Abstract Microtubules are central components of cytoskeletal transport systems and have been widely repurposed as active elements in motor-driven nanodevices. However, site-specific functionalization of stabilized microtubules remains a fundamental challenge, as the tubulin lattice presents chemically indistinguishable binding sites along its length. Here we report a strategy for selective end-functionalization of stabilized microtubules using DNA origami nanostructures. By coupling DNA origami to Fab fragments targeting acetylated α-tubulin Lys40 within the microtubule lumen, and exploiting steric exclusion of the origami from the lattice interior, binding is confined to accessible sites at microtubule ends and lattice defects. Using a six-helix bundle origami as a minimal construct, we demonstrate selective tip labelling of gliding microtubules without perturbing kinesin-driven motility. The same structures additionally mark lattice defects, enabling dynamic visualization of defect sites during transport. Furthermore, we show that tip-bound origami can hybridize with complementary DNA strands to capture cargo from surfaces in motion, establishing programmable, end-specific loading. This approach introduces a generalizable route to spatially controlled functionalization of cytoskeletal filaments, enabling new capabilities in molecular transport, nanoscale assembly, and the study of microtubule integrity and repair.

We demonstrate a triplex-based architecture for DNA-templated synthesis. This study is motivated by progress towards the development of a synthetic ribosome - autonomous, genetically programmable, molecular machinery for synthesis. Such schemes for the creation and evolution of chemically diverse DNA-tagged chemical libraries rely on hybridization reactions of oligonucleotide adapters to control sequential, DNA-templated reactions of covalently attached building blocks. To enable parallel one-pot library synthesis it is desirable that any building block can be incorporated at any position in a product oligomer: this is incompatible with geometries commonly used for DNA-templated synthesis which require alternate reactants to be attached to 3 ' and 5 ' termini of their adapters. Our triplex-based architecture overcomes this problem by templating reactions between building blocks attached to adapters with identical structures. It is intended to form the core of programmable molecular machinery for multistep synthesis. Here, we use single-step coupling reactions to characterize the triplex reaction template.

Tetrahedral DNA nanostructures (TDNs) are emerging as next-generation platforms for delivering therapeutic oligonucleotides. This study introduces a novel strategy embedding antisense oligonucleotide (ASO) sequences directly within the structural framework of TDNs, not requiring external extensions as hybridization handles and preserving flexibility for functionalization. The integration of a gapmer-based design enables structural reconfiguration upon cellular delivery, promoting ASO accessibility and efficient target engagement. To validate this approach, we engineered a TDN-gapmer targeting microRNA-21 (miR-21), a dysregulated biomarker linked to glioblastoma. Without requiring transfection agents, TDN-gapmer demonstrated autonomous delivery capacity into glioblastoma cells, leading to robust miR-21 inhibition. Mechanistic studies revealed that the integrated gapmer can potentially recruit ribonuclease (RNase) H, facilitating RNA cleavage and enhancing target suppression. Coarse-grained modeling provided a detailed view of the predicted structural transitions and thermodynamic parameters that demonstrate that cleavage initiates toehold formation, enabling strand displacement and potential catalytic reconfiguration of the nanostructures. The TDN-gapmer demonstrated stability in serum, withstanding degradation while maintaining its therapeutic potential. The ability to integrate active sequences into the structural framework increases availability for further potential multifunctionalization. This innovative TDN design establishes a versatile and transformative platform with promising implications for precision RNA-targeting therapeutics.

Construction of a molecular assembler from DNA that executes a programmed sequence of chemical reactions is a formidable challenge but worthwhile because it would allow assembly and evolution of functional polymers. We present a mechanism using parallel DNA and a DNA polymerase to address two challenges that currently block progress.

