A Versatile and Efficient Method to Isolate DNA–Polymer Conjugates

We present a facile and adaptable method to purify and isolate DNA–polymer conjugates from different uncharged homo, random, or block copolymer families. Anion exchange chromatography is used to separate the reaction solution and retrieve the excess unreacted polymer and oligonucleotide. The stationary phase has a high efficiency (25 nmol of DNA per run), facilitating the purification of large batches without compromising the peak shape and resolution. To demonstrate the versatility of this method, different types of polymers, including acrylates, methacrylates, and acrylamides containing hydrophilic and hydrophobic blocks, were purified with high yields. Additionally, DNA–polymer conjugates with various DNA block lengths were also successfully purified, further highlighting the broad applicability of this method.


POLYMERIZATION
The polymerization procedure was carried out as described by Alleva et al. 2022 1 For the polymerization of homo polymers or the first block of the block copolymers, the monomer, CTA and AIBN were dissolved in the polymerization solvent, purged with argon for 45-90 min and heated up to the respective temperature.The ratio of initiator to CTA was 1:10.After the reaction time, the reaction mixture was cooled down with an ice bath and a precipitate was formed in the respective precipitation solvent (Table S1).The collected solid was again dissolved and precipitated twice.The obtained solid was dried under vacuum.
The second block was obtained by dissolving the first block (macro CTA) in the polymerization solvent, adding AIBN and monomer, purged with argon for 45-90 min and heated up to respective temperature.After the reaction time, the reaction solution was cooled with an ice bath and precipitated in the precipitation solvent.The obtained solid was collected and dried under vacuum.

CTA REMOVAL
The polymerization procedure was carried out as described by Alleva et al. 2022 1 The obtained polymer was dissolved in dioxane and an excess of AIBN was added.The reaction solution was heated up to 80 °C.After the reaction time, the reaction solution was cooled in an ice bath, and the polymer was participated in the respective precipitation solvent (Table S1).The obtained polymer was dried under vacuum and analysed with SEC (DMF, PMMA standard).
For monitoring the conjugation reaction via PAGE, 1 µL of diluted reaction solution (25 pmol DNA, for SDNA 62.5 pmol due to its smaller size and less intercalation of the dye) was mixed with water and loading dye (1.7 µL, 6x Thermo Fisher) to a total volume of 10 µL and loaded onto the gel.The gel was run first at 100 V for 10 min and then at 150 V for 45-50 min on a Cell SureLockTM mini-cell electrophoresis system from Thermo Fisher using 0.5 × TBE buffer as the running buffer (44.5 mM Tris-Borate, 1 mM EDTA).Gene ruler ultra-low range DNA ladder (Thermo Fisher) was used as the DNA ladder.The gels are stained with SYBR Gold (2×, 50 mL) for 45 min at room temperature.The images were taken with ChemiDoc Touch Imaging System from Bio-Rad or G:BOX Chemi Gel Doc System from Syngene.For intensity measurement ImageJ was used to determine the intensity of the respective band.

SEC
Polymer measured with SEC: 10-20 mg of dry polymer was measured via the SEC method stated below.
Preparing the obtained DNA-polymer conjugate for SEC: 100-200 µL of the obtained conjugate solution was freeze dried.The obtained solid was measured via the SEC method stated below.
For the purification approach, 8 µL (1 nmol) were diluted with DMF to 100 µL and injected.
SEC experiments were performed on a PSS SECurity instrument comprising an auto sampler, a column oven with three 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 1 g/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 were fitted with OriginPro 2021.

SPIN FILTRATION (SF) FOR PURIFICATION
For the spin filtration, 200 µL of reaction solution, containing 25 nmol DNA were given to the respective spinfilter ((Amicon Ultra-6 mL Centrifugal Filters MWCO 10k or 30k cutoff) and were centrifuged 8 times for 1 hour.After each centrifugation the filtrate was discarded, the filter was refilled with 6 mL water and the solution was mixed.Centrifuged at 3900 rpm.

HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC) FOR PURIFICATION
HPLC instrument from Shimadzu was used including an auto sampler, a column oven and a fraction collector.The samples were purified by analytical HPLC using the column Jupiter 5 µm C18 300A (4.6 × 150 mm, 5 μm 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 to 15% B, then to 25% B and then increasing to 100% B, hold it for 20 min then decreasing to 5% B in 4 min.The absorbance was monitored at 240 nm and 260 nm. 8 µL (1 nmol) were injected.

