Surface Hybridization Chain Reaction of Binary Mixture DNA-PEG Corona Nanostructures Produced by Low-Volume RAFT-Mediated Photopolymerization-Induced Self-Assembly

DNA-polymer hybrids have been attracting interest as adaptable functional materials by combining the stability of polymers with DNA nanotechnology. Both research fields have in common the capacity to be precise, versatile, and tunable, a prerequisite for creating powerful tools which can be easily tailored and adapted for bio-related applications. However, the conjugation of hydrophilic DNA with hydrophobic polymers remains challenging. In recent years, polymerization-induced self-assembly (PISA) has attracted significant attention for constructing nano-objects of various morphologies owing to the one-step nature of the process, creating a beneficial method for the creation of amphiphilic DNA-polymer nanostructures. This process not only allows pure DNA-polymer-based systems to be produced but also enables the mixture of other polymeric species with DNA conjugates. Here, we present the first report of a DNA-PEG corona nano-object’s synthesis without the addition of an external photoinitiator or photocatalyst via photo-PISA. Furthermore, this work shows the use of DNA-macroCTA, which was first synthesized using a solid-support method resulting in high yields, easy upscaling, and no need for HPLC purification. In addition, to the formation of DNA-polymer structures, increasing the nucleic acid loading of assemblies is of great importance. One of the most intriguing phenomena of DNA is the hybridization of single-stranded DNA with a second strand, increasing the nucleic acid content. However, hybridization of DNA in a particle corona may destabilize the nanomaterial due to the electrostatic repulsive force on the DNA corona. Here, we have investigated how changing the DNA volume fraction in hybrid DNA-polymer self-assembled material affects the morphology. Moreover, the effect of the corona composition on the stability of the system during the hybridization was studied. Additionally, the hybridization chain reaction was successfully applied as a new method to increase the amount of DNA on a DNA-based nano-object without disturbing the morphology achieving a fluorescence signal amplification.


