Radiation and DNA Origami Nanotechnology: Probing Structural Integrity at the Nanoscale

Abstract

DNA nanotechnology has emerged as a groundbreaking field, using DNA as a scaffold to create nanostructures with customizable properties. These DNA nanostructures hold potential across various domains, from biomedicine to studying ionizing radiation‐matter interactions at the nanoscale. This review explores how the various types of radiation, covering a spectrum from electrons and photons at sub‐excitation energies to ion beams with high‐linear energy transfer influence the structural integrity of DNA origami nanostructures. We discuss both direct effects and those mediated by secondary species like low‐energy electrons (LEEs) and reactive oxygen species (ROS). Further we discuss the possibilities for applying radiation in modulating and controlling structural changes. Based on experimental insights, we identify current challenges in characterizing the responses of DNA nanostructures to radiation and outline further areas for investigation. This review not only clarifies the complex dynamics between ionizing radiation and DNA origami but also suggests new strategies for designing DNA nanostructures optimized for applications exposed to various qualities of ionizing radiation and their resulting byproducts.

This review explores the impact of ionizing radiation on DNA origami nanostructures, highlighting mechanisms of radiation‐induced damage, structural changes, and implications for nanotechnology and medicine. It compares experimental methods for probing structural integrity and discusses how chemical environments influence radiation effects, offering insights into designing resilient DNA nanostructures for advanced applications.

1. Introduction

DNA nanotechnology is a rapidly developing field with a growing range of applications in life sciences and materials sciences. At the same time, radiation is used in diverse contexts to analyze or modify materials, or as a medical treatment. Here, we review the most important interactions taking place between DNA origami nanostructures and radiation spanning various energies and qualities. Figure 1 illustrates the energy spectrum of radiation types discussed in this review, highlighting interactions with DNA origami nanostructures. We will discuss the implications and applications of these interactions across different fields. Notably, while some studies have investigated the effects of radiation on DNA origami, a comprehensive review summarizing the influence of radiation on these nanostructures is still lacking, highlighting the need for further investigation in this area.

Figure 1.

Energy spectrum illustrating the range of radiation types discussed in this review, ranging from 10⁻¹–10¹⁰ eV, and their interactions with DNA. Highlighted are photons (blue) including UV (A, B, C), VUV, XUV, high‐energy photons, and γ‐rays; electrons (green) encompassing low‐energy electrons (LEEs, <20 eV), high‐energy electrons, and β⁻ particles. Ion beams are shown in the bottom‐most block with nuclear stopping mechanisms typically dominating at the lower energy range (as highlighted in red) and electronic stopping mechanisms at high energy (in dark blue), although these ranges vary depending on the energy, size, and charge state of the ion projectile.

1.1. DNA Nanotechnology

DNA origami nanostructures are formed from a long‐single‐stranded scaffold strand by a set of short, artificial staple strands.[ ¹ , ² ] The scaffold strand is typically the viral DNA M13mp18 with 7249 nucleotides, but also other scaffold strands are used, or they can be even customized. ^[3] The scaffold strand is folded into 2D or 3D structures through hybridization with the staple strands at two or three positions of the scaffold strand and the overall structure is held together by crossovers formed between the scaffold and staples. DNA origami became extremely popular as a tool in nanotechnology, life sciences, chemistry, and physics because of its simple preparation, its well‐defined shape and its high degree of addressability.[ ⁴ , ⁵ , ⁶ , ⁷ , ⁸ ] Every single staple strand (and basically every nucleotide) can be individually addressed, i. e. modified by the very well‐developed DNA chemistry, e. g. exploiting hybridization between single‐stranded DNA overhangs, biotin‐streptavidin interactions or click chemistry. ^[2] In this way, functional moieties such as specific DNA structures, chromophores, proteins, nanoparticles, etc. can be placed with high precision on the DNA origami nanostructures making them attractive for a large variety of applications such as drug delivery, sensing, and as fundamental tool for biophysical and radiation‐induced damage studies.[ ⁹ , ¹⁰ , ¹¹ ] A recent review by Zhan et al. ^[12] provides a comprehensive overview of significant developments in DNA origami over the past five years, highlighting technological advancements in design and assembly methods for diverse applications.

The interaction of DNA origami with radiation plays a role in different contexts. As an example, interactions with photons occur in optical techniques where DNA origami serves as scaffold or platform to assemble components for antennas, optical biosensors, or for photochemical studies.[ ¹³ , ¹⁴ ] UV light can also enhance the structural integrity of DNA through UV‐welding or cross‐linking mechanisms involving covalent cyclobutane pyrimidine dimer formation between adjacent bases,[ ³ , ¹⁵ , ¹⁶ ] as discussed in more detail in Section 2. And finally, DNA origami serves as a tool in radiation sciences to study the interaction of different kinds of radiation with complex, but well‐defined DNA targets. ^[17]

1.2. Interaction of Radiation with DNA Origami Nanostructures

Understanding how radiation interacts with DNA is crucial from a fundamental point of view and becomes relevant for applications of DNA nanotechnology when considering especially DNA origami nanostructures. A key distinction lies in the energy levels of radiation: below and above the ionization threshold. For DNA, this threshold is approximately 8 eV for ionizing the G nucleobase and 9 eV for the sugar unit in gas phase measurements.[ ¹⁸ , ¹⁹ , ²⁰ , ²¹ ]

Above this ionization threshold, radiation can remove valence or core electrons from DNA, creating positively charged species on the DNA molecule. This direct ionization process can initiate DNA damage. However, the predominant damage in aqueous environments arises from secondary species generated by the ionization of water molecules, primarily low‐energy electrons and hydroxyl radicals.[ ²² , ²³ ] Detailed descriptions of these direct and indirect effects are provided in Section 2, along with explanations of different radiation qualities.

Below the ionization threshold, electronic excitation occurs in DNA. The most intense photon absorption band occurs at ~6 eV (200 nm) and 4.8 eV (260 nm) due to the π–π* transitions in the nucleobases. ^[24] This transition can induce photochemical transformations such as the formation of cyclobutane pyrimidine dimers (CPD), which are involved in the development of skin cancer. However, these processes are mitigated in DNA by efficiently returning to the electronic ground state through conical intersections.[ ²⁵ , ²⁶ ] Nonetheless, UV‐induced cross‐linking can be harnessed for targeted linking of DNA strands, thereby stabilizing DNA origami structures, as discussed further below. ^[27]

In the subionization regime, low‐energy electrons interact with DNA differently than photons of the same energy. Electrons transfer negative charges to DNA, facilitating efficient bond breaking through dissociative electron attachment (DEA), a phenomenon that has garnered detailed study in recent years.[ ²⁸ , ²⁹ ]

1.3. Experimental Methods for Probing Structural Integrity of DNA Origami Nanostructures

Investigating the structural integrity of DNA origami nanostructures under ionizing radiation involves various experimental techniques, each with its strengths and limitations. Here we compare several key methods used in this field, focusing on their principles, advantages, and disadvantages.

Tables 1 and 2 categorize and summarize the principles, analysis techniques, advantages, and limitations of various experimental methods used to investigate the structural integrity of DNA origami nanostructures, along with application examples. The methods are grouped into microscopy techniques (Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM) & Cryo‐TEM), electrophoretic techniques (Gel Electrophoresis), spectroscopic techniques (Mass Spectrometry (MS), fluorescence spectroscopy including Förster Resonance Energy Transfer (FRET)), scattering techniques (Small‐Angle X‐ray Scattering (SAXS), light scattering (static and dynamic)) and Raman techniques (Raman Scattering (RS) & Surface‐Enhanced Raman Scattering (SERS)).

Table 1.

Comparative analysis of experimental methods for probing structural integrity of DNA origami nanostructures: microscopy, electrophoretic and spectroscopic techniques.