ANION EXCHANGE COLUMN
Cytiva Capto TM HiRes Q 5/50 was used for the purification of DNA-polymer conjugates.The column was stored at 20% EtOH/water.

ÄKTA SYSTEM
The ÄKTA pure TM system was used as the device for using the anion exchange column.

PREPARING REACTION SOLUTION FOR AKTA PURIFICATION
The included DMF of the obtained reaction solutions were removed via spin filtration (Amicon Ultra-6 mL Centrifugal Filters MWCO 2k or 10k) by adding 5 mL nuclease-free water to the reaction solution and centrifuge for 1 h.This was repeated three times.

ÄKTA PURIFICATION METHOD
The following pictures/screenshots show the purification method, used with the ÄKTA pure TM with the anion exchange column Cytiva Capto TM HiRes Q 5/50 build in.The screenshots show the method applied for all conjugate purifications containing the 19 base oligonucleotide.The gradient is driven to 20% and held then isocratic.Please notice that for the short 10 base oligonucleotide the gradient is driven to 15% instead of 20% and for the long 40 base oligonucleotide the gradient is driven to 25%.All other parameters, pressures, flow rates etc. are the same.

POST-PREPARATION OF THE OBTAINED PURE CONJUGATE SOLUTION FROM ÄKTA
The obtained solutions contain NaCl due to the purification process.To remove the salt, the solutions were diluted with nuclease-free water and spin filtered with Amicon Ultra-6 mL Centrifugal Filters MWCO 10k for 20 min.This process is repeated three times.
Results and Discussion:         Table S1: Overview of the synthesized polymers, polymerization parameters and purification solvents. 1ble S2: Overview of the synthesized polymers, the conversion of the coupling reaction with the 19 base oligonucleotide 1 , the yield after purification and the molecular weight/dispersity of the conjugates given by SEC, measured in DMF.Yield after purification is calculated with conversion of the coupling reaction= 100%.

Figure S1 :
Figure S1: Overview of the single steps of the purification method

Figure S5 :
Figure S5: First column wash step to remove the uncharged, unreacted polymer excess.

Figure S6 :
Figure S6: Elution step.In this step the gradient is driven to the respective limit to elute the DNA-polymer conjugate and at higher concentrations the unreacted oligonucleotide.

Figure S7 :
Figure S7: Column wash step with 100% NaCl (2M) solution to remove all leftovers from the column.

Figure S8 :
Figure S8: Equilibration step to remove NaCl leftovers and set the milieu to water again.

Figure S15 :
Figure S15: Elution diagram of LCP2 from the first applied gradient.The diagram shows clearly, that the conjugate and also the unreacted oligonucleotide eluate at higher NaCl concentration.

Figure
Figure S18: HPLC purification method for the reaction solution CP2 after coupling.a) Applied stepwise gradient of the purification method with Acetonitrile (ACN) and water as solvents.b) elution diagram with absorption detection of the solvents as baseline.c) elution diagram of the reaction solution purification d) elution diagram with deducted solvent absorption.e) Magnification of the elution diagram in d) for better peak observation.f) PAGE gel (15%) of the respective fractions.Stained with SYBR Gold (2x).

Figure S20 :
Figure S20: Elution diagrams of the conjugates CP2 reaction solution measured by DMF SEC using polymethylmethacrylate (PMMA) as standard.
Figure S23: 1 H-NMR of P8 synthesized by RAFT polymerization.CTA group was removed with an excess of AIBN.

Table S3 :
Conversion determination using ImageJ intensity calculator to calculate conversion of the CP8 conjugation reaction.Used PAGE gels are shown in FigureS22

Table S4 :
Conversion determination using ImageJ intensity calculator to calculate conversion and the yield after ÄKTA of the LDNA, LCP2, SDNA and SCP2.Used PAGE gel is shown in Figure4a

Table S5 :
Intensity of the obtained conjugate CP2 from the respective fraction from the PAGE gels shown in FigureS18and S19 and the calculated proportion from the whole conjugate intensity.