DNA–Polymer Nanostructures by RAFT Polymerization and Polymerization‐Induced Self‐Assembly

Abstract Nanostructures derived from amphiphilic DNA–polymer conjugates have emerged prominently due to their rich self‐assembly behavior; however, their synthesis is traditionally challenging. Here, we report a novel platform technology towards DNA–polymer nanostructures of various shapes by leveraging polymerization‐induced self‐assembly (PISA) for polymerization from single‐stranded DNA (ssDNA). A “grafting from” protocol for thermal RAFT polymerization from ssDNA under ambient conditions was developed and utilized for the synthesis of functional DNA–polymer conjugates and DNA–diblock conjugates derived from acrylates and acrylamides. Using this method, PISA was applied to manufacture isotropic and anisotropic DNA–polymer nanostructures by varying the chain length of the polymer block. The resulting nanostructures were further functionalized by hybridization with a dye‐labelled complementary ssDNA, thus establishing PISA as a powerful route towards intrinsically functional DNA–polymer nanostructures.


Ultraviolet-Visible (UV-VIS) Spectroscopy)
UV-VIS spectra were recorded on a Spark ® 20M from Tecan Group Ltd. using either a NanoQuant PlateTM or a 384 well UV -Star microplate.
The sample preparation for FRET measurements was conducted by hybridizing Cy5-DNA (c = 10 μM) to the rhodamine Bcontaining DNA-polymer conjugate FP1 (c = 10 μM) by incubation in 2 × TAE/Mg buffer at 37 °C for 2 h. Cy5-DNA as well as the rhodamine B-containing DNA-polymer conjugate FP1 were incubated alone at identical conditions to account for possible photobleaching and served as reference samples. For UV-VIS measurements, each sample was diluted to 200 nM and was excited using an excitation filter at 485/20 nm.

Gel Permeation Chromatography (GPC)
GPC experiments were performed on a PSS SECcurity instrument comprising an autosampler, a column oven with 3 GRAM columns (10 3 , 10 3 and 10 2 Å, 300 × 8 mm, 10 μm particle size) and a RI as well as an UV detector (Agilent Technologies 1260 Infinity). DMF containing 1g/L lithium bromide was used as the eluent at a flowrate of 1 mL/min. Poly(methyl methacrylate) (1600 kDa-800 Da) served as the calibration standard for molecular weight measurements. The samples were filtered (0.4 µm) prior to injection. The data was processed with the software PSS WINGPC UniChrom.

Matrix-Assisted Laser Desorption/ionization-Time of Flight (MALDI-ToF) Mass Spectrometry
MALDI-ToF mass spectra were acquired on a rapiflexTM MALDI-ToF/ToF mass spectrometer from Bruker equipped with a 10 kHz scanning smartbeam 3D laser (Nd:YAG at 355 nm) and a 10 bit 5 GHz digitizer. Measurements were performed in the positive reflector mode using 3-hydroxypicolinic acid as the matrix. The samples were prepared by incubating a solution of 1 µL of DNA sample (100 μM in DNA) and 2 µL of matrix solution (95 µL of 3-hydroxypicolinic acid in acetonitrile/water (1:1, 50 g/L) + 10 µL of ammonium citrate dibasic in water (100 g/L)) with an ion exchange resin. The samples were then applied to the target plate and were left for crystallization. Prior to every measurement, the instrument was calibrated using 3 commercially purchased DNAs (3648 Da, 6120 Da, 9195 Da). The data was processed with mMass.
Electrophoresis was conducted at 175 V for 90 min (20 % gels) or at 135 V for 50 min (10 % gels). Each sample was prepared by first hybridizing 1 μL DNA sample (100 μM, 100 pmol) with the complementary Rh6G-DNA sequence (100 μM, 50 pmol) in 3.5 μL 0.5 × TBE buffer at 35 °C for 30 min in order to increase gel resolution as reported previously. [2] 1 μL DNA gel loading dye (6 × Thermo Fisher) was then added prior to running the gel. GeneRuler low Range DNA ladder (Thermo Fisher) was used as received. Gels were subsequently stained with SYBR Gold in 50 mL 0.5 × TBE buffer for 30 min at room temperature.
Purification of the polymers was accomplished by PAGE extraction as previously published. [3]

High-Performance Liquid Chromatography (HPLC)
Measurements were performed on a HPLC instrument from Shimadzu comprising an auto sampler, a column oven and a fraction collector. The samples were purified either by semi-prep HPLC using a ZORBAX Eclipse XDB-C18 HPLC column (9.4 × 250 mm, 5 µm) from Agilent at a flowrate of 4 mL/min or by analytical HPLC using the same column type (4.6 × 250 mm, 5 µm) and the identical elution protocol at a flowrate of 1 mL/min. The elution protocol started with the mobile phase from 5% solvent B (HPLC grade acetonitrile) and 95% solvent C (0.1 M triethylammonium acetate buffer), raising linearly first to 60 % B in 20 min, then to 100% B in 3 min, then decreasing to 5% B in 4 min and finally holding 5% B for 3 min. The absorbance was monitored at 310 nm and 254 nm.