S3
30:70 mixture of H 2 O and acetonitrile.The buffer gradient for analysis and purification was 1% buffer B for 5 minutes, 1% to 30% B over 15 minutes, 30% to 95% B over 5 min, 95% to 1% B over 1 min and finally 1% B for 3 min.
Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of oligonucleotides was performed on an Agilent 1200 HPLC system coupled to a Bruker AmazonX high resolution ion trap, in negative ion mode.The desalted oligonucleotide samples were eluted through a XBridge oligonucleotide BEH C18 column (130 Å, 2.5 µm, 4.6 x 50 mm) using a 5 vol% MeOH, 10 mM ammonium acetate (buffer A) and a 70 vol% MeOH, 10 mM ammonium acetate (buffer B) solvent system at 0.8 mL/min flow.The data was processed using Compass Data Analysis (Bruker) v.4.1 software, and the MaxEnt integrated deconvolution algorithm.Alternatively, LCMS analysis was performed on a Waters ACQUITY UPLC system coupled to a Xevo G2-XS QToF mass spectrometer in negative ion mode.The oligonucleotides were eluted through an AQUITY UPLC oligonucleotide BEH C18 column (130Å, 1.7 µm, 2.1 x 50 mm) using a 75 mM triethylammonium acetate (TEAA, pH 7.0) solution in H 2 O (buffer A) and a 75 mM TEAA solution in MeCN (buffer B) at 60 °C and a 0.2 mL/min flow.Leucine enkephalin was used as the reference for the LockSpray correction.The raw continuum data was deconvoluted to produce zero charge mass spectra using ProMass HR for MassLynx (Novatia) software.
Native polyacrylamide gels were run at room temperature in 1×TAE buffer at 180 V using a vertical nucleic acid electrophoresis cell connected to a PowerPack basic power supply (BioRad).Samples were combined with 20% loading buffer (0.05% bromophenol blue, 25% glycerol, 1x TAE) prior to running.Non-fluorescent DNA was stained using a 1:1000 aqueous SYBR® Gold nucleic acid gel stain (ThermoFisher) and visualized using a BioRad ChemiDoc™ MP Imaging system.The images were processed using ImageLab software v 6.0.1.
1 × TAE buffer consisted of 40 mM Tris-acetate and 1 mM EDTA. 1 × TAE buffer consisted of 10 mM Tris-HCl and 1 mM EDTA.The native loading buffer consisted of 25 % glycerol and 0.05 % bromophenol blue in 1 × TAE buffer, and was diluted five-fold before use.
Size exclusion chromatography (SEC) analysis was performed on a system composed of a Varian 390-LC-Multi detector suite equipped with a Varian Polymer Laboratories guard column (PLGel 5 μM, 50 × 7.5 mm), two Mixed-C Varian Polymer Laboratories columns (PLGel 5 μM, 300 × 7.5 mm) and a PLAST RT auto-sampler.Detection was conducted using a differential refractive index (RI) and an ultraviolet (UV) detector set to λ = 309 nm.The mobile phase used was DMF (HPLC grade) containing 5 mM NH4BF4 at 50 ˚C at a flow rate of 1.0 mL min-1.Poly(methyl methacrylate) (PMMA) standards were used for calibration.
Molecular weights and dispersities were determined using Cirrus v3.3 SEC software.
Zeta potential was measured by the technique of microelectrophoresis, using a Malvern Zetasizer Nano ZS instrument, at room temperature at 633 nm.All reported zeta potential values were the average of at least three runs with at least 40 measurements recorded for 3 runs.Zeta potential was calculated from the corresponding electrophoretic mobilities (µE) by using the Henry's correction of the Smoluchowski equation (µE = 4π ε0 εr ζ (1+κr)/6π μ).
Hydrodynamic diameters (D h ) of particles were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS with a 4 mW He-Ne 633 nm laser module operating at 25 o C. Measurements were carried out at an angle of 173° (back scattering), and results were analyzed using Malvern DTS 7.03 software.All determinations were repeated 4 times with at least 10 measurements recorded for each run.D h values were calculated using the Stokes-Einstein equation where particles are assumed to be spherical, while for cylindrical particles DLS was used to detect multiple populations and obtain dispersity information.
Static Light Scattering (SLS).For the particles in deionized water, light scattering data was collected over the whole angular range, 30 < θ < 50° with the sample maintained at 25°C.Autocorrelation functions calculated by the ALV LSE-5004 correlator unit were recorded at each (θ, c) and the REPES algorithm was used to determine relaxation times, τ(θ, c).The data set τ(θ, c) was then analyzed to estimate the mean translational diffusion coefficient according to the Stokes-Einstein equation.An empirical measurement was made of the refractive index increment for the polymer in deionized water using a differential refractometer, model DnDc1260 supplied by PSS GmbH.The light scattering experiments were conducted at 0.01, 0.02, 0.05, 0.10 and 0.20 mg•mL -1 to account for concentration effects.Following Andersson et al. 1 , a Zimm plot was constructed using the Debye method (Equation 1 and Equation 2) to determine the R g of the nanoparticles.To do this, the R θ /Kc versus q 2 data were plotted and a third order polynomial model was used to extrapolate  → 0. The fit's intercept provided the molecular mass according to light scattering (M LS ) while the slope at q 2 = 0 can be utilized to retrieve R g at the different concentrations.A first order model was utilized for the  → 0 extrapolation yielding R g and M LS (Table S5).
( 1) The mean translational diffusion coefficient (D app ) was calculated from the relaxation times at each angle, τ(θ, c) determined from the autocorrelation functions at each angle (θ, c) by the REPES algorithm.The Stokes-Einstein equation was used to determine the hydrodynamic radius (R h ) of the particles.The R g /R h ratio gives information about the inside of the spherical particle.A value of 1 indicates a hollow sphere with all the mass in the outer shell (i.e. vesicle).The 50%DNA-PHPMA 400 nanoparticles have a R g /R h ratio of 1.02.The aggregation number (M w,theo /M LS ) was calculated to be 2.73 × 10 3 which is in line with a vesicle morphology. 2 Transmission Electron Microscopy (TEM) analysis was performed on a JEOL 2100 electron microscope at an acceleration voltage of 200 kV.All samples were diluted with deionized water and then deposited onto formvar-coated copper grids.After roughly 1 min, excess sample was blotted from the grid and the grid stained with an aqueous 1 wt% uranyl acetate (UA) solution for 1 min prior to blotting, drying and microscopic analysis.
Cryogenic Transmission Electron Microscopy (Cryo-TEM) imaging was performed on a JEOL JEM-2100 plus microscope operating at an acceleration voltage of 200 kV.
Samples for cryo-TEM were prepared on lacey carbon grids (EM Resolutions).After 200fold dilution with deionized water, 8 µL of sample were deposited onto the grid followed by blotting for approximately 5 s and plunging into a pool of liquid ethane, cooled using liquid nitrogen in order to vitrify the samples.Then, transfer into a pre-cooled cryo-TEM holder using liquid nitrogen, was performed prior to the microscopic analysis.
Confocal Laser Scanning Microscopy (CLSM) was performed on FV3000 (Olympus) confocal microscope and the 60x oil lens was used for imaging.Images were acquired using the 488 nm (green channel) and the 561 nm (red channel) excitation wavelengths.Freshly prepared and purified solutions of FAM-H2 (green-emitting dye), TAMRA-cDNA (redemitting dye) and mixed 50% DNA 14 -PHPMA 400 copolymer nano-objects at 100-fold dilution were deposited on a glass slide before imaged by CLSM.Images were processed using cellSens (Olympus) and ImageJ image processing software.The LAD-1 LED array driver was purchased from Bio Research Centre Co., Ltd.The array was composed of 96 405 nm LEDs which each output a light power of 20 mW at 13.5 V when measured with an LMP-100 light power sensor (sensor area: 5.5 mm x 4.8 mm), placed directly above an array LED.
For the Thermomixer setup, the LED-array was suspended over the sample holder via a clamp stand.For the Incubator setup the LED-array was placed upon a shaker plate, face up with a 96-microwell plate placed directly upon it, into which 150 µL eppendorfs containing sample were placed.
A solution of bis-(ethylsulfanylthiocarbonyl) disulfide (16.0 g, 0.058 mol, 1 eq) and 4,4′-azobis(4-cyanopentanoic acid) (ACVA) (24.5 g, 0.087 mol, 1.5 eq) in 500 mL of ethyl acetate was heated at reflux for 18 h under N 2 (g) atmosphere.Following rotary evaporation of the solvent, the crude CEPA was isolated by column chromatography using silica gel as the stationary phase and 75:25 DCM-petroleum ether as the eluent.The isolated product was precipitated out of solution by using hexane leaving a yellow-light orange solid.The final product was collected and dried under reduced pressure to afford pure CEPA CTA (10.95 g, 0.042 mol, 36%).Scheme S1.Synthesis of CEPA-CTA.