	Methods	Principle	Analysis	Advantages	Limitations	Application Examples
Microscopy Techniques	Atomic Force Microscopy (AFM)	Scans the surface of deposited DNA origami nanostructures, providing high‐resolution topographical images using a cantilever probe.	Direct visualization of nanostructure morphology and dimensions.	High spatial resolution (~1 nm in vertical direction) and quantitative data on size, shape, and flexibility. Ability to operate in different environments (air, liquid). Relatively fast (seconds to minutes scan time). High‐speed AFM imaging options provide dynamic information on second time scale.	Potential tip‐sample interactions altering structure. Limited to substrates and 2D structures.	Kielar et al. [44] Ke et al. [45]
	Transmission Electron Microscopy (TEM) & Cryo‐TEM	TEM transmits a high‐energy electron beam through DNA origami samples, generating high‐resolution images based on electron diffraction.	Visualization of nanostructure morphology and internal structure.	Extremely high resolution (~0.1 nm) or near‐atomic resolution. Possibility to probe native structure in solution through cryo‐TEM and tomography imaging for 3D structures with atomic precision.	Electron beam induced damage at long exposures. Complex sample preparation that can change or damage DNA origami (e. g., staining, dehydration). Operates under high vacuum conditions.	Bai et al. [46] Pumm et al. [47]
Electrophoretic Technique	Gel Electrophoresis	Separation of DNA origami nanostructures based on their size and conformation through a gel matrix by applying an electric field.	Appearance of distinct bands (migration pattern) corresponding to intact and damaged DNA nanostructures.	Simple and widely available.	Limited sensitivity for subtle structural defects, and lower resolution compared to imaging techniques. Typically provides only qualitative or semi‐quantitative data.	Bellot et al. [48] Douglas et al. [49]
Spectroscopic Techniques	Mass spectrometry (MS)	Analyzes the mass‐to‐charge ratio of DNA fragments obtained from exposing the nanostructure to radiation.	Provides particle size and charge distribution and insights into structural information of DNA fragments.	High sensitivity and accuracy, can identify modifications and structural changes.	Requires ionization methods, and sample preparation methods that can potentially alter the structure. Not suitable for real‐time monitoring and for detection of large DNA nanostructures, only for small molecules and oligonucleotides.	Miller et al. [50] van Dyck et al. [51]
	Fluorescence Spectroscopy	Measures the light emission from fluorescently labelled DNA within the nanostructures upon excitation.	Changes in fluorescence intensity or lifetime can indicate structural changes	Real‐time monitoring. Sensitive to local environment and conformational changes, multiplexing capability.	Requires fluorescent labelling, limited spatial resolution compared to AFM and TEM. Potential for fluorescence quenching and photobleaching, affecting accuracy and repeatability.	Schmied et al. [52]
	Förster Resonance Energy Transfer (FRET)	Measures non‐radiative energy transfer between donor and acceptor fluorophores bound to different parts of the structure upon excitation by a specific wavelength of light.	Efficiency of FRET reflects the distance and relative orientation of fluorophores placed on known sites on DNA origami, indicating conformational changes: – “molecular ruler”.	High sensitivity to nanometer‐scale distance changes, real‐time monitoring, dynamic information on structural changes.	Requires careful design and positioning of fluorophores, possible background signal from nonspecific interactions interfering with accurate distance measurements. Higher costs due to fluorophore modification.	Satyabola et al. [53] Olejko et al. [54] Russ Algar et al. [55]

Table 2.

Comparative analysis of experimental methods for probing structural integrity of DNA origami nanostructures: light scattering and Raman.

	Methods	Principle	Analysis	Advantages	Limitations	Application Examples
Light Scattering Techniques	Small‐Angle X‐ray Scattering (SAXS)	Analyzes the scattering pattern of X‐rays by the DNA origami sample. Relevant for studying crystal structures and higher‐order assemblies.	Indirect measure of structural integrity (size, shape) through deviations from expected scattering pattern	Solution‐based analysis, preserving native conditions. Ensemble averaging.	Lower resolution compared to AFM and TEM; less detailed structural information. Requires typically synchrotron radiation source. Data interpretation requires modeling.	Hartl et al. [56] Baker et al. [57] Ober et al. [58]
	Light scattering (Static and Dynamic)	Measures the intensity and time‐dependence of scattered light from the DNA origami sample to determine shape and size distribution.	Provides information about size distribution, shape, and dynamics of the nanostructures. Melting temperatures can also be assessed. Real‐time folding temperatures resolution.	Label‐free, non‐invasive technique. Can provide real‐time monitoring. Allows following degradation by nucleases.	Lower resolution compared to AFM and TEM, less detailed structural information.	SLS: Ijäs et al. [59] DLS: Yuan et al. [60] Burns et al. [61]
Raman Techniques	Raman Scattering & Surface‐Enhanced Raman Scattering (SERS)	Measures inelastic scattering of light to generate the molecular fingerprint (vibrational spectrum) of the sample. SERS uses nanostructured surfaces to enhance this effect.	Provides detailed molecular composition and structural information.	Basically non‐destructive, label‐free technique with high chemical specificity. SERS offers enhanced sensitivity.	Lower spatial resolution compared to AFM and TEM, requires nanoparticles as SERS substrates, not suitable to study DNA origami as a whole.	Pilo‐Pais et al. [62] Prinz et al. [63]

Each method offers unique insights into the size, shape, flexibility, and overall structural changes of DNA origami nanostructures upon exposure to ionizing radiation. This comprehensive toolkit in the field allows for probing the effects of radiation on these nanostructures and to optimize their design for specific applications. For example, a recent tutorial by Neyra et al. ^[30] offers an in‐depth overview of analytical chemistry techniques relevant to DNA nanotechnology.

2. Mechanisms of Radiation‐Induced Damage to DNA Origami Nanostructures

2.1. DNA Radiation Damage Induced by UV Radiation

UV radiation emerges as a potent modulator of DNA origami nanostructures, with its effects influenced by both wavelength and dosage. This section presents UV‐induced alterations in DNA origami, ranging from the formation of CPDs to the induction of single‐ and double‐strand breaks. By examining this complex relationship between UV radiation and DNA structures, we uncover challenges and opportunities in leveraging UV irradiation for DNA origami applications.

The wavelength dependence of structural changes of DNA origami upon UV irradiation was recently probed in the range of 193 nm–310 nm, showing a strong correlation between DNA absorption and resulting structural effects on DNA origami. ^[31] The UV spectrum, spanning from 30 nm–400 nm is categorized into four regions based on photon energy: UVA (315–400 nm), UVB (280–315 nm), UVC (100–280 nm) as well as vacuum ultraviolet (VUV) and extreme ultraviolet (XUV) radiation (1–200 nm), each of which will be discussed separately.

Higher‐energy UV radiation, including UVB, UVC and VUV can induce DNA lesions through photochemical reactions, often resulting in the formation of CPDs between adjacent pyrimidine nucleobases, alongside 6‐4 photoproducts (6‐4PPs).[ ³² , ³³ ] The presence of CPDs in DNA origami nanostructures can disrupt base‐stacking within single‐strands and base‐pairing between double‐strands, leading to changes in shape, size, or height, which can be monitored using techniques such as AFM.[ ³⁴ , ³⁵ ] Additionally, the presence of CPDs in DNA origami nanostructures can be detected at the dimer‐level by DNA photolyases[ ⁴ , ⁵ ] which attach to CPD sites or by imaging techniques, such as cryo‐electron microscopy (cryo‐EM) ^[27] or transmission electron microscopy (TEM). ^[36] While an optimal density of CPDs can enhance the thermal stability of DNA origami nanostructures, an excessive density may lead to structural disruptions or even disintegration, a phenomenon often described as the “Goldilocks effect”.[ ³⁷ , ³⁸ , ³⁹ ]

Beyond CPD formation, UV radiation can also induce single‐ and double‐strand breaks in DNA. Bald and co‐workers developed an experimental scheme for detecting and quantifying UV‐induced single‐strand breaks using AFM imaging.[ ⁴⁰ , ⁴¹ , ⁴² ] Gel electrophoresis analysis and mass spectrometry studies are also commonly employed to assess photodegradation of DNA origami nanostructures, allowing for the differentiation of damaged staples from the scaffold strand.[ ² , ¹⁰ , ⁴³ ]

2.1.1. UVA Radiation (315–400 nm; 3.10–3.94 eV)

UVA radiation, while generally not known to induce direct damage in DNA origami nanostructures, can serve as an activator of embedded photoresponsive molecules.[ ² , ³⁹ ] This section explores the effects of UVA radiation on DNA origami and its potential applications when combined with photoresponsive molecules.