Fluorescence Correlation Spectroscopy (FCS)
FCS experiments were performed on a commercial setup (Carl Zeiss, Germany) consisting of the module Confocor 2 and an inverted microscope Axiovert 200 using a C-Apochromat 40×/1.2W water immersion objective. The excitation was done by the 543 nm line of a HeNe laser and the collected fluorescence signal was filtered through a LP560 long pass emission filter before r eaching the detector, an avalanche photodiode that enables single-photon counting. An eight-well, polystyrene chambered cover-glass (Lab-Tek, Nalge Nunc International) was used as a sample cell. For each solution, a series of five measurements with a total durat ion of five min were performed. The confocal observation volume was calibrated using a reference dye with known diffusion coefficients, i.e. Alexa 546. The experimentally measured autocorrelation curves were fitted with the model function for an ensemble of m different types of freely diffusing fluorescent species: In all cases, the experimental autocorrelation curves were fitted with two component models (m=2 in eq. S1) to account for the small amounts of freely diffusing Rho6G remaining from the ssDNA labelling or rhodamine B-containing monomer remaining after purification.

Atomic Force Microscopy (AFM)
AFM measurements were conducted on a Dimension FastScan Bio TM atomic force microscope from Bruker, which was operated in the PeakForce mode. AFM probes with a nominal spring constant of 0.25 Nm 1 were employed (FastScan-D, Bruker) for measurement in liquid. A circular mica disc (15 mm) was used as the substrate. Measurements were performed at scan rates between 0.8 and 2 Hz. Different areas of the mica substrate were scanned in order to ensure the integrity of the shown images. The images were finally processed by the software NanoScope Analysis 1.8.
For sample preparation, 50 μL 1 × TAE/Mg buffer (12 mM Mg 2+ ) containing the DNA-polymer nanostructure at [DNA] = 4 μM was applied onto a freshly cleaved mica substrate. The solution was left to incubate for 10 min in order to deposit the desired species on the mica surface. After successful adsorption, the supernatant was removed and fresh 1 × TAE/Mg buffer (200 μL) was added for the measurement.

Dynamic Light Scattering (DLS)
Dynamic light scattering (DLS) were recorded at 25 °C on a Zetasizer Nano S (Malvern Instruments Ltd, Malvern, U.K.) equipped with a HeNe laser (λ = 633 nm) and detected at a scattering angle of 173°. All measurements were performed in triplicate. The obtained data was processed by cumulant fitting for z-average and PDI, or by CONTIN fitting for intensity/ volume/ number weighted particle size distribution.
Samples were prepared at ~ [DNA] = 10 µM in 1 × TAE/Mg buffer and dust could be removed from the sample prior to each measurement by filtration through a GHP syringe filter (0.22 μm pore size, Acrodisc).

Cryogenic Transmission Electron Microscopy (Cryo-TEM)
Cryo-TEM measurements were performed on a FEI Talos 120C electron microscope at 120 kV operating voltage. Cryo-TEM samples were prepared on holey carbon films with various hole sizes using a FEI Vitrobot Mk IV. The samples were freshly prep ared in Mg 2+ -containing (12 mM) 1 × TAE buffer prior to the measurements. Image analysis was performed with the Fiji plug-in version of Imagej. For the synthesis of DNA-b-polymer conjugates, the identical protocol was pursued with the exception that BTPA-DNA stock was used instead of BTPA stock.

Experimental Procedures
DNA-diblock copolymer conjugates were achieved by removing 20 μL of the reaction mixture after polymerization of the first block and adding 20 μL of freshly prepared solution containing the monomer of the second block at the appropriate concentration as well as glucose at 100 mM, glucose oxidase at 1 μM and sodium pyruvate at 50 mM.
For the synthesis of rhodamine B-containing DNA-polymer conjugates, the general polymerization protocol was followed with the exception that RA stock was additionally added at the appropriate concentration.
For the PISA experiments, the general polymerization protocol was followed with the exception that DAAm and DMA stocks were added at the appropriate concentrations. Also, the solvent was changed to pure DPBS by preparing VA-044 stock (2 mM) in DPBS instead of t-butanol/DPBS (30/70, v/v).                         Figure S19. AFM images of the DNA-polymer worms before (left) and after functionalization (right) with the complementary Rh6G-terminated DNA sequence. A moderate change in uniformity of the structures could be seen by AFM after hybridization, however, their elongated morphologies were maintained.