Synthesis of poly(ethylene glycol) 113 -CEPA macro-CTA (PEG 113 macroCTA)
PEG 113 macroCTA was synthesized according to a previously reported method with slight modification. 4Poly(ethylene glycol) methyl ether (average M n =5,000 g mol -1 , PEG 113 -OH) (4.75 g, 0.98 mmol, 1 eq) was dissolved in 150 mL of dry DCM.The resulting solution was then purged with N 2 (g) for 30 min.After complete dissolution, CEPA CTA (1 g, 3.8 mmol, 4 eq), DCC (392 mg, 1.9 mmol, 2 eq) and DMAP (23 mg, 0.19 mmol, 0.2 eq) were added to the reaction mixture.The esterification reaction proceeded with stirring at room temperature for 18 h under continuous N 2 (g) flow.After this period, further DCC (392 mg, 1.9 mmol, 2 eq) and DMAP (23 mg, 0.19 mmol, 0.2 eq) were added to the reaction mixture and then stirred at room temperature for an additional period of 6 h under continuous N 2 (g) flow.The solution was then filtered to remove unreacted DCC and DMAP.The product was collected by 5 times of precipitation using cold diethyl ether as non-solvent, redissolved in deionized water and dialyzed against nanopure water using a 1,000 kDa MWCO membrane for 1 day (yield = 58%).The received PEG 113 macroCTA solution was lyophilized to give a light yellow powder as the final product (2.90 g, 0.55 mmol, 58%).
After centrifugation at 15000 rpm for 30 min at 4 °C the supernatant was removed, and the DNA pellet washed with ice-cold 70% ethanol followed by a repeated centrifugations using the same settings as described above.The supernatant of the washing solution was removed and the DNA pellet was dried on air.
Synthesis of ssDNA 14 macroCTA by solid support approach 250 µL of DEAE Sepharose suspension was used as solid support and pipetted into an empty Glen Research column housing and washed with 20 ml of H 2 O followed by 12 ml of DEAE binding buffer (10 mM acetic acid and 0.005% Triton X-100) using a syringe.The DNA-NH 2 (10 µM, 1 ml, 1 eq) was loaded onto the column after dissolving in 1 ml of DEAE binding buffer.The column was then washed with 3 ml of DEAE binding buffer, followed by 1 mL of H 2 O and 4 ml of DMF to switch the solvent system from water to DMF.At least 50 nmol of oligonucleotide can be loaded onto one 250-µL DEAE Sepharose column.The activated ester solution was composed of CEPA (0.5 M, 100 µL, 500 eq), EDC•HCl (1M, 50 µL.500 eq), NHS (1 M, 50 µL.500 eq), and DIPEA (1 M, 50 µL.500 eq) in solvent (DMF) and was incubated at 21 °C for 30 min before use.The CEPA active ester solution was loaded onto the solid support column in 1 ml of DEAE bind buffer and incubated for 10 min.
After the reaction was completed, the column was washed with 4 ml of the reaction solvent Scheme S4.Synthesis of ssDNA 14 macroCTA by solid support approach.

