Room temperature and selective triggering of supramolecular DNA

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Room temperature and selective triggering of supramolecular DNA assembly/disassembly by non-ionizing radiation Andrea A Greschner, Xavier Ropagnol, Mohamed Kort, Nabilah Zuberi, Jonathan Perreault, Luca Razzari, Tsuneyuki Ozaki, and Marc A. Gauthier J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10355 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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Journal of the American Chemical Society

Room temperature and selective triggering of supramolecular DNA assembly/disassembly by non-ionizing radiation Andrea A Greschner1, Xavier Ropagnol1, Mohamed Kort1,2, Nabilah Zuberi1, Jonathan Perreault3, Luca Razzari1, Tsuneyuki Ozaki1, Marc A Gauthier1,* Institut National de la Recherche Scientifique (INRS), EMT Research Center, Varennes, Qc, J3X 1S2, Canada Université Pierre et Marie Curie (UPMC), Paris, France 3 Institut National de la Rechercher Scientifique (INRS), Institut Armand Frappier, Laval, Qc, H7V 1B7, Canada KEYWORDS terahertz, far infrared, DNA, plasmid, protein, microwave, simultaneous cooling, enzyme, nanostructure, nonionizing radiation, supramolecular, denaturation, thermal melting, antibody 1 2

ABSTRACT: Recent observations have suggested that non-ionizing radiation in the microwave and terahertz (THz; far infrared) regimes could have an effect on double-stranded DNA (dsDNA). These observations are of significance owing to the omnipresence of microwave emitters in our daily lives (e.g., food preparation, telecommunication, wireless internet) and the increasing prevalence of THz emitters for imaging (e.g., concealed weapon detection in airports, screening skin cancer) and communication technologies. By examining multiple DNA nanostructures as well as two plasmid DNA, microwaves were shown to promote the repair and assembly of DNA nanostructures and single-stranded regions of plasmid DNA, while intense THz pulses had the opposite effect (in particular for short dsDNA). Both effects occurred at room temperature within minutes, showed a DNA-length dependence, and did not affect the chemical integrity of the DNA. Intriguingly, the function of seven proteins (enzymes and antibodies) was not affected by exposure to either forms of radiation under the conditions examined. This particularity was exploited to assemble a fully-functional hybrid DNA–protein nanostructure in a bottom-up manner. This study therefore provides entirely new perspectives for the effects, on a molecular level, of non-ionizing radiation on biomolecules. Moreover, the proposed structure–activity relationships could be exploited in the field of DNA nanotechnology, which paves the way for designing a new range of functional DNA nanomaterials that are currently inaccessible to state-of-the-art assembly protocols.

Introduction Deoxyribonucleic acid (DNA) carries the information blueprint for all living organisms, and serves several roles that are essential for life. Over the past two decades, DNA has advanced well beyond this natural role as storage medium for genetic information to that of a programmable design material and building block for ‘designer’ nanoscale structures.1-2 Furthermore, since seminal work in 90s,3 the complexity of these nanostructures has greatly evolved to display non-DNA appendages such as dendrimers, polymers, lipids, metal-binding sites, fluorophores, etc.4-6 Each of these classes of ligands provides new opportunities for creating functional nanomaterials, with applications ranging from drug delivery,7-8 in vivo chlorine9 and pH sensing,10 to molecular machines,11-12 nanowires,13 and surface patterning of polymer amphiphiles.14 Considering the pluripotency of these structures, the field has abundantly investigated approaches to manipulate the assembly and disassembly of DNA nanostructures of increasing complexity, using chemical or thermal cues. However, current tools for provoking the disassembly of DNA nanostructures are limited. Overall, general tools for externally triggering either DNA assembly or disassembly under mild and additive-free conditions that are compatible with sensitive functional ligands such as

proteins (i.e., room temperature without addition of denaturants, etc.) do not currently exist. Such a discovery would greatly expand the complexity and opportunities available for DNA-templated nanomaterials, and simultaneously expand their applications by providing a new degree of freedom over their assembly/disassembly processes. Intriguingly, recent observations have suggested that non-ionizing radiation in the microwave and terahertz (THz; far infrared) regimes could possibly have an effect on double-stranded DNA (dsDNA). For instance, while the denaturing thermal effect of microwave radiation on dsDNA solutions/samples is well-known, one study has suggested that microwaves can dissociate short dsDNA at very low temperature (–20 ºC to +20 ºC).15 Moreover, intense pulses of THz radiation have been shown to alter cellular differentiation and gene expression profiles. These effects were indirectly attributed to an effect on DNA although the underlying molecular phenomenon and characteristics of the nucleic acid(s) implicated remain to be established.16-18 In a broader biological context, these observations are of significance owing to the omnipresence of microwave emitters in our daily lives (e.g., food preparation, telecommunication, wireless internet). Moreover, the use of THz radiation is increasingly becoming commonplace, in particular for imaging (e.g., concealed weapon detection in airports, screening skin cancer) and communication technologies. In fact, the strong

