muscle in vivo and localized in their endosomes. Treatment with Dex nanotubes in vitro significantly reduced the LPS-induced TNF secretion by macrophages, as compared to equivalent amounts of free dexamethasone without affecting cell viability. Microinjection of Dex nanotubes into postischemic muscle tissue of anesthetized mice resulted in a marked reduction of ischemia-reperfusion-elicited leukocyte transmigration and diminished vascular expression of the endothelial adhesion molecules VCAM-1 and ICAM-1. Taken together, our results demonstrate that DNA nanotubes can be used as a platform for the targeted delivery of glucocorticoids and could thus foster the development of nanomedical therapeutics with reduced off-target effects.

Independent natural genetic variation of punishment- versus relief-memory.

A painful event establishes two opponent memories: cues that are associated with pain onset are remembered negatively, whereas cues that coincide with the relief at pain offset acquire positive valence. Such punishment- versus relief-memories are conserved across species, including humans, and the balance between them is critical for adaptive behaviour with respect to pain and trauma. In the fruit fly, Drosophila melanogaster as a study case, we found that both punishment- and relief-memories display natural variation across wild-derived inbred strains, but they do not covary, suggesting a considerable level of dissociation in their genetic effectors. This provokes the question whether there may be heritable inter-individual differences in the balance between these opponent memories in man, with potential psycho-clinical implications.

DNA nanotubes as intracellular delivery vehicles in vivo.

DNA-based nanoconstructs possess great potential for biomedical applications. However, the in vivo behavior of such constructs at the microscopic tissue/cell level as well as their inflammatory potential is largely unknown. Unmethylated CpG sequences of DNA are recognized by Toll-like receptor 9 (TLR9), and thus initiate an innate immune response. In this study, we investigated the use of DNA-based nanotubes as carrier systems for CpG delivery and their effect on immune cells in vivo and in real time. DNA nanotubes were microinjected into skeletal muscle of anesthetized mice. Using in vivo microscopy, we observed that the DNA tubes were internalized within minutes by tissue-resident macrophages and localized in their endosomes. Only microinjection of CpG-decorated DNA nanotubes but not of plain DNA nanotubes or CpG oligonucleotides induced a significant recruitment of leukocytes into the muscle tissue as well as activation of the NF-ĸB pathway in surrounding cells. These results suggest that DNA nanotubes are promising delivery vehicles to target tissue macrophages, whereupon the immunogenic potential depends on the decoration with CpG oligonucleotides.

Membrane-assisted growth of DNA origami nanostructure arrays.

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors--a three-layered rectangular block and a Y-shaped DNA structure--to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.

DNA-Tile Structures Induce Ionic Currents through Lipid Membranes.

Self-assembled DNA nanostructures have been used to create man-made transmembrane channels in lipid bilayers. Here, we present a DNA-tile structure with a nominal subnanometer channel and cholesterol-tags for membrane anchoring. With an outer diameter of 5 nm and a molecular weight of 45 kDa, the dimensions of our synthetic nanostructure are comparable to biological ion channels. Because of its simple design, the structure self-assembles within a minute, making its creation scalable for applications in biology. Ionic current recordings demonstrate that the tile structures enable ion conduction through lipid bilayers and show gating and voltage-switching behavior. By demonstrating the design of DNA-based membrane channels with openings much smaller than that of the archetypical six-helix bundle, our work showcases their versatility inspired by the rich diversity of natural membrane components.

Bone-like mineral nucleating peptide nanofibers induce differentiation of human mesenchymal stem cells into mature osteoblasts.

A bone implant should integrate to the tissue through a bone-like mineralized interface, which requires increased osteoblast activity at the implant-tissue boundary. Modification of the implant surface with synthetic bioinstructive cues facilitates on-site differentiation of progenitor stem cells to functional mature osteoblasts and results in subsequent mineralization. Inspired by the bioactive domains of the bone extracellular matrix proteins and the mussel adhesive proteins, we synthesized peptide nanofibers to promote bone-like mineralization on the implant surface. Nanofibers functionalized with osteoinductive collagen I derived Asp-Gly-Glu-Ala (DGEA) peptide sequence provide an advantage in initial adhesion, spreading, and early commitment to osteogenic differentiation for mesenchymal stem cells (hMSCs). In this study, we demonstrated that this early osteogenic commitment, however, does not necessarily guarantee a priority for maturation into functional osteoblasts. Similar to natural biological cascades, early commitment should be further supported with additional signals to provide a long-term effect on differentiation. Here, we showed that peptide nanofibers functionalized with Glu-Glu-Glu (EEE) sequence enhanced mineralization abilities due to osteoinductive properties for late-stage differentiation of hMSCs. Mussel-inspired functionalization not only enables robust immobilization on metal surfaces, but also improves bone-like mineralization under physiologically simulated conditions. The multifunctional osteoinductive peptide nanofiber biointerfaces presented here facilitate osseointegration for long-term clinical stability.

Cellular Uptake of Tile-Assembled DNA Nanotubes.

DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

Glycosaminoglycan mimetic peptide nanofibers promote mineralization by osteogenic cells.

Bone tissue regeneration is accomplished by concerted regulation of protein-based extracellular matrix components, glycosaminoglycans (GAGs) and inductive growth factors. GAGs constitute a significant portion of the extracellular matrix and have a significant impact on regulating cellular behavior, either directly or through encapsulation and presentation of growth factors to the cells. In this study we utilized a supramolecular peptide nanofiber system that can emulate both the nanofibrous architecture of collagenous extracellular matrix and the major chemical composition found on GAGs. GAGs and collagen mimetic peptide nanofibers were designed and synthesized with sulfonate and carboxylate groups on the peptide scaffold. The GAG mimetic peptide nanofibers interact with bone morphogenetic protein-2 (BMP-2), which is a critical growth factor for osteogenic activity. The GAG mimicking ability of the peptide nanofibers and their interaction with BMP-2 promoted osteogenic activity and mineralization by osteoblastic cells. Alkaline phosphatase activity, Alizarin red staining and energy dispersive X-ray analysis spectroscopy indicated the efficacy of the peptide nanofibers in inducing mineralization. The multifunctional and bioactive microenvironment presented here provides osteoblastic cells with osteogenic stimuli similar to those observed in native bone tissue.

Peptide nanofiber scaffolds for multipotent stromal cell culturing.

Self-assembled peptide nanofibers are versatile materials providing suitable platforms for regenerative medicine applications. This chapter describes the use of peptide nanofibers as extracellular matrix mimetic scaffolds for two-dimensional (2D) and three-dimensional (3D) multipotent stromal cell culture systems and procedures for in vitro experiments using these scaffolds. Preparation of 2D and 3D peptide nanofiber scaffolds and cell culturing procedures are presented as part of in vitro experiments including cell adhesion, viability, and spreading analysis. Analysis of cellular differentiation on peptide nanofiber scaffolds is described through immunocytochemistry, qRT-PCR, and other biochemical experiments towards osteogenic and chondrogenic lineage.