Controlling the self-assembly of complex nanostructures is one of the central goals in DNA nanotechnology, where DNA origami provides a versatile platform for designing programmable interactions. While polyhedral structures like the icosahedron have been successfully assembled both computationally and experimentally, more complex targets such as the snub cube pose additional challenges due to...
DNA origami is a powerful bottom-up technique that leverages specific Watson-Crick-Franklin base pairing to build complex and reproducible nanostructures with precise size and shape control. These biocompatible nanostructures offer diverse applications in biomedicine and nanobiotechnology, such as therapeutic delivery, biosensing, and biomimetics. However, many of these applications do...
Cell-free platforms are multi-component systems comprising Transcription and Translation (TX-TL) machinery liberated from cellular metabolism [1,2]. These systems are composed of three main compartments, cell lysate, Energy Buffer, and DNA template. The nature of multiplicity in cell-free systems bring along a number of variety in terms of TX-TL yield. The underlying cause unfortunately cannot...
Continuous biosensors rely on affinity-based nanoswitches that undergo target-induced conformational changes and as such enable real-time biomarker detection in healthcare and biotechnology applications1. However, developing sensitive and fully reversible affinity-based nanoswitches for continuous biosensing is hindered by limited understanding of their mechanism of action.
We recently...
Antigen-triggered Activation of a DNA Origami Nanopore
D. Putz and H. Dietz
Laboratory for Biomolecular Nanotechnology, School of Natural Sciences, Technical University of Munich, Germany
Email: dominik.putz@tum.de
Fabricating nanostructures capable of reacting to physiological stimuli by dynamic reconfiguration is of great interest when considering new diagnostics and...
Adenosine triphosphate (ATP) plays a crucial role in a wide range of cellular processes.
It provides cells with a source of chemical energy to perform essential functions necessary to sustain life, from the molecular transport across cell membranes to the biosynthesis of macromolecules [1].
One of the main goals of synthetic biology is to design artificial systems with life-like...
Starting several decades ago, our digital revolution has resulted in a continuous and ongoing exponential increase in digital data being produced, with estimates indicating that this data will soon outpace the storage capacity of current technologies [1,2]. This impending data storage crisis has led researchers to investigate alternative storage methods. DNA has been proposed as a promising...
Molecular motors are biological machines that convert chemical energy into mechanical work. Constructing synthetic molecular machinery is essential for understanding their natural counterparts and advancing the development of artificial biological systems. Although significant progress has been made in the realm of synthetic motors, the development of a chemically fueled, rotational motor...
DNA nanostructures are promising multifunctional carriers for therapeutic applications, but their use in biological environments is challenged by their limited stability [1]. We address this by cross-linking DNA nanostructures with cisplatin [2], exploiting its DNA-binding, cytotoxic, and radiosensitizing properties to create more robust structures for combined chemo- and radiotherapy....
Living systems are an inspiring example of how fundamental functions can emerge from networks of interacting molecules. Competitive dimerization networks, for example, are composed by families of proteins that bind to each other to form a combinatorial library of dimers that play a crucial role in the downstream activation of specific signalling pathways. In recent years, synthetic DNA has...
The ability to precisely control DNA origami orientation holds immense potential for a wide range of applications.1 This includes the development of advanced metamaterials, highly sensitive chiral sensing platforms, high-density data storage devices, and sophisticated drug delivery systems. Any method to achieve DNA origami orientation control is therefore attractive for both fundamental...
Many biochemical signal-processing pathways rely on families of proteins that competitively dimerize in diverse combinations. Such competitive dimerization networks (CDNs) enable complex input-output computations and context-specific adaptability by varying component expression levels. Inspired by this biological occurring paradigm and introducing the predictability and sequence specificity of...
Antibiotic resistance is posing a serious threat to human health. Therefore, innovative therapeutic tools are needed to efficiently treat resistant bacteria. Aptamers are small synthetic oligonucleotides which show high specificity and affinity for a designated target. They have multiple applications in the biotechnological field and can be considered as biological drugs to target resistant...
Membraneless organelles, such as DNA/RNA condensates formed via liquid-liquid phase separation (LLPS), provide a flexible and dynamic platform for biosensing applications in therapeutic advancements.1, 2 The DNA condensates may offer a biomimetic environment for molecular recognition, enabling the detection of disease biomarkers with high sensitivity and specificity. By leveraging multivalent...
Biomolecular condensates ̶ dynamic, biomolecule-enriched droplets which arise owing to Liquid-Liquid Phase Separation (LLPS) ̶ have been recently described to be implicated in many key vital processes within cells [1,2]. Although intracellular LLPS is extremely complex, the use of nucleic acids’ high programmability, governed by Watson-Crick-Franklin base-pairing, offers a straightforward...
