Luminescent DNA-origami nano-rods dispersed in a lyotropic chromonic liquid crystal

ABSTRACT Anisometric DNA nanoparticles with precisely tailored size and shape are prepared by three-dimensional folding of DNA – DNA origami – marked with a fluorescent intercalating dye (YOYO-1), stabilised with silica, and dispersed in a lyotropic chromonic liquid crystal (an aqueous solution of disodium cromoglycate). Observations by total internal reflection fluorescence microscopy indicate that the luminescent nanoparticles are stable in the liquid crystal dispersion and accumulate at the positions of point defects in the liquid crystal director field. The observations indicate that silica-stabilisation of the nanoparticles prevents the dye from leaking into the liquid crystal host and that the luminescent particles assemble at disclinations. GRAPHICAL ABSTRACT


Introduction
Deoxyribonucleic acid (DNA), the carrier molecule of genetic information, is one of the most versatile liquid crystal (LC) components, since it can appear in any major class of LCs.Lyotropic LCs can be formed by aqueous solutions of DNA [1,2], thermotropic LCs by DNA and DNA-surfactant complexes [3], and colloidal LCs by DNA nanoparticles fabricated using the tailored folding of DNA ('DNA origami') [4].The self-assembly of short DNA strands to larger aggregates may have even played an important role in the origin of life [5].The unique versatility of DNA has also triggered research at the borders between thermotropic, lyotropic and colloidal LCs.For example, the mutual aligning effects of lyotropic and thermotropic LCs [6,7], the influence of anisometric colloidal DNA nanoparticles on a lyotropic chromonic liquid crystal (LCLC) [8][9][10] and the optical properties of a lyotropic liquid crystal containing plasmonic DNA nanoparticles with switchable chirality [11] were investigated.In this work, DNA nanoparticles fabricated by DNA origami, stabilised with silica, and subsequently suspended in a LCLC, are described.
DNA origami is a very powerful, versatile method to form nanostructured monomolecular sheets [12], complex three-dimensional functional nanoparticles [13] and superstructures composed thereof [14].For this purpose, a single-stranded DNA scaffold with known base sequence (typically containing 7000-9000 nucleotides) is mixed with a few hundred kinds of singlestranded oligonucleotides ('staples', typically containing 20-40 nucleotides each) the sequence of which is complementary to two specific sections of the DNA scaffold [15][16][17][18][19]. Through base pairing via hydrogen bonds, a single staple facilitates the connection of two sections of the scaffold, several staples govern the formation of a planar sheet [12], and further DNA staples may lead to the connection of specific points on the sheet, thereby causing a folding of the DNA scaffold to a tailored three dimensional object [13].The sequences of the staples that are necessary to achieve a specific shape of the object are found by computer aided design [15][16][17][18][19].Further oligonucleotides ('handles'), the sequence of which is in part complementary to the base sequence of the scaffold, can be used to attach functional moieties to the tailored DNA nanoparticles, for example fluorescent marker molecules, metallic (plasmonic or magnetic) nanoparticles, biological antigens or antibodies et cetera.Consequently, possible applications in mechanics (nanorulers, cages that open or close owing to external stimuli, molecular force clamps, nano-robots), nanophotonics, analytics and biology have been envisaged [20][21][22][23][24][25].In this work, we used rod-like nanoparticles that are formed by 24 parallel double stranded DNA helices [24-helix bundle (24 HB), Figure 1].
Recently, Nguyen et al. [26] have developed a method to stabilise DNA nanoparticles by the formation of silica in a sol-gel process.The silica component enhances the thermal stability of DNA nano-objects fabricated by the DNA origami method and may be used for further functionalization.In this work, the method of stabilisation by silica [26] is extended to DNA particles that are labelled with an intercalating fluorescent dye.Our study was motivated by our earlier observation [10] that the luminescent dye acridine orange (AO) can be incorporated in the molecular aggregates of disodium cromoglycate (DSCG) (Figure 2, top), the building blocks of a well-known LCLC [27][28][29][30].This property enabled us to use the fluorescence dichroism of AO to measure the order parameter of an LCLC formed by DSCG [10].Encouraged by this finding, we were looking for a way to label DNA nanoparticles suspended in a LCLC specifically by a fluorescent dye, that indicates the location and potentially the orientation of the nanoparticles as opposed to their LCLC solvent.In the present paper, the intercalating fluorescent dye YOYO-1 iodide (Figure 2, below) was used to mark DNA nano-rods fabricated by the DNA origami technique.We report our finding that silica may protect dye-labelled DNA particles from losing the dye by its distribution to a LCLC environment.This finding may be very useful for further studies, as discussed in the last section of this article.