DNA-RNA hybrid strand displacement underpins the function of many natural and engineered systems. Understanding and controlling factors affecting DNA-RNA strand displacement reactions is necessary to enable control of processes such as CRISPR-Cas9 gene editing. By combining multiscale modeling with strand displacement experiments, we show that the distribution of bases within the displacement domain has a very strong effect on reaction kinetics, a feature unique to DNA-RNA hybrid strand displacement. Merely by redistributing bases within a displacement domain of fixed base composition, we are able to design sequences whose reaction rates span more than four orders of magnitude. We extensively characterize this effect in reactions involving the invasion of dsDNA by an RNA strand, as well as the invasion of a hybrid duplex by a DNA strand. In all-DNA strand displacement reactions, we find a predictable but relatively weak sequence dependence, confirming that DNA-RNA strand displacement permits far more thermodynamic and kinetic control than its all-DNA counterpart. We show that oxNA, a recently introduced coarse-grained model of DNA-RNA hybrids, can reproduce trends in experimentally observed reaction rates. We also develop a simple kinetic model for predicting strand displacement rates. On the basis of these results, we argue that base distribution effects may play an important role in natural R-loop formation and in the function of the guide RNAs that direct CRISPR-Cas systems.

Molecular electronics and other technologies whose components comprise individual molecules have been pursued for half a century because the molecular scale represents the limit of miniaturisation of objects whose structure is tuneable for function. Despite the promise, practical progress has been hindered by the lack of methodologies for directed assembly of arbitrary structures applicable at the molecular scale.DNA nanotechnology is an emerging framework that uses programmed synthetic oligomers to encode the design of self-assembling structures with atomic precision at the nanoscale. Here, we leverage DNA-directed self-assembly to construct single-molecule electrical transport devices in high yield, precisely positioning a metal-porphyrin between two 60 nm gold nanoparticles. Following deposition on SiO2 substrates, we image and establish electrical contact via established nanofabrication techniques. Each step of the process has a high probability of success and we demonstrate device yields dramatically better than is possible using conventional approaches. Our approach is inherently scalable and adaptable to devices incorporating multiple heterogenous functional molecular components, finally offering a realistic framework for the realisation of classical and quantum molecular technologies.

Fundamental investigations of ice nucleation, a key process in fields from environmental science to cryobiology, require model systems with chemical and physical structures that are well defined and easily varied. DNA origami is an especially promising model because of the exquisite control that it offers over the physical geometry of the nucleating agent at the nano-scale. Here we compare ice nucleation by solutions of a rectangular DNA origami tile, formed by annealing a 2.6 kbase single-stranded DNA scaffold with ninety shorter 'staple' oligonucleotides, to ice nucleation when these components are mixed at the same concentrations but not annealed. Isothermal measurements show that the molecular conformation has a dramatic effect on the ice nucleating efficiency. For an array of droplets containing annealed, well-folded tiles the freezing rate is constant, whereas for unannealed DNA the freezing rate decreases with time. Despite the freezing rate measured at low temperature being higher for the annealed DNA origami samples than for a significant proportion of the unannealed ones, in slow temperature-ramp measurements the latter generally freeze at higher temperatures. We show that this behaviour is consistent with the formation of small numbers of highly efficient nucleating agents in the unannealed samples, likely through molecular aggregation.

Nucleic acid nanostructures offer unique opportunities for biomedical applications due to their sequence-programmable structures and functions, which enable the design of complex responses to molecular cues. Control of the biological activity of therapeutic cargoes based on endogenous molecular signatures holds the potential to overcome major hurdles in translational research: cell specificity and off-target effects. Endogenous microRNAs (miRNAs) can be used to profile cell type and cell state, and are ideal inputs for RNA nanodevices. Here, we present CRISPR MiRAGE (miRNA-activated genome editing), a tool comprising a dynamic single-guide RNA that senses miRNA complexed with Argonaute proteins and controls downstream CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) activity based on the detected miRNA signature. We study the operation of the miRNA-sensing single-guide RNA and attain muscle-specific activation of gene editing through CRISPR MiRAGE in models of Duchenne muscular dystrophy. By enabling RNA-controlled gene editing activity, this technology creates opportunities to advance tissue-specific CRISPR treatments for human diseases.