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atmospheric attenuation of THz is driving the field to develop higher intensity sources, suggesting that humans will be increasingly exposed to this form of radiation. In light of this context, it is extremely timely to investigate the effects of microwave and THz radiation on DNA, in particular for long dsDNA as well as DNA nanostructures (highly modular in nature and offer the possibility of establishing structure–activity relationships).

under the conditions examined. To demonstrate new opportunities offered by this knowledge, microwave radiation was used to assemble, in a bottom-up manner, a DNA nanostructure bearing three different ligands, including a temperature-sensitive monoclonal antibody fragment. Our findings therefore present entirely new perspectives for the effect of non-ionizing radiation on biomolecules in solution, and propose guidelines for exploiting these effects in the field of DNA nanotechnology.

From a molecular standpoint, THz radiation resonates with lowfrequency vibrational modes such as long-range intra/intermolecular interactions. They are expected to excite the hydrogenbond network of water (2.4 – 6 THz ≈ 80–200 cm–1) and stretching modes dsDNA (0.2, 0.5, 1, and 6 THz).19 In addition, computational studies have predicted that dsDNA possesses a high density of vibrational modes (several hundred) between 0.1 – 6 THz (0 – 200 cm–1). Indeed, strong resonances around 0.25 and 0.6 THz have been predicted and later confirmed experimentally.1921 Simulations by two independent groups suggest that THz radiation can excite the dsDNA chain by inducing oscillations of the base pairs within the double helix, which can produce point compressions of the double-strand yielding localized reversible dissociation into so-called “breathers”. Similarly, microwaves act as a continuous source of high frequency electric fields that heat polar molecules in a solvent, such as water, by forcing them to rotate with the field and lose energy in collisions (2.45 GHz ≈ 0.08 cm–1).22 The resonant absorption of microwaves by DNA has been predicted theoretically, though experimental evidence still stimulates debate.23-25 Interestingly, because microwave and THz energy are transferred kinetically to a solvent or molecule, molecular dynamics can in principle be enhanced while bulk heating is prevented by quenching ‘hotspots’ with an appropriate heat sink (for microwaves; a process some have referred to as ‘simultaneous cooling’26), or avoided altogether due to the short duration of the THz pulses. As such, both sources of non-ionizing radiation have the potential to be used in a complementary manner (i.e., continuous vs. pulsed) to investigate interactions with DNA in solution at room temperature.

Results

This study unequivocally demonstrates that electromagnetic radiation in the microwave and THz regimes predictably affect the conformation of DNA in solution. By studying multiple small DNA nanostructures as well as two plasmid DNA, microwave radiation is shown to stimulate strand exchange and repair ‘defective’ (i.e., misassembled) DNA structures. In contrast, the opposite effect was observed for intense THz pulses. Both effects occurred at room temperature within minutes, showed a DNA-length dependence, and did not affect the chemical integrity of the DNA. For comparison to another class of biomolecule (one that undergoes irreversible unfolding processes), several proteins were found to be completely insensitive to these forms of non-ionizing radiation,