Chen et al. ^[64] demonstrated through AFM imaging that DNA origami ribbons irradiated with UVA light (365 nm, ~3.4 eV) retain their structural integrity and fold correctly, indicating that UVA radiation alone does not affect the nanostructures’ morphology. This stability makes UVA radiation suitable for applications involving light‐controlled processes, such as photodynamic therapy (PDT) using DNA origami combined with photosensitizers. PDT is a promising cancer treatment that uses the synergy between UV or visible light in the presence of a photosensitizer and molecular oxygen to generate ROS that kill cancer cells. However, the efficacy of PDT with UVA radiation is limited e. g. by the shallow penetration depth of UVA, the hypoxic environment in solid tumors, and potential photobleaching of photosensitizers. Zhuang et al. ^[65] have designed a photosensitizer‐loaded DNA origami nanostructure based on the Rothemund triangle to enhance intracellular imaging techniques and PDT efficiency. They used a carbazole derivative, 6‐bis[2‐(1‐methylpyridinium) ethynyl]‐9‐pentylcarbazole diiodide (BMEPC), as both an imaging agent and a photosensitizer. ^[66] AFM analysis showed that DNA nanostructures loaded with BMEPC maintained their morphology but slightly increased in size. Subsequent UVA irradiation (365 nm) caused partial degradation of these BMEPC‐loaded nanostructures, as observed by AFM (Figure 2A, B.I) and confirmed by agarose gel electrophoresis (AGE), showing a dose‐dependent increase in DNA damage. For instance, at a fixed ratio of 20000 BMEPC molecules per DNA origami, the exposure to 365 nm UV light at gradually longer times leads to evident fading of the AGE bands.

Figure 2.

(A) A summary of commonly observed UV‐induced structural damages and modifications to DNA origami nanostructures spanning the UVA‐UVC range. (B.I) AFM images of the control sample (a) and UVA‐irradiated DNA nanotriangles loaded with BMEPC which acts as a photosensitizer. Adapted with permission from Ref. [65] Copyright 2016 American Chemical Society. (b). (B.II) AFM scans in liquid of plain origami tiles annealed to 60 °C (a) and those which were cross‐linked by 8‐methoxypsoralen (8‐MOP) at proximal thymine residues upon activation by UVA light and then also annealed to 60 °C (b), demonstrating enhanced thermal stability by cross‐linking. Adapted with permission from Ref. [67] Copyright 2011 American Chemical Society. (B.III) Electrophoresis results of UVB cross‐linked and uncross‐linked DNA origami bundles heated to different temperatures. The bundles contain strategically placed proximal thymidine residues that link into CPDs upon UVB irradiation. Reprinted with permission from Ref. [27] Copyright 2018 American Association for the Advancement of Science. An average 2D particle TEM micrograph of the CPD‐cross‐linked sample in double‐distilled water is shown on the rightmost image. (B. IV) AFM images of DNA origami nanoribbons formed from rectangular DNA origami tiles before (a) and after mild irradiation using UVC light showing induced conformational changes that take advantage of the stress relaxation from the nicking of DNA strands by (b) UVC. Adapted with permission from Ref. [64] Copyright 2024 American Chemical Society.

Photon‐induced crosslinking offers another way for enhancing the thermal stability of DNA origami nanostructures. Psoralen derivatives, such as 8‐methoxypsoralen (8‐MOP), are effective photo‐activated crosslinkers under UVA radiation, improving structural integrity and expanding the application horizons of DNA origami nanostructures (Figure 2A, B.II). Rajendran et al. ^[67] incubated DNA origami with 8‐MOP and irradiated them with UVA light (365 nm), achieving optimal crosslinking after 60 minutes, which significantly increased the melting point of the DNA origami. AFM imaging revealed that shorter irradiation times were insufficient for effective crosslinking, while longer exposure caused structural damage.

Another psoralen derivative, 4’‐aminomethyltrioxsalen, was used by Liu et al. ^[68] for reinforcing DNA origami structures at 345 nm, facilitating the preparation of metallic nanostructures. Li et al. ^[39] used triarylpyridinium (TP1), a photoresponsive molecule, to induce conformational changes in DNA origami ribbons. Upon UVA irradiation, TP1 cyclized into TP2, a polycyclic form with DNA intercalator properties. AFM images showed dose‐dependent conformational changes, including flattening and twisting of the ribbons.

More recently, Gerling et al. ^[36] developed a mechanism for reversible stabilization of DNA origami nanostructures based on photochemical crosslinking using 3‐cyanovinylcarbazole (^cnvK). Upon irradiation with 365 nm light, ^cnvK‐modified blunt ends covalently bond to thymine residues at adjacent blunt ends. This covalent bond can then be cleaved upon irradiation with shorter‐wavelength 310 nm light, with bond formation and cleavage occurring on the second timescale. This reversible stabilization method offers unprecedented precise control over the structural dynamics of DNA origami nanostructures, enhancing their potential applications in various fields.

In conclusion, UVA radiation itself may not cause direct damage to DNA origami nanostructures, but it can effectively activate embedded photoresponsive molecules, underscoring its importance in driving light‐controlled applications at the nanoscale. This capability enables the development of novel nanostructures for applications such as PDT, drug delivery, additive nanofabrication, and DNA metallization, expanding the potential uses of DNA origami beyond fundamental research. ^[69]

2.1.2. UVB Radiation (280–315 nm; 3.94–4.43 eV)

UVB radiation plays a significant role in DNA damage and stability. This section examines studies that utilize DNA origami nanostructures to investigate the effects and mechanisms of UVB‐induced DNA modifications.

Gerling et al. ^[27] pioneered efforts to enhance the structural stability of brick‐like DNA origami nanostructures through site‐selective modifications by incorporating thymidine moieties at all strand termini and crossover positions. Upon irradiation with 310 nm (~4.0 eV) UV light, CPD bonds form between adjacent thymidines, linking single‐strand breaks within the folded DNA origami nanostructure. After a 2 hour long irradiation, the melting point of these crosslinked nanostructures increased to 90 °C, as determined by gel electrophoresis analysis, compared to 45–50 °C for non‐reinforced DNA origami nanostructures (Figure 2A, B.III). The structural integrity of these nanostructures was monitored using transmission electron microscopy (TEM) and cryo‐electron microscopy (cryo‐EM).

In line with a previous study by Rajendran et al., ^[67] which reported an optimal irradiation time of 1 hour for crosslinking using 8‐MOP, Gerling et al. observed that irradiation times shorter than 2 hours were insufficient for complete crosslinking, as indicated by only slight increases in the melting points compared to non‐irradiated DNA origamis. Conversely, longer irradiation times resulted in damage to the DNA origami nanostructures. The reinforced nanostructures demonstrated stability in sodium chloride buffers (up to 300 mM) and phosphate‐buffered saline (PBS) solution, mimicking physiological conditions.

In conclusion, UVB radiation effectively induces CPD crosslinks in DNA origami nanostructures, enhancing their thermal stability and robustness under physiological conditions, opening new prospects for DNA origami applications in biotechnological and biomedical areas.

2.1.3. UVC Radiation (100–280 nm; 4.43–12.40 eV)

UVC represents one of the most energetic forms of UV light, capable of causing significant damage to DNA molecules. This section explores studies using DNA origami nanostructures to investigate the specific effects of UVC radiation on DNA.

Chen et al. ^[39] demonstrated that DNA origami nanostructures, specifically rectangle and shaft, are converted into a flat design upon irradiation with either UVB (312 nm, 3.97 eV) or UVC (254 nm, 4.88 eV) light. Despite being designed flat, the nanorectangle tends to curve due to internal stresses from repulsive electrostatic forces. Similarly, the shaft, formed from three rectangles and intended to be in a cis‐form, often exhibits a trans‐form twisted shape caused by the intrinsic curvature of the DNA origami rectangles. UV irradiation induces a flattening effect on these structures, as evidenced by AFM images (Figure 2B.IV), which show that UV light creates DNA damage leading to the flattening of nanorectangles and the transformation of trans‐form shafts into cis‐form. This effect is mediated by the amount of photon‐induced CPDs and is dose‐dependent, as quantified by gel electrophoresis and confirmed by AFM imaging. For instance, the dose of either UVB or UVC radiation causing fragmentation of the DNA origami shaft is about 6–7 times higher than the respective onsets of the flattening effect observed at 2.5 KJ/m² (UVC) or 6.8 KJ/m² (UVB).