The synthesis of artificial sequence-defined polymers that match and extend the functionality of proteins is an important goal in materials science. One way of achieving this is to program a sequence of chemical reactions between precursor building blocks by means of attached oligonucleotide adapters. However, hydrolysis of the reactive building blocks has so far limited the length and yield of product that can be obtained using DNA-templated reactions. Here, we report an architecture for DNA-templated synthesis in which reactants are tethered at internal abasic sites on opposite strands of a DNA duplex. We show that an abasic site within a DNA duplex can protect a nearby thioester from degradation, significantly increasing the yield of a DNA-templated reaction. This protective effect has the potential to overcome the challenges associated with programmable, sequence-controlled synthesis of long non-natural polymers by extending the lifetime of the reactive building blocks.

Modular reconfigurable systems can be achieved with DNA origami, demonstrating the potential to generate molecular robots.

Mechanicalforces are relevant for many biological processes, fromwound healing and tumor formation to cell migration and differentiation.Cytoskeletal actin is largely responsible for responding to forcesand transmitting them in cells, while also maintaining cell shapeand integrity. Here, we describe a FRET-based hybrid DNA-protein tensionsensor that is designed to sample transient forces in actin networksby employing two actin-binding motifs with a fast off-rate attachedto a central DNA hairpin loop. Such a sensor will be useful to monitorrapidly changing stresses in the cell cytoskeleton. We use fluorescencelifetime imaging to determine the FRET efficiency and thereby theconformational state of the sensor, which makes the measurement robustagainst intensity variations. We demonstrate the applicability ofthe sensor by confocal microscopy and by monitoring crosslinking activityin in vitro actin networks by bulk rheology.

Nucleic acid therapeutics require delivery systems to reach their targets. Key challenges to be overcome include avoidance of accumulation in cells of the mononuclear phagocyte system and escape from the endosomal pathway. Spherical nucleic acids (SNAs), in which a gold nanoparticle supports a corona of oligonucleotides, are promising carriers for nucleic acids with valuable properties including nuclease resistance, sequence-specific loading and control of receptor-mediated endocytosis. However, SNAs accumulate in the endosomal pathway and are thus vulnerable to lysosomal degradation or recycling exocytosis. Here, an alternative SNA core based on diblock copolymer PMPC25-PDPA(72) is investigated. This pH-sensitive polymer self-assembles into vesicles with an intrinsic ability to escape endosomes via osmotic shock triggered by acidification-induced disassembly. DNA oligos conjugated to PMPC25-PDPA(72) molecules form vesicles, or polymersomes, with DNA coronae on luminal and external surfaces. Nucleic acid cargoes or nucleic acid-tagged targeting moieties can be attached by hybridization to the coronal DNA. These polymeric SNAs are used to deliver siRNA duplexes against C9orf72, a genetic target with therapeutic potential for amyotrophic lateral sclerosis, to motor neuron-like cells. By attaching a neuron-specific targeting peptide to the PSNA corona, effective knock-down is achieved at doses of 2 particles per cell.

Nanoscale manipulation and patterning usually require costly and sensitive top-down techniques such as those used in scanning probe microscopies or in semiconductor lithography. DNA nanotechnology enables exploration of bottom-up fabrication and has previously been used to design self-assembling components capable of linear and rotary motion. In this work, we combine three independently controllable DNA origami linear actuators to create a nanoscale robotic printer. The two-axis positioning mechanism comprises a moveable gantry, running on parallel rails, threading a mobile sleeve. We show that the device is capable of reversibly positioning a write head over a canvas through the addition of signaling oligonucleotides. We demonstrate "write" functionality by using the head to catalyze a local DNA strand-exchange reaction, selectively modifying pixels on a canvas. This work demonstrates the power of DNA nanotechnology for creating nanoscale robotic components and could find application in surface manufacturing, biophysical studies, and templated chemistry.

Mechanical forces are relevant for many biological processes, from wound healing or tumour formation to cell migration and differentiation. Cytoskeletal actin is largely responsible for responding to forces and transmitting them in cells, while also maintaining cell shape and integrity. Here, we describe a novel approach to employ a FRET-based DNA force sensor in vitro and in cellulo for non-invasive optical monitoring of intracellular mechanical forces. We use fluorescence lifetime imaging to determine the FRET efficiency of the sensor, which makes the measurement robust against intensity variations. We demonstrate the applicability of the sensor by monitoring cross-linking activity in in vitro actin networks by bulk rheology and confocal microscopy. We further demonstrate that the sensor readily attaches to stress fibers in living cells which opens up the possibility of live-cell force measurements.