Microwave induced assembly of DNA nanostructures: To initially gauge the effect of microwave radiation, a model DNA nanostructure initially proposed by Sleiman and co-workers was selected.27 This system is formed of three single-stranded DNA (ssDNA) strands (red, green, and blue in Fig. 1A) containing precisely positioned complementary A/A’, B/B’, and C/C’ sequence stretches that can assemble into a number of possible structures.27 Mixing the strands together at room temperature produces a large range of higher-order and partially assembled structures. However, following a thermal anneal, two predominant dsDNA structures, a ‘CUBE’ and a ‘NINJA STAR’, are formed along with a few other fully complementary thermodynamic constructs. Thus, this system is highly appropriate for monitoring assembly towards the most thermodynamically stable structures, exchange between thermodynamic structures, as well as disassembly resulting in kinetic structures (kinetic traps) or, ultimately, ssDNA. Microwave power programs were created to mimic the typical temperature programs used for DNA nanostructure annealing (Fig 1B). To control temperature, a heat sink comprised of a jacketed reaction vessel and microwavetransparent cooling media was used, ensuring full energy transmission directly to the reactants. A fiber-optic temperature probe submerged directly in the sample solution (~180 µL) provided feedback to ensure that the maximum set temperature was not exceeded. To the extent of our knowledge, only one other study has explored the effect of microwaves on DNA at low temperature.15 While interesting, the temperature range examined (–20 ºC → +20 ºC → – 20 ºC) involved sample freezing, and the study focused on dsDNA disassembly, rather than assembly. To stimulate dsDNA assembly, it was rationalized herein that maintaining a constant bulk temperature near room temperature would be more advantageous. Further, gradually decreasing the microwave power would mimic the progressive decrease of molecular dynamics expected of thermal anneals (slow cooling of hot solutions). To achieve this profile, the microwave reactor was programmed to rapidly heat the sample (initial temperature ~6 – 8 ºC using a maximum power setting (85 – 105 W) to a defined maximum temperature of 20 ºC. The desired temperature was


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Figure 1. Microwave induced assembly of DNA nanostructures at room temperature. (A) Equilibria of DNA nanostructures assembled from three ssDNA strands used to gauge the effect of non-ionizing radiation on assembly/disassembly in solution. (B) Microwave power program used to guide the assembly of DNA towards their most stable thermodynamic nanostructures. Inset, relative distribution of fully assembled structures following a 95 W (max.) anneal. Mean + SD (n = 3). Star denotes statistically significant difference (ANOVA, Tukey, p = 0.05; Full comparison of all species in Supplementary Table S1). (C) Example of the evolution of the composition of a reaction mixture during microwave exposure (n = 1). (D) Native gel electrophoresis of randomly assembled (left) and thermally-assembled (right) DNA nanostructures before and after exposure to microwave radiation. (E) Scheme of the fluorescent strand exchange experiment, adapted from reference 15. (F) Effect of microwave power and dsDNA length on fluorescence recovery from strand exchange experiment. Mean ± SD (n = 3). reached within ~5 – 15 min, then remained constant. Based on the characteristics of the heat sink (initial temperature of the coolant and its flow rate through the jack eted reaction vessel), the microwave power remained constant at its maximum setting for 5 – 20 min, then slowly decreased to 0 W over ~65 min as the coolant slowly warmed (Fig 1B). This is comparable to the length of the thermal anneal. At the conclusion of an anneal performed at 95 W, 82 ± 8 % of the DNA structures consisted of distinct, thermodynamic, fully complementary structures, without bulk

temperature ever exceeding 25 ºC (Fig 1B). In comparison, a control sample not exposed to microwaves possessed 20.6 ± 0.6 % of such species. Intriguingly, using a slightly lower maximum power setting (85 W rather than 95 W), lead to significantly less self-assembly (Fig 1B), possibly indicating the existence of a threshold power necessary for initial DNA duplex dissociation (akin to the thermal denaturation temperature (TM) of dsDNA). Interestingly, this result is consistent with



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Figure 2. THz-induced disassembly of DNA nanostructures at room temperature. (A) Temporal waveform and spectrum of the THz source. (B) Gel electrophoresis of randomly assembled (left) and thermally-assembled (right) DNA nanostructures before and after exposure to THz radiation. (C) Change in product distribution of thermally annealed tetrahedron and dedicated ninja star nanostructures after exposure to THz. Mean + SD (n = 3) (D) Effect of THz radiation and dsDNA length on fluorescence recovery from strand exchange experiment. Mean + SD (n = 3). Statistically significant differences (for panes C and D) are indicated by an asterisk (ANOVA, Tukey, p = 0.05). observations from Edwards et al. for shorter DNA strands.15 Increasing the maximum microwave power to 105 W did not change the yield of thermodynamically assembled products, but altered the distribution of these to favor the NINJA STAR and CUBE (Fig 1B), the most stable amongst these. However, because further increasing power (150 W; vide infra) showed signs of DNA damage, additional anneals in the 105 – 150 W range were not examined. To determine if the microwave anneal followed a reaction path similar to a thermal anneal (Fig 1C), aliquots were taken from a sample mixture at various time points throughout microwave exposure. Based on analysis by gel electrophoresis, microwave irradiation appears to first correct defects in misfolded structures. Indeed, a variety of sharp bands associated with the various thermodynamic structures accessible to the system were observed by gel electrophoresis at early time points. Thereafter, more slowly, the distribution of thermodynamic structures shifted to the most stable ones (NINJA STAR and CUBE, Fig 1C). The relative proportion of the NINJA STAR and CUBE gradually increased at a rate that appeared to be independent of the progressively decreasing microwave power, but plateaued once a power of ~15 W was reached. Exposure of a thermally-preassembled NINJA STAR to microwaves under the same conditions