Fang et al. ^[70] monitored the morphological response of DNA origami nanostructures exposed to UVC radiation (254 nm, 4.88 eV) using AFM imaging. They demonstrated that a combination of five different DNA origami shapes – cross, hexagon, rectangle, six‐helix bundle and triangle – can serve as a radiometer for measuring UV exposure over doses from 6.48–25.92 kJ/m². Regardless of the shape or size, AFM assessment of UVC‐irradiated DNA origami nanostructures revealed a general pattern of expansion, distortion, and eventual disintegration. These observations indicate a clear dose‐dependent effect of UVC light on the structural integrity of DNA origami nanostructures. At shorter exposure times, UVC light induces DNA single‐ and double‐strand breaks, leading to a relaxation of well‐packed structure. With longer exposure times and the accumulation of DNA strand breaks, complete disintegration of the DNA origami nanostructures occurs. Complementary electrophoresis gel analysis and mass spectrometry studies of the irradiated samples further demonstrated that DNA single‐strand breaks (nicks) increase with the UVC dose. The hypothesis that DNA radiation damage by UV light is efficiently induced by free radicals, such as reactive oxygen species (ROS), was confirmed since intact DNA origami nanostructures were observed in AFM analysis of irradiated samples containing 2 % or 6 % of dimethyl sulfoxide (DMSO), a known free radical scavenger. The effects of UV‐induced crosslinking, which can improve the stability of DNA origami nanostructures, were not considered by the authors.

A similar study extended the UV irradiation damage profiling to the wavelengths of 193 nm (6.4 eV), 225 nm (5.5 eV), 260 nm (4.8 eV), and 310 nm (4 eV), using AFM and UV‐Vis spectroscopy to characterize the damage. ^[31] The extent of structural damage to DNA origami nanostructures correlated with the UV‐Vis absorption spectrum of DNA, which has a local maximum at 260 nm. Following 193 nm irradiation, structural damage was more abrupt due to additional damage from ⋅OH radicals, as evidenced by the dominance of tris reaction products absorbing at 268 nm in the UV‐Vis absorption spectrum of the irradiated sample in tris‐containing buffer. This presents an additional factor challenging the stability of DNA origami nanostructures at shorter UVC wavelengths as well as the influence of optical properties of the buffer components and their associated photolysis products on spectroscopic analyses of irradiated DNA origami solutions.

In summary, the findings from these studies offer valuable insights into the dose‐dependent nature of UVC‐induced DNA damage and highlight the utility of DNA origami as a tool for understanding radiation effects at the nanoscale.

2.1.4. VUV and XUV Radiation (1–220 nm; 6.20–41.3 eV)

In the near UV range, DNA exhibits its most intense electronic excitation band, peaking at 190 nm. This band has been extensively studied due to the availability of the ArF laser sources emitting at 193 nm, which has been shown to cause significant damage to DNA in various forms, including plasmid DNA, ^[71] chromosomal DNA, ^[72] as well as DNA origami scaffolds. ^[31] This pronounced damage is logical given the numerous possible dissociation channels available at this wavelength.

Although a single 193 nm photon is insufficient to directly ionize DNA components through vertical processes, ^[73] it can surpass the adiabatic ionization potential of the guanine base. ^[74] Consequently, this wavelength can induce ionization of DNA bases in their natural environment with relatively high quantum yields.[ ²⁹ , ⁷⁵ ] While the damage to plasmid DNA in the deeper VUV and XUV regions has been already explored by, e. g. Yokoya et al.. ^[76] Nováková et al., ^[77] and Alizadeh et al., ^[78] there are no studies of our knowledge focusing on DNA origami structures. Although, DNA origami nanotriangles have been used as platforms for single‐molecule evaluation of VUV‐induced DNA strand break studies in dry conditions, no significant structural damages to the DNA origami platforms were observed within the utilized range of fluences for VUV energies between 6.5–9 eV. ^[41] Still, there is a gap in determining the effects of VUV and XUV on DNA origami which presents an interesting research direction, particularly in the context of DNA‐assisted lithography.[ ⁷⁹ , ⁸⁰ ] Beyond this, DNA‐based nanofabrication methods offer a wide range of approaches for constructing nanostructures, where radiation like VUV and XUV could play a pivotal role.[ ⁸¹ , ⁸² ] Techniques such as DNA‐templated synthesis and DNA‐directed self‐assembly enable precise control over the placement and patterning of nanomaterials, including DNA‐metal complexes, DNA‐templated conductive polymer nanowires and DNA‐templated carbon nanotubes, which are relevant for nanoelectronics applications. ^[83]

DNA origami platforms have been used to quantify VUV‐induced strand breaks using the same technique as is described in detail in Section 2.4 as a function of energy and for a range of different sequences. The advantage of this technique is the absolute quantification in terms of strand break cross sections, which allow in conjunction with photoabsorption cross sections the determination of strand break quantum yields. The quantum yields have been determined to be 0.5 at 8.94 eV, which is remarkably high. ^[41] Additionally, sequence‐ and substrate‐dependent effects on VUV‐induced DNA strand breakage have been determined using this technique.[ ⁴⁰ , ⁴² ]

In summary, while much remains to be understood regarding the complex interaction of UV radiation and DNA origami nanostructures, recent advancements underscore the transformative potential of radiation‐induced structural alterations in shaping the future landscape of DNA nanotechnology and biotechnology.

2.2. Radiation‐Induced Structural Alterations: γ‐ and β‐ Rays

This section delves into investigations utilizing atomic force microscopy (AFM) to investigate the effect of γ‐rays and β‐rays on DNA origami nanostructures, elucidating analogous damage patterns induced by both forms of irradiation.

γ‐rays are the most energetic photons in the electromagnetic spectrum, with energies ranging from a few hundred keV up to several MeV. While UV radiation, which damages DNA primarily through excitation and ionization processes of valence electrons, γ‐rays interact directly with inner‐shell electrons or even nuclei. In general, primary interactions between γ‐rays and matter include the photoelectric effect, Compton scattering and electron‐positron pair production. ^[84] β‐rays, or high‐energy electrons, cause comparable structural alterations in DNA origami nanostructures akin to those induced by γ‐rays.

Sala et al. ^[85] have investigated the direct and indirect effects of ⁶⁰Co γ‐rays (1.17 and 1.33 MeV) on DNA origami nanostructures, specifically the Rothemund triangle. To assess structural damage, AFM images were recorded from DNA origami nanostructures (i) adsorbed onto Si substrates and (ii) in buffer solutions, exposed to progressively longer irradiation times. Complementary agarose gel electrophoresis (AGE) studies were also conducted to analyze the damage to the staples and scaffold strands of the DNA origami (Figure 3A).

Figure 3.

(A) A scheme of typical damages caused by low to medium LET radiation such as γ‐rays and proton beams to 2D DNA origami nanotriangles in solution. The single‐stranded domains on the corners of the 2D DNA origami triangle are compromised and although the overall structure is preserved, unraveling the structure in electrophoresis show absorbed‐dose‐dependent strand breaking in the origami scaffold. Reproduced from Ref. [85] with permission from the Royal Society of Chemistry. (B) A summary of typical damages on 2D DNA origami triangles in dry conditions on Si substrates irradiated by low to medium LET radiation (I) and high‐LET radiation in the form of ion beams (II) presenting opportunities for localized nanoscale processing of DNA origami nanostructures. Adapted with permission from Ref. [93]

For γ‐irradiation of dry DNA origami nanostructures deposited on silica, AFM images comparing unirradiated and irradiated DNA triangles indicated that even at the highest dose of 300 Gy, the shape of the DNA triangles is preserved. A linear fit to the number of remaining intact triangles as function of the dose indicates that only 0.014 % of the DNA origami triangles were destroyed per Gy of γ‐radiation under dry conditions with most of the observed damages concentration on the corners of the triangles where poly‐T single‐stranded domains are present (Figure 3B.I). Similarly, irradiation of DNA origami nanostructures in buffer solution did not alter their shape at the dose of 300 Gy. The percentage of intact DNA triangles slightly increased to 0.03 % Gy⁻¹ compared to the dry case. This apparent stiffness of the Rothemund triangle can be due to the absence of secondary reactive species, as tris scavenges most radicals in the buffer solution. However, in water (without tris), the damage rate increased to about 0.08 % per Gy, approximately six times higher than under dry conditions.