DNA nanostructures can be made by combining a long single-strand DNA scaffold with more than 200 short DNA oligonucleotides (staples) that will direct the folding of the scaffold to any pre-designed shape [1]. DNA nanostructure products have numerous functions, such as studying nanoscale biological interactions and facilitating drug delivery. It has been shown that DNA nanostructures can bind...
Metabolic compartmentalization enables the separation of pathways and components in the cell and is a key feature found throughout biology [1]. Compartments offer two primary advantages: isolation and condensation [2]. While the former shields the reaction from the environment, the latter typically increases the local concentration of reactants. In this study, we use a DNA origami compartment...
MicroRNAs (miRs) control protein expression in cells and some have been found to be deregulated in cells and bodily fluids of cancer patients compared to healthy individuals. Despite their great potential as minimally invasive biomarkers, there is currently no cancer diagnostic test based on circulating miR detection, mainly due to their low endogenous concentration. Prostate cancer (PCa) is...
Dynamic behavior is a key feature of biological supramolecular assemblies that is essential for environmental adaptability and functional regulation. The reproduction of such non-equilibrium dynamics in synthetic materials is promising for the creation of adaptive systems with life-like properties. Dissipative DNA nanotechnology has recently gained attention as a powerful approach to develop...
Bottom-up synthetic biology aims to create biomimetic synthetic cells to perform out-of-equilibrium, ‘life-like’ functions such as growth and division.$^1$ Giant unilamellar vesicles (GUVs) are good cell mimics due to having sizes similar to living cells, and faithfully imitating the properties of their membranes.$^1$ Recently, Fabrini et al. have developed branched RNA constructs, or...
We develop a colorimetric sensing platform compatible with single-molecule detection by assembling gold-nanosphere dimers on a Y-shaped DNA origami that acts as a nanoscale actuator (Fig.1-a). DNA origamis are a highly programmable and flexible platform that can be precisely engineered to undergo controlled conformational changes in response to specific molecular targets, such as DNA/RNA...
Here, we develop orthogonal enzyme-driven DNA transcriptional timers capable of rationally programming a tunable delay of in vitro transcription onset. These timers are based on blocker strands that, by binding to the promoter region of the DNA template, prevent transcription initiation. The blockers can subsequently be removed via specific enzymatic reactions. Once the blocker strand is...
In recent decades, advances in synthetic biology have enabled the transition from cell-based to cell-free biosensors, significantly expanding their application range from environmental monitoring [1] to medical diagnostics [2]. Indeed, cell-free biosensors are emerging as cost-effective, user-friendly and field-deployable platforms for the detection of viral nucleic acids [3] and small...
KEYWORDS: RNA based therapeutics, DNA nanotechnology, nucleic acid nanostructures
(NANs), cell penetrating peptides (CPPs), non-viral vectors, RNA therapies
RNA-based therapeutics, including small interfering RNAs (siRNAs) and microRNAs (miRs), have emerged as promising tools to modulate regenerative pathways in ischemia-damaged cardiac tissue. However, their clinical application...
Understanding the mechanisms behind structure-switching in aptamer-modified nanopores remains limited, particularly in how conformational dynamics influence ionic conductance [1]. To address this gap, we investigated two aptamer-functionalized quartz nanopipette sensors designed to detect the neurotransmitters dopamine and serotonin. The two molecules with nearly identical size and charge yet...
Precise spatial control of proteins is critical for the construction of synthetic biological systems and the investigation of dynamic molecular processes. DNA origami offers a powerful platform for organizing biomolecules with nanometer precision; however, most existing reconfiguration strategies depend on thermal cycling, enzymatic reactions, or highly customized designs, which limit their...
Nucleic acids are versatile biomolecules containing specific sequences of bases which define their properties and reactivity. The use of nucleic acids, and in particular DNA, is a promising strategy in the designing of advanced materials when selective responsivity is required. In this context, the fabrication of DNA-responsive hydrogels has been recently reported, exploiting standard...
The programmability of CRISPR-based techniques, combined with the advantages of nucleic acid nanotechnology, has enabled the development of innovative diagnostic tools.
Here we present a two-step CRISPR-based immunoassay, named MAIGRET (Molecular Assay based on antibody-Induced Guide-RNA Enzymatic Transcription). This platform enables the sensitive and versatile detection of specific...
Communication is one of the fundamental properties of living systems. Cells constantly exchange information using small molecules; however, understanding the molecular pathways remains difficult due to the overall complexity of living organisms. One of the ways to address this problem is a bottom-up approach, which uses simplified model systems to study individual biological processes. The aim...
mRNA therapy represents a paradigm shift in therapeutics by enabling transient expression of target proteins, facilitating rapid and scalable treatment modalities with high specificity. Leveraging endogenous cellular machinery for translation, mRNA-based therapeutics bypass traditional gene therapy's integration risks and offer controlled protein production. This approach is particularly...
Micro- and nanoscale compartments are essential in living organisms. They offer a unique environment promoting chemical reactions with high specificity and efficiency due to high enzyme and substrate concentrations while protecting possible intermediates and reducing competing cross-reactions. [1] Protein cages and custom DNA origami structures have emerged as artificial nanocontainers to...