did not alter product distribution, further supporting that this microwave program directs equilibria towards the most thermodynamically stable structures, at temperatures significantly below their TM. Considering these results, it appeared plausible that, analogous to TM, the microwave power required to open dsDNA is sequence and length-dependent. To investigate this hypothesis, a modified version of the strand exchange experiment by Edwards et al. was performed (Fig 1E; i.e., using the present isothermal protocol and longer dsDNA). As illustrated in Fig. 1E, dsDNA of differing lengths (15, 20, 30, and 40 base pairs; bp) were exposed to increasingly high microwave power in the presence of a second dsDNA of same length and sequence but bearing 5’-Cy3 (a fluorophore) on one strand and 3’-BHQ2 (a quencher) on the other. While all dsDNA are stable at room temperature, microwaveinduced denaturation/renaturation permits strand exchange, leading to recovery of fluorescence. As seen in Fig. 1F, a correlation was observed between microwave power and dsDNA length. Strand exchange was most readily observed for 15-bp dsDNA, beyond a threshold of ~97 W. Twenty and 30-bp dsDNA exhibited limited strand exchange between 95–115


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Figure 3. Effect of microwaves and THz on pDNA. (A) Conformational equilibria of pDNA, and effect of ssDNA and dsDNA breaks. (B) Conformational distribution of pDNA before and after exposure to microwave radiation. (Inset: example of gel electrophoresis used for analysis) Mean + SD (n = 3). (C) Conformational distribution of pDNA before and after exposure to THz radiation. (Inset: example of gel electrophoresis used for analysis) Mean + SD (n = 3). (D) Example of profiles obtained for the digestion of pDNA by T7 endonuclease before and after microwave exposure. Mean ± SD (n = 3). (E) Digestion half-lives (from profiles such as in D) for pDNA by nucleases before and after and THz exposure. Mean + SD (n = 3). Statistically significant differences (for panes B, C, and E) is indicated by an asterisk (ANOVA, Tukey, p = 0.05). W, but became substantially more pronounced at higher powers. Strand exchange for 40-bp dsDNA was limited at 125 W, suggesting that even higher microwave power would be required to denature these duplexes. The observation that shorter dsDNA are more easily denatured than longer ones is consistent with the known thermodynamics of duplex DNA – longer strands contain more base-pairing hydrogen bonds, and therefore require more

energy to denature. Considered in the context of the NINJA STAR system above, which has 10 bp per edge, the microwave power required for denaturation should be below that of a simple 15 bp dsDNA (i.e., 97 W), even accounting for increased stability through cooperativity between neighbouring strands. The observed effect of microwaves on the NINJA STAR between 85 – 105 W correlates well with this prediction. While the NINJA STAR system is an interesting investigative tool for determining the effect of