Agarose gel electrophoresis (AGE) analysis was performed to determine whether the stability of DNA origami nanostructure was due to its well‐packed design, or if the structure shielded the scaffold DNA strand from reactive radicals. At low tris and Mg²⁺ concentrations, allowing for uncoupling of staples from the scaffold strands, AGE analysis revealed that damage induced to the scaffold DNA strand was dose‐dependent, with the respective band broadening and shifting downwards due to nonspecific fragmentation as the absorbed dose increased. The AGE results complemented AFM imaging, as strand breaks in the scaffold DNA strand were detected by AFM only if locally clustered on the DNA origami nanostructure.

Additionally, comparing radiation‐induced DNA damage on the scaffold strand of the DNA origami to a single‐stranded and double‐stranded DNA with the same lead sequence, in the presence of different amounts of OH⋅ radical scavengers, Sala et al. observed significantly higher damage in exposed ss and dsM13mp18 compared to the folded scaffold strand. This finding confirmed that the DNA origami nanostructure provides protection to the scaffold strand against radical species.

Furthermore, DNA origami nanoframes with two parallel target sequences were used to study the incorporation of halogenated nucleosides and their effects on high‐energy electron‐induced DNA damage. After irradiating buffer solutions containing DNA nanoframes with 16 MeV electrons, Sala et al. ^[86] directly counted DNA double‐strand breaks (DSBs) using AFM and evaluated total DNA damage with real‐time polymerase chain reaction (RT‐PCR). Four different target sequences were investigated: A10 and G10 systems with respectively higher AT and GC contents, and sequences incorporating halogenated nucleosides 8‐bromoadenosine (^8BrA) and 2’‐deoxy‐2’‐fluorocytidine (^2’FC), which serve as models for radiosensitizing agents with halogens at the nucleobase or sugar moiety, such as gemcitabine used in pancreatic cancer treatment.[ ⁸⁷ , ⁸⁸ ]

All DNA origami nanostructures were designed with target sequences placed in the top position and a control strand comprised of a scrambled DNA sequence (R(A10)) in the bottom position. The study showed that the A : T and G : C ratio did not affect DNA DSB yields, while RT‐PCR indicated similar total lesions in sensitized and control strands. Incorporating three ^2’FC nucleosides increased DNA DSB yields, while RT‐PCR indicated similar total lesions in sensitized and control strands. This suggests that clustered ^2’FC‐damaged strands are more prone to DSBs, whereas control strands evolve into non‐DSB damage forms. For ^8BrA‐modified DNA, both total lesions and DNA DSB yields increased, although clustered damages did not predominantly result in DSBs. Proximity effects were observed, as DSBs in control strands increased when near sensitized strands with ^2’FC and ^8BrA, likely due to fragmentation products from sensitized strands causing damage in adjacent control strands. The interstrand distance was ∼12 nm, but new designs could explore radiation‐induced proximity effects by varying DNA sensitizing compounds and interstrand distances.

With regards to DNA origami stability upon exposure to high‐energy electrons within the irradiation and solution conditions employed in the work, structural damage to the nanoframes leading to fragmentation already begin at 6 kGy of absorbed dose which already influences the DSB analysis at such high doses. Nonetheless, such high doses already open a wide window for the use of DNA origami in high‐energy irradiation studies.

In summary, investigations into γ‐ray and β‐ray interactions with DNA origami pave the way for tailored strategies to fortify nanostructure resilience and leverage radiation effects for diverse biotechnological applications.

2.3. High‐LET Particle Interactions with DNA Origami

High‐energy ionic projectiles, characterized by their high linear energy transfer (LET), exhibit greater biological effectiveness compared to low‐LET radiation. However, this does not directly translate to increased DNA damage, as the number of lesions induced by heavy ions exhibits a similar dose dependence as that of low‐LET radiation. ^[89] The key difference lies in the complexity of the damaged sites. ^[90] The common explanation is that the intricate spurs created by high‐LET radiation facilitate recombination of the formed ⋅OH radicals, thus mitigating their damaging effects. ^[90] This is supported by the observation that the decrease in OH radical concentration with increasing LET in solution is significantly steeper than the decrease in overall damage. ^[40] This implies that ⋅OH radicals likely play a less dominant role in DNA damage at high LET. While several hypotheses exist, the most compelling one suggests an increasing contribution of secondary species, like LEEs, to radiation damage at high LET. ^[91] This will be further explored in the following section. A comprehensive understanding of these fundamental effects requires more intricate experimental and theoretical investigations. ^[92] This section focuses on structural changes caused by high‐LET particles in DNA origami nanostructures.

Sala et al. ^[85] have investigated the effects of high‐LET radiation by irradiating DNA origami nanostructures with a 30 MeV proton beam. Analysis using AFM revealed a damage rate of (0.12±0.06) DNA triangles per Gy of absorbed dose in a buffer solution, which is about 4 times higher than that observed in dry conditions. Furthermore, the damage induced by protons was about doubled that caused by γ‐rays in dry conditions and 1.5 times higher in buffer solutions. Notably, the damage caused by proton beam to single‐stranded and double‐stranded M13mp18 DNA was significantly enhanced compared to the folded scaffold DNA strand in the presence of tris (⋅OH radical scavenger). This observation implies a substantially lower susceptibility of DNA origami nanostructures to ⋅OH radical attack compared to supercoiled plasmid DNA.

The enhanced stability of scaffolded structures towards ionizing radiation motivated a follow‐up study by Sala et al. ^[93] focusing on ion projectiles commonly used in nanotechnology. The findings, summarized in Figure 3B.II (adapted from Sala et al. ^[93] ) illustrate the structural changes induced by ions with different energies.

Gallium (Ga) ion beams, commonly used for material milling in SEM lead to the complete removal of DNA origami nanostructures from the substrate. However, this effect is highly localized to the irradiated area. This demonstrates the potential of low‐energy heavy ion beams, readily available in commercial equipment for precise cutting of deposited DNA origami nanostructures.

Irradiation with high‐energy Fe ions in a vacuum environment, corresponding to typical ion implantation energies, results in the formation of well‐defined craters within a 10 nm radius around the ion impact point. This suggests the potential for combining ion implantation with DNA origami nanostructure patterning or use in complex multistep semiconductor preparation. However, high fluences can also lead to a decrease in the height of the deposited nanostructures.

At even higher LET values, the size of the nanostructures becomes insufficient to dissipate all the incoming energy. Consequently, stochastic processes such as heating can induce shape transformations, e. g., the liftoffs in the inner seams of the DNA origami triangles. Such design‐controlled shape deformation is one of the promising fields to explore in the future.

In summary, the exploration of high‐LET particle interactions with DNA origami nanostructures can pave the way for tailored strategies to harness their effects in diverse technological applications.

2.4. Low‐Energy Electron Induced DNA Damage: Mechanisms and Implications

This section explores the mechanisms through which low‐energy electrons (LEEs) induce damage to DNA molecules. With energies typically below 20 eV, LEEs are recognized as potent contributors to DNA radiation damage due to their efficient interaction with molecular targets. Upon interacting with DNA, LEEs can initiate a cascade of electron‐driven reactions, resulting in various forms of damage, including single‐and double‐strand breaks, base damage, and cross‐linking.[ ⁹⁴ , ⁹⁵ ]

The Ameixa and Bald account ^[11] provides a comprehensive overview of fundamental mechanisms underlying DNA radiation damage caused by photons and low‐energy electrons. The primary interaction mode between LEEs and DNA involves the process of electron attachment, where an electron is temporarily captured by a DNA molecule, forming a transient negative ion (TNI), also known as resonance. ^[96] Additionally, LEEs can trigger secondary processes like dissociative electron attachment (DEA) and auto‐detachment, generating additional reactive species such as transient anions and neutral radicals, which contribute to DNA damage. These secondary processes significantly amplify the overall impact of LEE‐induced DNA damage, especially in genomic regions with increased electron interaction.[ ²⁸ , ⁹⁷ ]

