A Janus-Type Phthalocyanine for the Assembly of Photoactive DNA Origami Coatings

Design and synthesis of novel photosensitizer architectures is a key step toward new multifunctional molecular materials. Photoactive Janus-type molecules provide interesting building blocks for such systems by presenting two well-defined chemical functionalities that can be utilized orthogonally. Herein a multifunctional phthalocyanine is reported, bearing a bulky and positively charged moiety that hinders their aggregation while providing the ability to adhere on DNA origami nanostructures via reversible electrostatic interactions. On the other hand, triethylene glycol moieties render a water-soluble and chemically inert corona that can stabilize the structures. This approach provides insight into the molecular design and synthesis of Janus-type sensitizers that can be combined with biomolecules, rendering optically active biohybrids.


Chemicals
All chemical reagents were purchased from commercial sources (Sigma Aldrich, Alfa Aesar, Acros Organic, TCI) and used without further purification. All reactions were performed in standard glassware under argon atmosphere. Microwave reactions were carried out in a CEM Discovery Apparatus, in closed glass vials (Biotage 2 -5ml) under argon atmosphere. Anhydrous solvents were dried with molecular sieves. Reactions were monitored using thin-layer chromatography (TLC) plates pre-coated with silica gel 60-F254 (Merck). Column chromatography was performed on Merck silica gel 60, 40-63 m (230-400 mesh), and Biorad Biobeads SX-1 (200-400 mesh) were used as stationary phase for Size Exclusion Chromatography (SEC). The single-stranded scaffolds (p7249 and extended p7560) were obtained from Tilibit Nanosystems and the short single-stranded staple strands were bought from Integrated DNA Technologies. The 50× TAE Buffer (2 M tris(hydroxymethyl)aminomethane (Tris), 50 mM ethylenediaminetetraacetic acid (EDTA) and 1 M acetic acid for adjusting the pH to 8.0) was purchased from Thermo Fisher Scientific. Agarose was purchased from Bioline, 6× Gel Loading Dye Blue from New England Biolabs, sodium chloride from VWR Chemicals, magnesium chloride hexahydrate, dimethyl sulfoxide and ethidium bromide from Sigma-Aldrich. The water used in all the experiment was Milli-Q grade. Mass experiments were done at the Servicio Interdepartamental de Investigación (SIdI) of the Universidad Autónoma de Madrid, by Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), that was recorded using a Bruker Ultrareflex III spectrometer; Electrospray Ionization Source (ESI) in positive or negative mode, that was performed using an ultra-high-resolution QTOF instrument (MAXIS II, Bruker), and Fast Atom Bombardment (FAB) was recorded using a Waters -VG AutoSpec spectrometer. UV-Vis absorbance and fluorescence were recorded using JASCO V-660 and JASCO FP-8600 spectrophotometers, respectively. Infrared spectra (IR) were recorded on a Cary630 FTIR spectrophotometer from Agilent Technologies, using solid sample (Diamond ATR); spectra intensity was corrected by ATR algorism and the subsequent baseline correction.

Equipment
The DNA origami structures were folded using G-storm G1 Thermal Cycler. For purification purpose, Eppendorf 5424R microcentrifuge was utilized. The absorbance for determining the concentration of DNA origami was measured using a BioTek Eon Microplate spectrophotometer and a Take3™ micro-volume plate. The Electrophoretic mobility shift assay were done using BioRad horizontal wide Mini-Sub GT electrophoresis system and a BioRad PowerPac™ Basic power supply. The gels were visualized under UV light using a BioRad Gel Doc™ XR+ Documentation system. The transmission electron microscopy images of the biohybrids were taken using an FEI Tecnai 12 Bio-Twin instrument operated at an acceleration voltage of 120 kV and were processed using Gatan Digital Micrograph software. For UV-Vis spectroscopy, Cytation 3 plate reader (BioTek) was used. Figure S1. Absorption and fluorescence spectra for fluorescence quantum yield calculations. λexc = 665 nm.

Synthesis of DNA origami
The 24-helix bundle (24HB) and 60-helix bundle (60HB) synthesis have been adapted from Ijäs et al. 1 and Linko et al. 2 respectively. The scaffold used for folding 24HB and 60HB is 7560 and 7249 nucleotides respectively. For 24HB, there are 202 oligonucleotides staple strands with 8× poly-T overhang whereas, the 60HB design is composed of 141 oligonucleotides staple strands. The exact sequence can be extracted from the above-mentioned references. The folding buffer (FOB) utilized for 24HB was in 1x TAE, 17.5 mM MgCl2 and while, 60HB was folded in buffer containing 1x TAE, 20 mM MgCl2 and 5 mM NaCl. The volume and final concentration have been tabulated in Table 1. Same thermal annealing was used for both the DNA origami which was initiated after mixing all the ingredients in PCR tubes. The protocol was as follows: • Heating the mixture to 65 °C, • followed by, cooling the mixture from 65 °C to 60 °C over 90 minutes • and then cooling slowly from 60 °C to 39 °C over 60 hours.
After the folding procedure, the DNA origami structures were kept at 12 °C until the program was manually stopped.

Purification of DNA origami
The folded DNA origami structures were purified by a poly(ethylene glycol) (PEG) purification method. The method was adapted from Stahl et al. 3 Two folding reactions (i.e. 200 µL) were then mixed with 600 µL of 1× FOB to obtain a starting volume of 800 µL. The solution was mixed thoroughly with 800 µL of PEG precipitation buffer (15 % PEG 8000 (w/v), 1× TAE and 505 mM NaCl), after which the mixture was centrifuged at 14000 rcf for 30 minutes at room. After the centrifugation, the supernatant was removed, and the DNA origami pellet was redissolved in 1× FOB (same as described above for 24HB and 60HB). Depending on the required DNA concentration, the pellet was redissolved in a different amount of 1× FOB to get final concentrations between 30 nM and 50 nM. The DNA origami solution was incubated at room temperature overnight to ensure complete dissolution of the pellet before refrigerated.