microwaves on a variety of thermodynamic structures simultaneously, and exchange between these, most DNA nanostructures in the literature are designed to assemble into a unique structure. Thus, the ability of microwaves to induce assembly of two other DNA nanostructures with either different sequence or structure to the system above was investigated. For the first, the sequences of the constituent ssDNA strands for the NINJA STAR system were redesigned so that the system only possesses a single thermodynamic structure, termed the DEDICATED NINJA STAR. The second model structure investigated, the Turberfield Tetrahedron, consists of four ssDNA strands that can assemble into a TETRAHEDRON with edges 17 bp in length (vs. 10 bp for the NINJA STAR system above), and into a dimer (Supplementary Fig S1).28 Based on strand exchange experiments similar to those performed above (Supplementary Fig S2), a 105 W (max.) microwave anneal of these systems was selected. For both systems, conversion to the thermodynamic structure was comparable to that obtained by a thermal anneal, and was ~51% and ~75% higher (for the DEDICATED NINJA STAR and TETRAHEDRON, respectively) than that observed for controls assembled at room temperature (Supplementary Fig S1 and S3). THz pulse-induced disassembly of DNA nanostructures: In contrast to the microwave source above, the high-intensity THz source employed here produced picosecond pulses (at a repetition rate of 2.5 kHz) with a peak electric field of ~70 kV·cm–1 at the focus of the THz beam, which fully encompassed the sample. The spectrum extended from 0.1 – 3 THz with a maximum at 0.9 THz (Fig 2A). Considering the energy (~0.4 µJ·pulse–1), average power (~1 mW) and duration of each pulse, sample heating is not expected. To initially investigate possible effects of intense THz radiation on DNA strands, an unassembled (i.e., non-annealed) mixture of the three component ssDNA strands of the NINJA STAR system was exposed to THz radiation for a duration of 10 min. Analysis by gel electrophoresis showed a large distribution of ~93% randomly assembled structures, which was essentially identical to that obtained for the unexposed control sample. Thereafter, a partially (thermally) pre-assembled mixture of DNA nanostructures, containing 43% distinct, thermodynamic products (NINJA STAR, CUBE, and others) was irradiated with THz pulses, to gauge the effect of this radiation on multiple structures simultaneously. Interestingly, a 10-min exposure of the mixture to THz radiation shifted the mixture well away from all the thermodynamic products towards higher-order structures (Fig 2B). The composition of the resulting mixture was reminiscent of the unassembled controls, with ~96% randomly assembled products. These data not only provide the first direct experimental evidence for an effect of intense THz radiation on DNA nanostructures in solution, but also confirms that this interaction, the nature of which is discussed later, provokes opening of the double-helical structure of dsDNA. Exposure of assembled DEDICATED NINJA STAR and TETRAHEDRON nanostructures to THz radiation had a similar effect. A decrease in the amount of thermodynamic

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structures of ~21% and ~58% was observed for the DEDICATED NINJA STAR and TETRAHEDRON, respectively (Fig 2C and D). The observed distribution of nanostructures following exposure to the picosecond THz pulses was consistent with the formation of kinetic rather than thermodynamic structures. That is, because the duration of the THz pulses was ~1 ps, the disruptive force is rapidly switched ON/OFF, leading to the recurrent production of partially unfolded structures (or ssDNA strands) that then pair with the nearest complementary strands. Further, because this process occurs at room temperature, the randomly assembled structures do not have sufficient energy to reorganize towards thermodynamic products. To assess the sequence-length dependence of the effect of THz on DNA nanostructures, the strand exchange experiment from the previous section was employed. Intriguingly, while an increase in fluorescence might have been expected due to, at minimum, disruption of the fluorescence-quenched dsDNA upon exposure to THz radiation, the opposite effect was observed. Indeed, for 10 – 20 bp dsDNA, a small yet statistically significant decrease in fluorescence was observed (Fig 2C). This result is consistent with dsDNA rapidly dissociating under the influence of the THz pulses and then rapidly re-hybridizing with itself. The small change in fluorescence intensity suggests that the ssDNA strands bearing the fluorophore and quencher do not undergo strand exchange with the unlabeled duplex but rather re-hybridize in a misaligned manner, leading to an altered efficiency of the quenching process. Indeed, it is well-known that the fluorescence properties of cyanine dyes are sensitive to interactions with DNA, including between the fluorophore and neighboring bases.29-30 The fluorescence of the longer dsDNA (30 and 40 bp) was not significantly affected by THz, suggesting that THz-induced denaturation, if occurring, is partial and that re-hybridization occurs without misalignment of the strands. This result also suggests that, similar to microwave energy, there is an upper duplex length limit for denaturation using THz radiation. This limit was in the 20 – 30 bp range for the THz exposure conditions tested. Overall, the microwave and THz exposure conditions evaluated here drive the equilibrium of DNA nanostructures in opposite directions, that is, towards thermodynamic or kinetic structures, respectively. Effect of microwave and THz radiation on larger DNA structures: Because microwave and THz radiation both had a pronounced effect on ‘small’ DNA nanostructures (six 10 bp sections for NINJA STAR and DEDICATED NINJA STAR, six 17 bp edges for the TETRAHEDRON), even at short exposure times, significantly larger structures relevant in a biological context were investigated next. Two different plasmid DNAs (pDNA) of differing length, pUC18 (2868 bp), and pBR322 (4361 bp) were selected. pUC18 and pBR322 are circular dsDNA that can undergo conformational re-organization in the form of supercoiling, which refers to the winding of the circular DNA about itself. The tightly wound nature of supercoiled DNA has several purposes, including