In cancer therapy, the simultaneous administration of radiosensitizing agents alongside high‐energy radiation is a common practice. Halogenated nucleosides and platinum‐derivatives, when incorporated into DNA via biological pathways, can render specific regions of the DNA molecule more susceptible to electron interactions due to their chemical composition, such as higher concentrations of certain chemical groups (halogens or pseudo‐halogens), or structural characteristics prone to DNA strand breakage. ^[17]

DNA origami has emerged as a powerful tool for studying DNA radiation damage induced by LEEs. Bald and co‐workers developed an experimental technique for determining absolute cross sections for LEE‐induced DNA single‐ and double‐strand breaks using DNA origami nanostructures. This approach, schematically outlined in Figure 4A, allows for controlled experiments focusing on model DNA molecules, probing the effects of chemical composition, size, shape and spatial arrangement on LEE interactions. This technique allows for the direct comparison of different DNA sequences within a single irradiation experiment, providing insights into sequence effects, among others. For instance, figure 4B shows AFM images comparing (i) a control sample and (ii) samples exposed to LEEs. This experimental approach achieves absolute quantification of DNA strand breakage, high sensitivity, and investigation of sequence dependencies have been achieved, making DNA origami a valuable tool in DNA radiation damage research. Schürmann et al. ^[98] studied the effect of incorporating 8‐bromoadenine, a potential radiosensitizer, into a DNA sequence, quantifying electron‐induced DNA strand breakage. AFM analysis of DNA origami nanostructures yielded an average strand break enhancement factor of 1.9±0.6.

Figure 4.

(A) Schematic outlining the experimental procedure for absolute quantification of DNA single‐strand breaks induced by radiation. Adapted with permission from Ref. [11] Copyright 2024 American Chemical Society. (B) AFM images comparing (I.) a control sample, and (II.) samples exposed to 10 eV LEEs under high‐vacuum conditions for 40 seconds. Adapted with permission from Ref. [99] Copyright 2017 Springer Nature. (C) Effects of 8‐bromoadenine (^8BrA) modifications on DNA sequences, namely: (I.) low‐energy electron‐induced single strand breaks in sequences TT(XTA)₃TT, where X=^8BrA, A, and (II.) comparison of obtained EF_SB as a function of the electron energy along with the average EF_SB(^8BrA). Adapted with permission from Ref. [98] Copyright 2017 Wiley‐VCH.

In summary, the mechanisms by which low‐energy electrons (LEEs) induce DNA damage underscore their significance in radiation biology and cancer therapy. In this regard, the continued exploration of LEE‐induced DNA damage and its implications have potential for the development of novel therapeutic strategies and the optimization of existing treatment modalities.

2.5. ROS‐Induced DNA Damage Mechanisms Using DNA Origami Nanostructures

This section focuses on the mechanisms by which reactive oxygen species (ROS) induce DNA damage. ROS, including hydroxyl radicals (⋅OH), superoxide radicals (O₂⋅⁻), and singlet oxygen (¹O₂), are byproducts of cellular metabolism and can also be generated by water radiolysis. Upon interacting with DNA, ROS can cause various lesions such as base modifications, DNA strand breaks, and DNA‐protein cross‐links. Hydroxyl radicals, in particular, are highly reactive, capable of abstracting hydrogen atoms from DNA sugar moieties or directly attacking the DNA backbone, resulting in strand breaks. Additionally, superoxide radicals contribute to DNA damage through the formation of secondary reactive species like hydrogen peroxide (H₂O₂), which can diffuse into the nucleus and cause oxidative damage to DNA bases. Singlet oxygen, generated during photosensitization reactions, can oxidize DNA bases, forming mutagenic lesions. ROS‐induced DNA damage triggers cellular responses, including activation of DNA repair pathways and potentially leading to apoptosis.

By employing DNA origami nanostructures as experimental platforms, DNA molecules can be exposed to ROS in controlled environments. This approach enables the study of ROS‐DNA interactions at the nanoscale, providing insights into the spatial distribution of DNA damage and the role of DNA structure in modulating ROS‐induced lesions.

Ray et al.[ ¹⁰⁰ , ¹⁰¹ ] used AFM imaging of DNA origami nanostructures to investigate DNA damage by ROS at the single‐molecule level. In one study, ^[100] DNA origami nanostructures, such as waffles and chopsticks, were adsorbed on mica substrates and exposed to singlet oxygen ¹O₂ generated from C₆₀ under green light (528 nm) irradiation. AFM imaging revealed structural changes, including reduced and broader height distribution and disintegration of DNA origami nanostructures, particularly in ROS ‐exposed regions. These structural transformations suggest that DNA single‐strand breaks cause a relaxation of internal stresses and flattening of the DNA nanostructure. Subsequent experiments with quenchers (L‐histidine) and electron donors (NADH) confirmed the involvement of ¹O₂ and other ROS species, such as O₂⋅⁻ and ⋅OH.

In another study, Ray et al. ^[101] used a chemically modified Au‐coated AFM tip with a tripod molecule containing C₆₀ to locally produce ¹O₂ upon green light exposure. The “tripod‐C₆₀” was attached to the Au‐coated AFM tip by multiple S−Au bonds between the rigid tripod scaffold and the gold coating of the tip, placing the C₆₀ molecule close to the substrate. AFM imaging demonstrated localized DNA damage, providing further evidence of ¹O₂ involvement, as DNA waffles were cut by the action of the photoexcited C₆₀ molecule upon green light irradiation. Complementary studies confirmed that ¹O₂ is the precursor for oxidative DNA damage by the “tripod‐C60” AFM tip. For example, AFM imaging in a D₂O solution, where the lifetime of ¹O₂ is longer than in H₂O, showed more extensive DNA damage in the area scanned by the “tripod‐C60” AFM tip in relation to unscanned areas.

The formation of ¹O₂ from photosensitizers has significant applications in biology and medicine, particularly in PDT for cancer treatment. In PDT, photosensitizers administered to the patient preferentially accumulate in tumor tissues. Upon irradiation with light, these photosensitizers produce ¹O₂, which causes localized oxidative damage and selectively kills targeted cells. DNA nanostructures have been utilized to control and investigate the generation, lifetime and reactions of ¹O₂. ^[103]

In a pioneering study by Helmig et al., ^[102] DNA nanostructures were designed to monitor the reactions of ¹O₂ produced upon irradiation of the photosensitizer indium pyropheophorbide (IPS, Figure 5A) on the DNA origami surface. The experimental approach, depicted in Figure 5A, involved creating DNA origami nanostructures as platforms for strategically placing biotinylated oligonucleotides (ssDNA) containing a ¹O₂ cleavable (SOC) linker at four positions. A non‐cleavable biotinylated ssDNA was positioned at the corner of the DNA nanostructure as a reference. Figure 5B shows AFM images before and after irradiation. The decrease of bright streptavidin (SAv) spots in the irradiated sample, compared to the control sample, indicates the single‐molecule level response to ¹O₂. Figure 5C illustrates the DNA origami template used for studying distance‐dependent ¹O₂ production by IPS, with interior and peripheral groups placed at 18 nm and 36 nm from the IPS, respectively. Upon photoexcitation, the DNA strand breaks at the interior and peripheral positions were observed to be 81 % and 70 % of that of the reference DNA, respectively.

Figure 5.

(A) Schematic representation of the experimental approach for studying ¹O₂ production and diffusion from a centrally positioned photosensitizer (IPS) towards SOC linkers upon exposure to red light. (B) AFM images comparing (1) a non‐irradiated control sample and (2) a sample after irradiation, showing the effects of photoexcitation. (C) Distance‐dependent analysis using (1) a DNA origami structure with DNA groups placed at three distinct positions and (2) the relative DNA strand breakage upon photoexcitation as a function of the distance from the IPS. Adapted with permission from Ref. [102] Copyright 2010 American Chemical Society.