Experiments
DNA nano-rods based on the 24-helix bundle design (Figure 1) were fabricated by the DNA origami method using a scaffold with 8064 nucleotides.The aqueous folding mixture based on TE buffer containing 30 nM scaffold, ≈150 nM of each of the 213 staples, and 14 mM MgCl 2 was heated in a thermocycler to 65°C and slowly cooled within 24 hours down to room temperature (Supporting information: For the composition of the folding mixture, see Table S1 and Table S2; for the 24HB design, see Figure S1 and Figure S2).After the folding reaction, a small part of the sample was investigated using electrophoresis to control the success of the reaction.For further synthetic steps, the main part of the folding mixture was purified from residual staples by repeated precipitation using poly(ethylene glycol) (PEG), centrifugation (30 min at 16,000 rcf) and resuspension.Alternatively, we tested purification by washing using Millipore filters (type Amicon Ultracel-100k, Merck), which lead to similar yields.Yet, the results reported here describe DNA nanoparticles that were purified by PEG precipitation.
For the purpose of subsequent silica deposition, the buffer was replaced by smaller amounts of deionised water with a rather small concentration of MgCl 2 during the purification process, to yield a mixture with a concentration exceeding 100 nM DNA origami nano-rods and a salt concentration of only 0.5 mM MgCl 2 .Subsequently, silica was grown on the 24HB nano-rods according to the sol-gel procedure described previously [26].After adding solutions of N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (TMAPS) and tetraethoxysilane (TEOS), the respective mixture was rigorously stirred for 15 min.and allowed to rest for a few days.Afterwards, the silica-stabilised DNA nanoparticles were separated from the reaction mixture by centrifugation and redispersed in deionised water.
Fluorescent DNA nano-rods were prepared by mixing the purified, folded DNA particles prior to the silica growth with a dye solution.For this purpose, a small volume of a 1 mM solution of the dye YOYO TM -1 iodide in DMSO (ThermoFisher, catalogue # Y3601, in short 'YOYO-1' [31]) was added and allowed to interact with DNA nanoparticles for 50 minutes, before the silica growth reaction was started.After adding the dye, the samples were protected from light exposure to avoid bleaching.
For studying the behaviour of luminescent DNA nano-rods in a liquid crystalline solvent, a small volume of an aqueous suspension of these nano-rods was added to a solution of 15% (by weight) DSCG in water, which is known to exhibit a nematic LCLC phase at room temperature.This composite was sandwiched between two cover slides, which were uniformly rubbed with an abrasive (3 M) to promote a parallel alignment of the director [32].The sample was sealed with Parafilm foil to avoid evaporation of the solvent and studied by total internal reflection fluorescence (TIRF) microscopy using a commercial microscope, type Nikon Eclipse Ti, operating at 474 nm, using a microscope lens with 60× magnification.