Concentration of DNA origami
The concentration was measured via UV/Vis spectroscopy and determined using Beer-Lambert law.
where A260 is the absorbance at 260 nm, ε is the molar extinction coefficient (for the 24HB and 60HB structure 1.05×10 8 M -1 cm -1 and 0.9×10 8 M -1 cm -1 respectively) and l is the path length through the solution in centimeters (0.05 cm). A sample size of 2 µL was used for the measurements.

Electrophoretic mobility shift assay (EMSA)
Agarose gel electrophoresis was used to analyze the folded 24HB and 60HB structures, and to verify the removal of excess amounts of staple strands after the PEG purification. A 2% (w/v) agarose gel was prepared by mixing 2 g of agarose into 90 mL of 1× TAE buffer and heated until a clear solution was obtained. After cooling it slightly, 10 mL of 110 mM MgCl2 was added. The gel was stained with 80 μL of ethidium bromide (EtBr) solution (0.58 mg/mL) before being poured into the casting tray. For the DNA origami samples, 10 μL of DNA origami solution was used, whereas the scaffold reference sample was prepared by diluting 4 μL of the scaffold (type p7249 and p7560, c = 100 nM) in 6 μL of their respective 1× FOB. The samples were mixed with 2 μL of Gel Loading Dye Blue (6×) and the whole sample of 12 μL was loaded into the gel pocket. The gel was run at a constant voltage of 90 V for 40 minutes. The running buffer utilized for EMSA was 1× TAE buffer containing 11 mM MgCl2 and the gel electrophoresis chamber was kept on an ice bath for the run. Figure S2. Qualitative analysis of 24HB and 60HB using agarose gel electrophoresis. 1) Scaffold (type p7560). 2) Scaffold (type p7249) 3) Unpurified, but folded 24HB. 4) Unpurified, but folded 60HB. 5) PEG-purified 24HB. 6) PEG-purified 60HB.

Spin filtering
The folded DNA origami were spin filtered to remove the folding buffer and redispersed in the milli-Q water. The Amicon® Ultra 0.5mL filters (100K) were first rinsed with 500 µL milli-Q water and centrifuged at 14000 rcf for 5 minutes at room temperature using an Eppendorf 5424R microcentrifuge. The follow-through is discarded and the filter is filled with 250 µL of DNA origami and 250 µL of milliQ water. The filter is then centrifuged at 6000 rcf for 10 mins. The follow-through is discarded and 480 µL milliQ water is added. The filter is then centrifuged at 6000 rcf for 10 mins. The follow-through is discarded and the filter is inverted into a new outer shell and centrifuged for 150 seconds at 1000 rcf. The filtrate is then mixed with milliQ water and the concentration is checked by the previously mentioned protocol (2.3).

Transmission Electron Microscopy (TEM)
The folded 24HB and 60HB structures were imaged using TEM. The sample was negatively stained with uranyl formate (2 % (w/v)) containing 25 mM NaOH according to the method described by Castro et al. 4 The sample was prepared by diluting the DNA origami solution in 1× FOB to a final concentration of 2 nM. The sample was observed using Formvar carbon-coated copper grids (400 mesh, Electron Microscopy Sciences) and were plasma cleaned (20 seconds oxygen plasma flash using a Gatan Solarus) before applying the sample. The sample volume of 3 µL was applied on the carbon-coated side of the grid for 3-3.5 minutes. After the incubation, the excess solution was drained from the edge using filter paper. First, the grid was immersed into a 5 µL droplet of uranyl formate solution, and excess liquid was directly drained off from the edge using filter paper, followed by a subsequent immersion into a 20 µL droplet of uranyl formate solution. The excess amount of stain was removed after 45 seconds from the edge of the grid using filter paper. After this process, the grid was left at room temperature for at least 30 minutes before imaging.

EMSA
The ZnPc-DNA origami complexes were prepared by mixing the DNA origami solution (final concentration of 2.0 nM) with increasing amounts of ZnPc, rendering ZnPc/DNA origami ratios from 500 to 50 000. The final DMSO concentration was adjusted at 10% for all samples. The mixtures were incubated at room temperature for 45 minutes. The total sample volume was 20 µL and 4 µL of gel loading dye (6×) was added before loading 22 µL of the total sample into the gel pockets. The agarose gel was prepared and ran using the protocol described in Section 2.4. The gel was visualized using a BioRad ChemiDoc™ MP Imaging system and the gel was excited at 532 nm (Alexa 546) and 633 nm (Alexa 647).

4.2
Reversible ZnPc-DNA origami binding Figure S4. Agarose gel EMSA of the a-b) 1-24HB complex and c-d) 1-60HB complex (100 μM and 2 nM, respectively) at increasing NaCl concentrations. In a) and c) the NaCl was added after binding. In b) and d) the NaCl was added before binding.The EMSA gels show the EtBr (top) and ZnPc (bottom) emission channels recorded at 532 and 633 nm, respectively.

UV-Vis spectroscopy
The NaCl crowding effect on the ZnPc 1 was studied by absorbance, recorded on a Cytation 3 plate reader ( Figure S8).       Figure S15. MALDI-TOF mass spectra of ZnPc 1 left), and high-resolution MALDI-TOF spectrum of selected peak (bottom right) and calculated distribution (top right). S11 Figure S16. High-Resolution MALDI-TOF mass spectrum of selected ZnPc 1 peak (bottom) and calculated distribution (top). Figure S17. High-Resolution MALDI-TOF mass spectrum of selected ZnPc 1 peak (bottom) and calculated distribution (top).