We have recently constructed a modular chimera that mimics the functional architecture of the 26S proteasome, coupling protein unfolding and proteolysis.[1] The nanomachine consists of two sequentially aligned compartments, A(p97) and B(aCt), made by scaffolded DNA origami cages. The first compartment contains p97, an ATP-dependent unfoldase, that mechanically unfolds the substrate. This then...
Super-resolution microscopy has transformed biological imaging by enabling visualization of sub-cellular structures at nanometer scales beyond the diffraction limit, while also opening new possibilities for single-molecule sensing, nanomaterials characterization, and the study of molecular interactions with unprecedented spatial precision. Single-molecule localization microscopy (SMLM)...
The applicability of CRISPR-Cas has gone beyond genome editing and has found expansive use in molecular diagnostics. While there have been many fluorescent sensors, electrochemical sensors for CRISPR-Cas are now rapidly gaining attention as they reduce the need for expensive instruments, are low-cost, and can be used in low-resource settings, satisfying the requirements for an ideal...
As global data storage demands surge, conventional technologies like magnetic tape, SSD and hard drive face critical limitations in scalability and sustainability. DNA offers a promising alternative due to its exceptional density and durability. Current sequence-based DNA storage methods, however, are hindered by the high cost of DNA synthesis, which requires unique sequences for each bit of...
Self-assembled deoxyribonucleic acid (DNA) origami nanostructures (DONs) are highly versatile, possessing unique structural and mechanical properties that make them promising candidates for diverse applications such as biosensing, drug delivery, and advanced nanodevices. However, realizing their full potential often requires precise pre- or post-assembly processing/ modification for...
In recent years, DNA ability to self-assemble into predetermined geometries, following Watson−Crick base-pairing rules, allowed the tailoring of functional supramolecular systems, which opened several opportunities in the fields of nanotechnology and material science. In this context, the DNA origami technique represents a general way to construct nano-objects with defined static 3D shapes,...
Dynamic DNA nanostructures (DONs) can be used as triggerable switches in novel chemical and biotechnological applications, including electrochemical sensing. One such example is a so-called Zipper DON [1] that can adopt either a closed or open conformation based on the formation of pH-sensitive DNA triplexes. This property makes the Zippers excellent sensor components for pH-dependent sensing...
Precise control of biomolecular interactions is central to synthetic biology, therapeutics, and biosensing [1,2]. Enzyme-inhibitor complexes, in particular, represent a powerful but underutilized axis for programmable regulation due to their inherent reversibility and specificity [3]. However, conventional approaches to modulate these systems often lack dynamic tunability and contextual...
ABSTRACT
DNA topology is an intrinsic property of DNA molecules that influences fundamental cellular processes such as chromatin architecture and gene expression. Genomic DNA is organised into topological domains, with supercoiling levels regulated by DNA topoisomerases. These enzymes modify the DNA supercoiling of covalently closed DNA molecules by either removing or introducing supercoils....
Sensing and quantifying nucleic acids is of fundamental importance for diagnostics, development of new drugs and the application of personalized therapies. Nanotechnology allows the creation of biosensors that are cost-effective, fast, sensitive and easy to use.
We have realized a reusable multi-readout biosensor that is able to detect hybridization between two complementary sequences by...
Nature's enzymatic reaction networks rely on intricate communication pathways to coordinate complex biochemical functions. Translating such communication to synthetic systems remains challenging, particularly between unrelated enzymes. Here, we present DNA-based molecular transducers (DNA Transducer) that enable artificial communication between DNA repair enzymes and CRISPR-Cas12a (Figure 1)....
The development of rapid, cost-effective, and highly selective biosensing platforms remains a pressing challenge across environmental, medical, and industrial sectors [1-3]. To address this, we introduce a general in silico aptamer discovery strategy designed for small-molecule detection, replacing the laborious SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process with a...
Insulin receptors (IRs) organize into nanoclusters on the cell membrane, as demonstrated by our group and others using super-resolution microscopy. The nanocluster arrangements of IRs provide the ideal environment for multivalent interactions with insulin. We designed an insulin-DNA nanostructure that offers precise control over the number and spatial distribution of insulin molecules within...
Aptamers are short, single-stranded DNA or RNA oligonucleotides that exhibit high specificity and affinity for target molecules. Their potential applications range from medical diagnostics to cancer therapy. Aptamers are synthetically generated through a selection process in which large libraries of random oligonucleotide sequences are screened to identify those with the strongest binding...
Immunotherapy has revolutionized the treatment of cancer and immune-related diseases by harnessing the body’s own immune system. A key factor in modulating immune cell activation, particularly T-cells [1], is the precise spatial presentation of ligands [1], [2]. However, controlling this at the nanoscale remains a major challenge [3], [4]. In this study, we investigated how spatial patterning...