Results and discussion
In agreement with our earlier studies [8][9][10], the folding of the single stranded DNA scaffold through base pairing with 213 specifically tailored DNA oligomers (as described in the Supporting Information) yields well defined nano-rods with a length of roughly 100 nm (Figure 3).These nano-rods can be stabilised with silica using the sol-gel-process described in Ref. [26].For reaction times up to 48 hours, separated nano-rods appeared.However, it could not be completely avoided that nano-rods neighbouring or touching each other became commonly overgrown by silica, thereby stabilising also larger agglomerates.For the concentrations tested in this study, the formation of larger aggregates overgrown by silica became dominant for reaction times exceeding 72 hours.
Before using the fluorescent dye YOYO-1 for the purpose of studying a very complex LCLC/nanoparticle composite, the interactions of this dye with DNA nano-rods and its interactions with DSCG were investigated, separately.Comparison of the fluorescence intensities of the dichroic dye acridine orange (AO, studied earlier in Ref. [10]) and of YOYO-1 in different solvents reveals striking differences (Figure 4).The overview given in Figure 4 shows how the fluorescence intensities of aqueous solutions of the dyes acridine orange (AO, used in Ref. [10]) and YOYO-1 (studied in this work) are affected when (a), (d) DNA nano-rods {fabricated using the 14-helix bundle (14HB) or 18-helix bundle (18HB) design [10]}, or (b), (e) the mesogenic compound DSCG, or (c), (f) both DSCG and DNA nano-rods are added.Dispersing DNA nanorods in an isotropic solution of AO has almost no effect on the fluorescence intensity (Figure 4(a)).In striking contrast, the fluorescence intensity is enhanced by a factor of ≈20, when YOYO-1 is used (Figure 4(d,g)).This characteristic fluorescence enhancement is typical for intercalating dyes and frequently used in DNA analytics.The behaviour observed here confirms the well-known sensitivity of YOYO-1 with respect to molecules that may form hydrogen bonds [31].Interestingly, both dyes (AO and YOYO-1) show an enhanced fluorescence intensity, when they are dissolved in a uniformly aligned nematic liquid crystal based on DSCG (Figure 4(b,e)), which indicates that the dye molecules interact strongly with DSCG molecules.However, the interaction is again different for the two dyes.Measuring the linearly polarised components of the fluorescence intensity I ⊥ (plane of polarisation perpendicular to the director n) and I || (plane of polarisation parallel to n) [10] reveals a larger orientational order parameter S = 2 (I ⊥ -I || )/(2I ⊥ + I || ) for AO (S AO = 0.56 ± 0.03) than for YOYO-1 (S YOYO-1 = 0.32 ± 0.02).Since the orientational order parameter of the molecular DSCG aggregates is probably very similar at room temperature, this difference seems to indicate that the transition dipole moments of AO molecules are aligned along the long axes of the neighbouring DSCG molecules, whereas the orientations of YOYO-1 molecules incorporated in DSCG aggregates are much less ordered.This different behaviour of AO and YOYO-1 may be attributed to the similarity and dissimilarity of the respective dye molecules to the molecular structure of DSCG.Adding DNA nano-rods to a dyed nematic DSCG mixture has only a small effect on the linearly polarised fluorescence intensities observed perpendicular and parallel to the director (Figure 4(c,f)).In the case of AO, the presence of DNA nano-rods can be neglected (like in a isotropic mixture (Figure 4(a))).However, the dye molecules of YOYO-1 show similar fluorescence  enhancements in both cases, when they interact with DSCG or DNA.
The fluorescence enhancement of YOYO-1 when interacting with DNA may be not very sensitive to the spatial conformation of the DNA molecules.This raises the question whether the shape of folded DNA nano-rods remains stable, when many intercalating dye molecules are incorporated through hydrogen bonding.Thus, the stability of the shape of the particles was investigated at concentrations of YOYO-1 that are of interest for the TIRF experiment intended.For this purpose, 60 µL of a sample containing a concentration of 156 nM of DNA nano-rods (9.36 pmol) was mixed with 3 µL of the 1 mM solution of the dye YOYO-1.The resulting mixture exhibits a concentration of 47.6 µM YOYO-1 and a total amount of 3 nmol YOYO-1.Since the DNA nanoparticles are formed from a scaffold with 8064 nucleotides, the concentration of base pairs (bp) is 8064 ⋅ 156 nM = 1.258 mM, corresponding to a total amount of 79 nmol of base pairs.So, on average, every 26 th to 27 th bridge by hydrogen bonds between the DNA nucleotides may be disturbed by interference with an intercalating dye molecule.In spite of this potential stress, TEM observations of the dye-labelled DNA origamis (Figure 5) indicate that rod-like shape of the folded nanoparticles is preserved.
A dye-labelled sample as shown in Figure 5 was used for the sol-gel process that leads to silica deposition on the nanoparticles.The growth process (performed as described in Ref. [26]) was stopped after 48 hours by separating the nano-rods from the reaction mixture using centrifugation.This procedure yielded a yellowish fluorescent pellet and a colourless supernatant solution, indicating that a decent fraction of the dye molecules is indeed incorporated in the silicastabilised DNA nano-rods.The pellet was dispersed in 36 µL water yielding a concentration of 190 nM DNA nano-rods.The total amount of 6.84 pmol corresponds to a yield of 73%.
Finally, a volume of 1 µL of the fluorescent, silicastabilised DNA-nano-rods solution was mixed with 30 µL of a nematic DSCG mixture (15% by weight DSCG in water).Observation of a uniformly aligned sample of this composite in the TIRF microscope shows luminescent spots on a non-luminescent background.The latter exhibits ellipsoidal islands with cusps.These islands can be identified as the well-known tactoids (Figure 6) which are frequently seen for LCLC samples in the polarising microscope [33] and have also been observed in DNA/DSCG composites previously [8].It is obvious from the TIRF observation (Figure 7) that the fluorescent nanoparticles  accumulate at the cusps of the tactoid islands, i.e. at point defects of the director field.By inserting a quarter-wave plate, the state of polarisation of the probe beam was changed between linear and circular polarisation.This change did not result in a variation of the fluorescence intensity, which indicates that the sample showed no dichroic behaviour.Obviously, larger agglomerates of the fluorescent DNA nanoparticles are accumulated at the disclinations of the director field.They dominate the fluorescence and show no preferred alignment.In comparison, the fluorescence from single nano-rods dispersed in the liquid crystal (if any) is too weak to be detected.In any case, the sole appearance of luminescent spots at the cusps confirms that the YOYO-1 dye is is fixed in the silica-stabilised particles and not dispensed into the liquid crystal solvent.