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Figure 4. Chemical damage to DNA induced by microwaves or THz. (A) Effect of microwave power on a pre-assembled DNA ninja star. No change of product distribution was observed, albeit smearing was observed above ca. 150 W; (B) Denaturing PAGE of DNA nanostructures exposed to THz radiation. Lane 1: Preassembled nanostructure control. Lane 2: Pre-assembled nanostructures post-THz exposure. Lane 3: Randomly-assembled nanostructures post-THz exposure. The larger-order structures observed in native analysis are no longer present, indicating that no chemical crosslinking has occurred. (C) Conformational distribution of pUC18 and pBR322 after microwave exposure. (inset: denaturing agarose gel electrophoresis (AGE) results) Mean + SD (n = 3). (D) Conformational distribution of pUC18 and pBR322 following THz exposure. (inset: Representative AGE results) Mean + SD (n = 3). (E) Schematic of an E. coli transformation experiment. (F) Number of colony forming units (cfu) after transformation for both pUC18 and pBR322 plasmids after left microwaving, and right THz exposure. For panes C, D, and F, stars denote statistically significant differences (ANOVA, Tukey, p = 0.05). compaction of strands within the cell and regulating the accessibility of gene sequences. Interestingly, supercoiling also induces strain that can cause the dsDNA to locally open to ssDNA regions (‘breathers’), which relieves stress on the DNA strand (Fig 3A). ssDNA nicks and dsDNA breaks also relieve strain, and unwind supercoiled pDNA to its circular or linear forms, respectively. Thus, analysis of pDNA conformation, from supercoiled to linear, provides a wealth of information for studying

the influence of microwaves and THz pulses on these much larger DNA structures. Solutions of as-received pDNA (70 – 75% supercoiled) were exposed to 80 W of microwave



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0.05). For pane B, activity was normalized to that of the unexposed control. radiation for 20 min, based on the experiments of the preceding section. Macroscopic temperature, as measured in situ using a fiber optic probe, was maintained below 30 °C, and averaged 16 – 18 °C (Power – Temperature profiles in Supplementary Fig S4). Results of native gel electrophoresis of the plasmids post-microwave exposure were unchanged compared to the controls, and remained 74 ± 4 % supercoiled for pUC18 and 69 ± 2 % supercoiled for pBR322 (Fig 3B). Thus, because these microwave exposure conditions did not appear to induce nicks or breaks to produce circular/linear pDNA, the characteristics of breathers were more carefully scrutinized. This was accomplished by analyzing the susceptibility of pDNA to nucleases that selectively cut the ssDNA expected within the breathers. T7 endonuclease I (T7) cleaves mismatched and ssDNA, cruciform structures, and Holliday junctions. Faster digestion times would indicate an increase in this type of DNA defect. Bal-31, which possesses both exo- and endonuclease (much slower) activity, has many targets, including nicks, gaps, and ssDNA regions. It has been demonstrated that under-wound and over-wound DNA inevitably possess ssDNA breathers along the duplex, making them more susceptible to Bal31 digestion.31-32 Finally, exonuclease VII targets both the 5′ and 3′ ends of ssDNA. It will therefore assay for the presence of large ssDNA overhangs that might be present on nicked DNA (digestion of small ssDNA overhangs would not produce a distinguishable difference in gel mobility). Remarkably, while unexposed pDNA was digested by T7 in a couple hours (τ½ ~ 80 min), microwaveexposed pDNA was entirely resistant (Fig 3D). This result suggests that microwaves provide sufficient energy to the system to ‘repair’ DNA breathers present in the as-received pDNA (i.e., rehybridization), in a manner that makes it inaccessible for digestion by T7. This effect was observed for both pDNA, with no obvious influence of size, and is consistent with the results obtained with microwaves above. Interestingly, digestion of pDNA by Bal-31 was unaffected by microwaving the plasmid, demonstrating no significant change in digestion half-life (Supplementary Fig S5). The results could be interpreted as the correction of defects that are selectively digested by T7. Alternatively, under the expectation that pDNA has a distribution of breathers of varying sizes, this observation could equally suggest that Bal-31 may have the ability to digest smaller breathers than T7 (though this is difficult to validate experimentally). Thus, if this hypothesis holds, then microwaves appear, at minimum, to reduce the size of large breathers to below the threshold that is digestible by T7.