Efforts to spatially control the generation of ¹O₂ have been extensive; ^[103] however, there has been limited exploration into quantitatively assessing DNA strand breaks induced by ¹O₂ or further ROS produced by photosensitizers. Wang et al. ^[104] utilized DNA origami nanotechnology to host both single‐stranded (ssDNA) and double‐stranded DNA (dsDNA), demonstrating that the molecular ruby [Cr(ddpd)₂][BF₄]₃ (Figure 6) induces DNA strand breaks via ¹O₂ production upon exposure to UVA/vis light, highlighting its potential as a photosensitizer for PDT. Using the experimental method illustrated in Figure 6A, DNA strand breaks induced by photoexcitation of this Cr^III complex at the single‐molecule level, enabling determination of respective quantum yields. AFM images in Figure 6B depict DNA origami before and after treatment with [Cr(ddpd)₂][BF₄]₃ and irradiation. Specifically, AFM analysis revealed minimal changes in height profile in the angstrom range when compared to non‐treated controls kept in darkness versus those exposed to light. Figure 6C illustrates results obtained with [Cr(ddpd)₂][BF₄]₃ at concentrations of 5, 20 and 50 μM, showing (1) absolute cross‐sections for DNA single‐ and double strand breaks, σ_SSB and σ_DSB , in the order of 10⁻¹⁹ cm², and (2) respective quantum yields, indicating the number of photons absorbed by Cr^III required for DNA strand breakage, observed at 1 %–4 % in this concentration range.

Figure 6.

(A) Schematic of the experimental procedure for the determination of absolute DNA strand break cross sections, σ_SB , upon photoexcitation of the water‐soluble photosensitizer [Cr(ddpd)₂][BF₄]₃.(B) AFM images depicting a) a DNA origami control sample kept in the dark and b) a DNA origami sample treated with the photosensitizer and exposed to UV light (365 nm, 0.60 mWcm⁻²) for 120 seconds. Insets show the results before and after exposure. (C) 1. Absolute DNA strand break cross sections for ssDNA (black) and dsDNA (red) and 2. Quantum yields as function of the photosensitizer's concentration. Adapted with permission from Ref. [104] Copyright 2023 Wiley‐VCH.

Rabbe et al. ^[105] have investigated how ionic composition affects the stability of DNA origami nanostructures, particularly triangles and six‐helix bundles (6HBs), when exposed to ROS generated during PDT. They found that ROS‐induced damage is more severe at low ionic strengths, while magnesium ions (Mg²⁺) offer protection and can even aid in repair after irradiation. Additionally, 6HBs show greater resistance to ROS compared to triangles, due to their mechanical properties.

Overall, these studies highlight the significance of DNA origami for investigating ROS‐induced DNA damage and suggest potential therapeutic strategies for targeting oxidative stress‐related pathways in disease treatment, including cancer and aging.

3. Applications

This section examines the potential of DNA origami nanostructures in ionizing radiation dosimetry and outlines future research directions.

3.1. Applications and Implications from Medicine to Nanotechnology

DNA origami nanostructures offer versatile applications in various fields. Its ability to self‐assemble into well‐defined structures makes it a valuable tool for high‐resolution lithography. Precise manipulation of light allows for high‐resolution patterning of DNA origami structures on surfaces, enabling the creation of intricate nanoscale circuits and devices Light can be also used to trigger specific reconfigurations within these structures, enabling control over the final pattern. This approach has the potential to transform miniaturization processes in fields like electronics and biosensors.

In medicine, light‐responsive DNA origami holds promise for PDT and photosensitive drug delivery. By incorporating therapeutic agents within its structure, DNA origami can be designed to target specific locations within cells. This targeted approach could significantly improve treatment efficacy while minimizing side effects. For instance, Zhuang et al. ^[65] demonstrated that DNA origami nanostructures loaded with the photosensitizer carbazole derivative BMEPC were effective for intracellular imaging and PDT. More recently, Garcia‐Diosa et al. ^[106] investigated the ability of DNA origami to quench singlet oxygen, showing that these nanostructures can effectively reduce singlet oxygen levels. This suggests that DNA origami could enhance PDT by mitigating oxidative damage to healthy tissues while increasing therapeutic effects on cancer cells. Additionally, electrostatic interactions between photosensitizers and DNA origami – affecting photosensitizer aggregation, stoichiometric ratios, and cation concentrations – may drastically influence the photosensitizer's photo(oxidative) activity, offering further potential to refine therapeutic outcomes. This highlights the dual role of DNA origami nanostructures as both a drug carrier and an active agent in managing oxidative stress in medical applications.

Furthermore, the sensitivity of DNA origami to radiation makes it a potential candidate for developing biosensors. Recent advancements in DNA‐modulated plasmon resonance ^[107] include multifunctional bioprobes made from DNA origami, which improve the sensitivity and specificity of plasmonic sensors. New designs, such as fiber‐optic sensors with gold nanoparticles conjugated to DNA, and enhanced surface plasmon resonance imaging (SPRi) allow for tailored applications. These developments are paving the way for innovative medical diagnostics and environmental monitoring solutions. For sensors and detectors, DNA origami's ability to respond to radiation can be harnessed to develop highly sensitive radiation detectors for medical applications and environmental monitoring. Specifically, in nano dosimetry, DNA origami can be utilized to measure radiation doses at the nanoscale, providing detailed information that is crucial for both medical treatments and radiation safety and protection assessments in various environments, such as hospitals, nuclear power plants and space missions.

3.2. DNA Based Dosimetry

There are several open challenges in ionizing radiation dosimetry that DNA origami nanostructures can address. One of the most important challenges is ensuring the biological relevance of the measurement, which makes DNA an attractive material for various types of dosimetry. Additionally, DNA‐based dosimetry provides an important benchmark for state‐of‐the‐art models of radiation damage, ranging from ab initio to multiscale approaches. ^[108]

However, similar to other dosimetric approaches, DNA‐based dosimetry faces several problems that could be mitigated by the application of scaffolded nanostructures. One significant challenge is the evaluation of DNA damage. Common approaches include gel electrophoresis combined with incubation with various endonuclease enzymes ^[109] and colorimetry based on immunochemical reactions. ^[110] For instance, Kirby and co‐workers have developed an elegant approach for DNA double‐strand break dosimetry, where double‐stranded DNA strands of 4 kbp were labeled with magnetic beads and fluorescein. Double strand breaks then result in disconnection of the fluorescent probe and decrease of the signal on magnetically separated bead. ^[111]

DNA origami presents opportunities for real‐time evaluation of damage. Recent approaches include using DNA origami as a photosensitive “dosimetric” modification of field‐effect transistors ^[112] and employing Förster Resonance Energy Transfer (FRET) techniques. DNA origami's precise positioning of FRET allows for sensitive detection of structural changes in the subnanometer range, which could be indicative of radiation damage, similar to works on DNA origami structural distortions.[ ¹¹³ , ¹¹⁴ ] While direct application of this approach in dosimetry is yet to be established, recent works on using FRET DNA origami to monitor pH changes ^[115] or DNA decomposition in in vitro experiments ^[116] suggest its potential utility.

Another challenge in dosimetry, particularly with tissue‐equivalent materials, is resistance to high doses of radiation and spatial resolution. ^[117] 2D DNA origami, as demonstrated in studies by Sala et al., ^[85] show promise by requiring high doses of radiation to induce damage, with localized effects within approximately 10 nm of the ionization track. However, extending DNA origami‐based detectors into the third dimension, such as through nanoarray DNA curtains, ^[118] presents challenges due to secondary species created on one strand potentially causing damage to neighbor strands, limiting spatial resolution to around 100 nm, ^[119] even though this will be still superior to most of the bulk‐based detectors.

Finally, a significant challenge lies in absolute measurements of dose estimates for nanoscopic matter, compounded by the complexity of environmental effects, especially with low‐concentration samples. This challenge hinders achieving absolute numbers and contributes to ongoing discussions regarding various effects on DNA damage and consequences for Relative Biological Effectiveness (RBE). We envisage a concept based on mixing and depositing DNA origami nanostructures of different shapes, where one shape is loaded with a radioisotope and other acts as a probe for ionizing radiation effects at the nanometer scale. This approach enables analyses through techniques such as AFM to search for radiation track effects and direct estimation of decay in solution. ^[90] Ongoing research efforts are directed towards the realization of proposed nano‐dosimetric architectures based on DNA origami.

3.3. DNA Computing

Various applications of DNA nanostructures have been envisaged for DNA‐based computing. Two challenging questions need to be addressed that are closely related to the interaction of DNA nanostructures with radiation.