Conclusion
In summary, our investigations indicate that fluorescent silica-stabilised DNA nano-rods can be fabricated using the DNA origami method.Even at relatively large concentrations, intercalation of the fluorescent dye does not affect the shape of the folded nano-rods.After the growth of silica, the purification process of the dyed nano-rods indicates that the resulting particles are stabilised against bleeding of the dye into the aqueous environment.This stabilisation is also confirmed by observation of LCLC composites containing the particles using TIRF microscopy.Although the YOYO-1 dye was found to show an enhanced fluorescence intensity in liquid crystalline DSCG solutions without DNA, the dispersion of fluorescent DNA nanoparticles in a DSCG-based LCLC solvent shows an enhanced fluorescence only at the cusps of the tactoids, which correspond to defects of the director field.This behaviour can be attributed to an accumulation of the fluorescent nanoparticles at these defect positions rather than a (hypothetical) dispersion of the fluorescent dye in the LCLC solvent.
It is very well known that colloidal particles dispersed in liquid crystals tend to accumulate at positions, where the director field exhibits a disclination.In particular, the accumulation of colloidal particles at the surface defects (boojums) of thermotropic LC droplets has been reported [34].Since anchoring of the director at the interface of a particle (with radius R) may cause an elastic deformation of the director field in the environment, this effect is driven by the competing influences of the anchoring energy per area w and the elastic energy of director field distortions, which increases linearly with an effective elastic coefficient K.This competition can be described by a dimensionless parameter w R/K [35].In the weak anchoring regime (w R/K << 1), i.e. for small particles with R << K/w, the director field remains almost undisturbed.Thus, only particles with sizes exceeding a critical value tend to migrate to the LC surface or to positions of inevitable defects.
LCLCs are particularly interesting systems, because their material properties can cover a much broader range than the typical range of respective quantities in thermotropic liquid crystals.Tactoids with defects at their tips are known to appear frequently in LCLCs.They were found to exhibit a twisted director field [36], the appearance of which is facilitated by unusually small values of the twist elastic coefficient.For a value of K 22 ≈ 0.64 pN [30] and typical values of w ≈ 0.2-0.6 µJ m −2 [32], one can estimate that particles of micron size (R > ≈ 1 µm) migrate to tips of the tactoids owing to interaction with the director field.This seems to indicate that the fluorescence observed by TIRF microscopy is caused by aggregates of the fluorescent DNA nano-rods rather than originating from single nano-rods.Of course, minor fluorescence of remaining well-dispersed single nano-rods cannot be excluded by the TIRF observation.In any case, the appearance of luminescent spots at the defect positions confirms the stability of the silica-stabilised DNA particles, since a weak uniform luminescence would appear instead, if the dye would spread in the DSCG solution.
Hopefully, our results may pave the way to other interesting experiments on liquid crystalline nanoparticle dispersions.For example, aggregates of silica-stabilised DNA nanoparticles may be dispersed in liquid crystal environments that show a regular array of disclinations, thereby leading to regular arrays of highly functionalised nanoparticles, in which any of the extraordinary opportunities [20][21][22][23][24][25] are used.The protocol for the stabilisation by silica may also be further optimised to make sure that only well-separated DNA-nano-rods without any clusters are obtained.If so, one could, for example study the orientational order parameter of a LCLC solvent (as in Ref. [8]) and the order parameter of nano-objects dispersed in this solvent, simultaneously.Recent attempts to optimise the silica growth process [37,38] are expected to facilitate further progress in this direction.In addition, it might be possible to make the silica surface hydrophobic, thereby opening the opportunity of dispersing highly functional DNA nanoparticles not only in water-based lyotropic, but also in thermotropic liquid crystals for both basic studies and applications.

Figure 1 .
Figure 1.(Colour online) Structure, size and shape of the rod-like DNA nanoparticles that are fabricated using the DNA origami method.A single stranded DNA loop is folded by hydrogen bonding with tailored, complementary DNA oligomers, thereby forming a bundle of 24 parallel double stranded DNA helices [24-helix bundle (24HB) design].

Figure 3 .
Figure 3. Images of DNA nano-rods observed by transmission electron microscopy (TEM) before and during the growth of silica.

Figure 7 .
Figure 7. (Colour online) Image observed by total internal reflection fluorescence (TIRF) microscopy: the LCLC exhibits a similar composition as the sample shown in Figure6and contains DNA nano-rods that are prepared using the 24 helix-bundle (24HB) design, dyed with YOYO-1, and stabilised by silica.