Figure 5. Effect of microwaves and THz on proteins. (A) Catalytic activity of microwave-exposed Bal-31 and human serum, as determined by their ability to digest pUC18. (B) Catalytic activity of microwave-exposed lysozyme and chymotrypsin. (C) Binding curves for anti-EpCAM before and after exposure to microwaves. (D) Effect of THz exposure on the activity of Bal-31 and (E) T7 endonuclease. Mean + SD (n = 3). For all panes, stars denote statistically significant differences (ANOVA, Tukey, p =

Having observed the stabilizing effect of microwaves on pDNA, the effect of THz radiation was then investigated. In contrast to the small nanostructures (previous section), the much larger plasmids exhibited no significant change in conformation following a 10 min THz exposure, as determined by native agarose gel electrophoresis (Fig 3C). This result may be attributed to higher stability or slower conformational dynamics of pDNA versus the DNA nanostructure systems, which both relate to double-helical strand length and bp number, among other factors.33-36 This is also consistent with data from the preceding section indicating a lack of effect of THz radiation on dsDNA longer than ~30 bp. Analysis of pDNA’s susceptibility to T7,


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Figure 6. Assembly of a DNA–protein hybrid nanostructure. A) Scheme for the bottom-up assembly of three DNA strands, each bearing a different ligand (DNA and fab’ drawn approx. to scale). B) Native PAGE to assess assembly of DNA components alone (without antibody fragment) by three different methods reveals defects in the absence of microwave or thermal annealing. Mean + SD (n = 3) C) Native agarose gel electrophoresis of the DNA–protein nanostructure prepared by the three assembly techniques, showing the co-localization of signals from Cy3 and Cy5. D) Binding curves towards immobilized tumour necrosis factor alpha obtained by surface plasmon resonance revealing that microwave assembly does not affect the antibody fragment attached to the DNA nanostructure. Thermal annealing resulted in loss of binding affinity. For pane B, stars denote statistically significant differences (ANOVA, Tukey, p = 0.05). Bal-31, and exonuclease VII digestion showed that THz radiation had only a slight or no effect on plasmid structure (Fig 3E and Supplementary Fig S6), albeit a transient effect cannot be excluded. For both plasmids, the rate of exonuclease VII digestion remained unchanged, i.e., very low, for both exposed and unexposed DNA, supporting the absence of major damage to the pDNA. These combined results indicate that the THz exposure conditions do not macroscopically affect base pairing for large DNA structures (pDNA), even though pronounced effects were observed for the substantially smaller nanostructures. Nevertheless, it should not be excluded that higher THz fields (obtained, for example, by exploiting the strong radiation confinement of plasmonic antennas37) or average power may have an effect on pDNA. Microwave or THz-induced damage to DNA: Because microwave and THz radiation provoked DNA nanostructure assembly or disassembly, respectively, a more detailed investigation into sidereactions, such as strand breaks and cross-linking, was undertaken. Firstly, a thermally pre-assembled NINJA STAR was exposed to microwaves with a fixed power setting between 10 – 250 W for 20 min. Sample temperature was controlled via coolant temperature and flow rate, and the average temperature was maintained between 14 – 22 ºC (Supplementary Fig S7). Analysis by native gel electrophoresis revealed the absence of additional bands (vs. control), albeit a smearing of the NINJA STAR band was observed above 150 W, representing ~20% of the total DNA in the lane. This result suggested minor damage to the NINJA STAR such as partial

disassembly, possibly caused by chemical modification to the constituent strands at higher microwave power, though the precise effect was not scrutinized. Nevertheless, cross-linking or breaks were not evidenced (Fig 4A). Because THz-exposed DNA nanostructures lead to a distribution of products, denaturing polyacrylamide gel electrophoresis (PAGE) was used to probe for strand breaks (production of higher-mobility bands) and crosslinking (production of lower-mobility bands). Irradiated DNA nanostructures qualitatively revealed a similar product distribution as the control samples, which correspond to the CUBE and NINJA STAR in varying degrees of disassembly as well as bands corresponding to the individual strands (Fig 4B). Only minor smearing of the bands was observed (

Room temperature and selective triggering of supramolecular DNA

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