The first question involves the integration of existing silicon‐based technology with DNA nanotechnology. Several strategies can be employed for this integration. One approach is the combination of lithographic techniques with DNA nanostructures. It has been shown that the strong binding of DNA origami to technology‐relevant surfaces such as silicon allows for the application of ion beam‐induced techniques to shape the underlying substrates or even the DNA nanostructures, while preserving their overall shape. ^[93] Another strategy involves the use of photoresponsive crosslinkers combined with DNA nanostructures. These crosslinkers can remain only in selectively irradiated areas when exposed to light.[ ³⁶ , ⁶⁷ ] A novel approach utilizes sequence‐dependent photosensitive monomers, offering an additional level of control over the photopolymerization process. ^[120]

The second question pertains to the evaluation of the stability of DNA nanostructures upon irradiation. The stability of DNA origami with respect to radiation is crucial not only during lithographic processes but also when DNA nanostructures serve as active components in devices. For example, if quantum computation is performed on DNA nanostructures using photoresponsive centers, ^[121] it is important to determine how many operations these structures can sustain.

Another topic is DNA‐based memory or data storage. DNA offers high writing density and long storage lifetime due to the high stability of DNA strands.[ ¹²² , ¹²³ ] Both sequence‐based and structure‐based approaches are under development for digital data storage. ^[124] Unlike conventional memories, where the effects of background ionizing radiation on the Earth's surface ^[125] are negligible due to the size of the memory cell and the temporary nature of such damage, DNA‐based memories may be significantly more sensitive to ionizing radiation due to their smaller size and higher writing density. ^[126] As a result, the stability and longevity of DNA‐based storage devices need systematic study and evaluation.

3.4. Radiation‐Resistant DNA Origami Nanostructures

Developing ionizing radiation‐resistant DNA origami nanostructures is crucial for their successful deployment in environments exposed to such radiation. This section examines potential strategies to enhance the resilience of DNA nanostructures against radiation damage, considering their integration into nanoscale devices under background radiation exposure on Earth's surface.

3.4.1. Sequence Engineering

DNA itself offers some inherent opportunities for optimization. Alternative backbones, such as phosphoramidite linkages, may potentially enhance resistance to strand breaks induced by radiation compared to the natural phosphodiester backbone.[ ¹²⁷ , ¹²⁸ ] For example, Kalinowski et al. ^[127] successfully synthesized a relatively small DNA origami sheet using phosphoramidite ligation, incorporating GC‐rich regions to enhance stability. Additionally, integrating redundant staple sequences and error correction mechanisms inspired by natural DNA repair systems within the origami can provide further protection against radiation damage. ^[129] For instance, Scheckenbach et al. ^[130] have demonstrated that self‐repair mechanisms, taking advantage of the self‐regeneration by a pool of intact building blocks replacing strands damaged by exposure to lasers or enzymatic attack and exchange under thermal equilibrium conditions.

3.4.2. Chemical Modifications

Introducing crosslinking agents and functional groups with antioxidant properties (e. g., thiols, catechols) can scavenge free radicals, thereby mitigating the damaging effects of low‐energy electrons (LEEs) and reactive oxygen species (ROS).

3.4.3. Supramolecular Assembly

Physically shielding nanoparticles or biocompatible polymers can create supramolecular DNA origami structures, ^[131] potentially absorbing radiation energy and protect the DNA core.

3.4.4. Electrostatic Interactions

Coating DNA origami nanostructures with polymers, proteins and other molecules through electrostatic interactions has proven to significantly enhance stability. This approach explores the binding of cationic molecules to the negatively charged DNA backbone, shielding the DNA from radiation damage and environmental factors. For instance, Kostiainen and colleagues demonstrated the use of cationic polymers, ^[132] protein‐dendron hybrids ^[133] and viral capsid‐like particles[ ¹³⁴ , ¹³⁵ ] to reinforce DNA origami nanostructures, enhancing their structural integrity in complex environments. Similarly, Shih's group has shown that cationic oligolysines can both bind and cross‐link DNA origami nanostructures, improving their resilience against enzymatic degradation and low‐salt denaturation.[ ¹³⁶ , ¹³⁷ ] LaBean's research further explores the use of peptides and proteins to enhance stability through similar electrostatic and cross‐linking mechanisms.[ ¹³⁸ , ¹³⁹ ] These strategies not only enhance structural support but also offer potential for increasing radiation resistance, as the coatings can absorb or deflect radiation energy before it reaches the DNA core.

3.4.5. Design Considerations

Minimizing exposed single‐stranded regions within the DNA origami structure is crucial, as these areas are more susceptible to radiation damage than double‐stranded segments. Smaller and more compact structures may experience less overall radiation exposure compared to larger designs. For example, Wang et al. ^[140] investigated 2D wireframe DNA origami structures. They introduced a method using honeycomb edges composed of six parallel duplexes, which significantly improves the structural fidelity of the origami compared to traditional methods that rely on fewer duplexes.

3.4.6. Combining Synergistic Strategies

The most effective approach likely involves exploring synergistic effects between these strategies to achieve optimal radiation resistance. The design could be customized based on the anticipated quality and intensity of ionizing radiation and intended application.

4. Summary and Outlook

In conclusion, this review has described the effect of irradiation on the structural integrity of DNA origami nanostructures, exploring fundamental processes in DNA molecules, and applications across various technological fields. Additionally, it has presented strategies for engineering radiation‐resistant DNA origami nanostructures. However, for these engineered DNA origami structures to realize their potential in enhancing resistance to radiation across diverse exposure scenarios, rigorous experimental validation and optimization are needed. Through interdisciplinary efforts, advancements in radiation‐resistant DNA nanostructures are expected to drive progress in fields such as radiation therapy, radiobiology and radiation detection. Applications in high radiation environments, including nuclear technology and space exploration, highlight the transformative impact of these developments. Overall, DNA origami nanostructures hold promise for pioneering novel solutions that will shape the future of scientific research and technological development in the years ahead.

Conflict of Interests

The authors declare no conflict of interest.

Biographical Information

João Ameixa holds an M.Sc. in Biomedical Engineering from the University NOVA of Lisbon. In 2020, he earned his Ph.D. in Physics from the University of Innsbruck and in Radiation Biology and Biochemistry – Applied Atomic and Molecular Physics from the University NOVA of Lisbon through a cotutelle agreement. From 2021 to 2023, he conducted postdoctoral research in Prof. Dr. Ilko Bald's Hybrid Nanostructures group at the University of Potsdam, using DNA origami nanostructures and AFM to investigate radiation damage to DNA. Since 2024, he has been acting as the scientific coordinator of the innoFSPEC Transfer Laboratory at the University of Potsdam, promoting technology and knowledge transfer.

Biographical Information

Leo Sala finished his Master's degree in Chemistry at the University of Porto and the University of Paris‐Saclay in 2015. He completed his Ph.D. in Chemistry also at the University of Paris‐Saclay in 2018. In 2019, he began his postdoctoral research at the J. Heyrovský Institute of Physical Chemistry, where he now continues as an associate researcher working on the preparation, design, and optimization of DNA nanostructures for a wide array of applications: fundamental DNA radiation damage studies, drug delivery, in vivo imaging, DNA data storage, among others.

Biographical Information

Jaroslav Kočišek holds Ph.D. degrees in Physics from Comenius University in Bratislava (2010) and Charles University in Prague (2013). After postdocs studying electron scattering at the University of Fribourg and collisions of multiply charged ions at GANIL, he moved to J. Heyrovský Institute of Physical Chemistry, where he is now leading a research group exploring the molecular mechanisms of radiation damage.

Biographical Information

Ilko Bald studied chemistry at the FU Berlin where he also obtained a PhD in Physical Chemistry. After a postdoc at the University of Iceland he moved to the Interdisciplinary Nanoscience Center (iNANO) at Aarhus University working on scanning probe microscopy and DNA nanotechnology. In 2013 he established a junior research group “Optical Spectroscopy and Chemical Imaging” at the University of Potsdam and the Federal Institute for Materials Research and Testing (BAM, Berlin). Since 2019 he is Professor for Hybrid Nanostructures at the University of Potsdam investigating nanomaterials, their optical properties, and electron induced processes.

Ameixa J., Sala L., Kocišek J., Bald I., ChemPhysChem 2025, 26, e202400863. 10.1002/cphc.202400863