Synthesis of DNA−Polymer
Conjugates by Photopolymerization-Induced Self Assembly ssDNA 14 -macroCTA (250.5 µL, 20 mg/mL, 1 eq) was added in a centrifuge tube containing HPMA (2.5 mg for 5%w/w, 5 mg for 10%w/w, 200,300,400 eq).Then, glucose solution (6 µL, 0.84 M), nanopure water (8.4 µL) and GOx solution (8 µL, 12.5 µM) were added into the mixing vial, respectively, resulting in a total volume of 50 μL.The mixture was shaken via vortexer to produce a clear colourless solution which was then transferred to pointed base PCR plate.Mineral oil was added around 200 µL on top of the mixture and the plate was covered by plate seal, and placed in LED array setup, which was contain in an incubator to maintain a temperature of 37 °C.The solution was then exposed to 405 nm light for 2 h, resulting in the solution turning opaque and milky white.
In order to study the DNA hybridization of the particle surface, a 10 mM MgCl 2 in 1×TAE solution was used as a buffer to provide optimal DNA hybridization conditions.The surface hybridisation of nanoobjects formed using different ratios of ssDNA and PHPMA containing diblock copolymers was investigated.Nanopure water (177 or 159 µL) was added into MgCl 2 (2 µL, 1M), nanoobject solution (1 µL, 100 mg/ml), TAE (20 µL, 10×) to prepare the particle solution under buffered condition (10 mM) in 1.5 mL tube.The tube was shaken at room temperature at least 30 min and then transferred to a DLS microcuvette.DLS was used to investigate particle size and size dispersity.Subsequently, varying systematic amounts of cDNA (100 µM) was added to the centrifuge tubes, containing the nanoobjects with varying ratios of ssDNA.The centrifuge tube was shaken at room temperature for at least 30 min before investigation by DLS, TEM and confocal microscopy.

Figure S1 .
Figure S1.RP-HPLC chromatograms of DNA macroCTA synthesized by solution method (blue line), solid support method (red line) using NH 2 -ssDNA as a starting material (black solid line).The chromatograms were investigated at detector wavelength 309 nm (dash line) and detector wavelength 260 nm (solid line).Products eluted with a gradient of buffer A, 0.1 M triethylammonium acetate (TEAA), in a 95:5 mixture of H 2 O and acetonitrile and buffer B, 0.1 M TEAA, in a 30:70 mixture of H 2 O and acetonitrile.

Figure S2 .
Figure S2.GPC traces of PEG-macroCTA (dot line) and the DNA/PEG polymer conjugates (solid lines) as measured by DMF GPC using polymethylmethacrylate (PMMA) calibration standards.

Table S1 .
List of ssDNA-copolymer nanoparticles obtained from RAFT aqueous dispersion Photopolymerization-Induced Self Assembly and Summary of Characterization Data 1 Calculated from1H NMR spectroscopy (400 MHz) in deuterated MeOD.b Determined by DMF SEC with poly(methyl methacrylate) (PMMA) standards. cDetermined by DLS using z-average data.d Determined by TEM. S = Spheres, LR = Lumpy Rod, and V = Vesicle.