Electron cryotomography (cryoET), an electron cryomicroscopy (cryoEM) modality, has changed our understanding of biological function by revealing the native molecular details of membranes, viruses, and cells. However, identification of individual molecules within tomograms from cryoET is challenging because of sample crowding and low signal-to-noise ratios. Here, we present a tagging strategy for cryoET that precisely identifies individual protein complexes in tomograms without relying on metal clusters. Our method makes use of DNA origami to produce "molecular signposts" that target molecules of interest, here via fluorescent fusion proteins, providing a platform generally applicable to biological surfaces. We demonstrate the specificity of signpost origami tags (SPOTs) in vitro as well as their suitability for cryoET of membrane vesicles, enveloped viruses, and the exterior of intact mammalian cells.

Linear actuators are ubiquitous components at all scales of engineering. DNA nanotechnology offers a unique opportunity for bottom-up assembly at the molecular scale, providing nanoscale precision with multiple methods for constructing and operating devices. In this paper, DNA origami linear actuators with up to 200 nm travel, based on a rail threading a topologically locked slider, are demonstrated. Two strategies, one- and two-pot assembly, are demonstrated whereby the two components are folded from one or two DNA scaffold strands, respectively. In order to control the position of the slider on the rail, the rail and the inside of the slider are decorated with single-stranded oligonucleotides with distinct sequences. Two positioning strategies, based on diffusion and capture of signaling strands, are used to link the slider reversibly to determined positions on the rail with high yield and precision. These machine components provide a basis for applications in molecular machinery and nanoscale manufacture including programmed chemical synthesis.

The success of DNA nanotechnology has been driven by the discovery of novel structural motifs with a wide range of shapes and uses. We present a comprehensive study of the T-motif, a 3-armed, planar, right-angled junction that has been used in the self-assembly of DNA polyhedra and periodic structures. The motif is formed through the interaction of a bulge loop in one duplex and a sticky end of another. The polarity of the sticky end has significant consequences for the thermodynamic and geometrical properties of the T-motif: different polarities create junctions spanning different grooves of the duplex. We compare experimental binding strengths with predictions of oxDNA, a coarse-grained model of DNA, for various loop sizes. We find that, although both sticky-end polarities can create stable junctions, junctions resulting from 5$'$ sticky ends are stable over a wider range of bulge loop sizes. We highlight the importance of possible coaxial stacking interactions within the motif and investigate how each coaxial stacking interaction stabilises the structure and favours a particular geometry.

DNA self-assembly allows the construction of nanometre-scale structures and devices. Structures with thousands of unique components are routinely assembled in good yield. Experimental progress has been rapid, based largely on empirical design rules. Herein, we demonstrate a DNA origami technique designed as a model system with which to explore the mechanism of assembly. The origami fold is controlled through single-stranded loops embedded in a double-stranded DNA template and is programmed by a set of double-stranded linkers that specify pairwise interactions between loop sequences. Assembly is via T-junctions formed by hybridization of single-stranded overhangs on the linkers with the loops. The sequence of loops on the template and the set of interaction rules embodied in the linkers can be reconfigured with ease. We show that a set of just two interaction rules can be used to assemble simple T-junction origami motifs and that assembly can be performed at room temperature.

Recent years have seen great advances in the development of synthetic self-assembling molecular systems. Designing out-of-equilibrium architectures, however, requires a more subtle control over the thermodynamics and kinetics of reactions. We propose a mechanism for enhancing the thermodynamic drive of DNA strand-displacement reactions whilst barely perturbing forward reaction rates: the introduction of mismatches within the initial duplex. Through a combination of experiment and simulation, we demonstrate that displacement rates are strongly sensitive to mismatch location and can be tuned by rational design. By placing mismatches away from duplex ends, the thermodynamic drive for a strand-displacement reaction can be varied without significantly affecting the forward reaction rate. This hidden thermodynamic driving motif is ideal for the engineering of non-equilibrium systems that rely on catalytic control and must be robust to leak reactions.