Transient Transfection of Rolling‐Circle Amplified DNA in Biomanufacturing‐Relevant Mammalian Cell Lines: A Comparison of Transfection Conditions for Optimal Protein Expression

ABSTRACT

Rolling‐circle amplification (RCA) is a completely synthetic and rapid mechanism for scaling‐up bulk DNA that eliminates requirements for large‐scale bacterial fermentation. Several reports have described the surprising ability of large unprocessed (hyperbranched) rolling circle‐amplified DNA (RCA DNA) to effectively transfect cultured cells; however, to our knowledge a comprehensive analysis of transfection conditions has not been conducted for this unique type of large synthetic DNA. Herein, we present comparative transfection data for two of the most common mammalian cell types used in biopharmaceutical manufacturing (suspension Chinese hamster ovary CHO cells and adherent HEK293 cells) and a panel of commercial transfection reagents that represent three general compositions (i.e., cationic lipid, cationic polymer, and complex blends). We show the efficiency of RCA DNA delivery is highly influenced by the transfection reagent and cell line, but expression patterns generally track with plasmid controls. Reagents comprising complex blends provide some of the best transfection results for RCA DNA, although additional reagent types work well. We also show it is possible to transfect two different RCA DNA products into CHO cells to produce monoclonal antibodies at high titer. We conclude that large and unique synthetic DNA produced by RCA is capable of being used in place of supercoiled plasmid for downstream transfection workflows.

Abbreviations

CHO

Chinese hamster ovary

MFI

mean fluorescence intensity

mRFP

monomeric red fluorescent protein

RCA

rolling‐circle amplification

RCA DNA

rolling circle‐amplified DNA

TEM

transmission electron microscopy

WPRE

woodchuck hepatitis virus posttranscriptional regulatory element

1. Introduction

Biopharmaceuticals are fundamentally therapeutic molecules produced from living cells using recombinant DNA technology. Delivering recombinant DNA into cells by transient or stable transfection is a critical rate‐limiting step for research, process development, and manufacturing‐scale production of biological drugs. A standard approach for obtaining recombinant DNA is by plasmid isolation from bacteria, which involves a complex process of microbial culture, cell extraction, and DNA purification that is expensive and time‐consuming. However, new approaches for obtaining recombinant DNA have been developed using cell‐free synthetic biology to eliminate traditional dependencies on fermentation‐based plasmid production [1, 2, 3]. Of these, enzymatic DNA production by rolling‐circle amplification (RCA) is a scalable solution for obtaining bulk synthetic DNA (milligrams–grams) that previously was only possible through bacterial fermentation.

Random‐primed RCA reactions produce double‐stranded DNA concatemers that comprise multiple linear tandem‐repeats of the original circular template supplied to the reaction [4]. Atomic force microscopy studies have shown that rolling circle‐amplified DNA (RCA DNA) products are microns in size with a hyperbranched structure, and gel electrophoresis confirms high‐molecular weight species >100 kb in length [5]. These physical characteristics are analogous to the size and structural complexity of chromosomal DNA. Consequently, several methods have emerged to cleave RCA concatemers into smaller uniform states, such as linear synDNA or closed doggyboneDNA (also known as dbDNA), for cell delivery [6], [7]. For example, enzymatically digested linear DNA fragments (derived from RCA reactions) have been transfected into cells to produce infectious viruses and gene therapy vectors [3], [8, 9, 10]. However, a few groups have successfully delivered large unprocessed (hyperbranched) RCA DNA concatemers directly into cultured cells for recombinant expression. For example, Mizuta et al. and Sato et al. reported transfection and expression of GFP‐encoding RCA DNA in COS7 and LLC‐PK1 mammalian cells [5], [11]. Separately, Marano et al. reported transfection of RCA DNA encoding viral cDNA for successful production of infectious virus from common viral production cell lines (Vero, HEK293T, and BHK21) [12]. These sporadic observations demonstrate feasibility for using unprocessed RCA DNA reaction products directly in cells; however, to our knowledge a systematic analysis of transfection parameters is lacking for this unique type of large synthetic DNA.

The mechanism of DNA transfection into cells is mediated by complex formation with reagents that mask the net charge of the DNA payload. Transfection reagents are typically positively charged molecules that electrostatically interact with negatively charged DNA, forming a net‐positive surface charge on the reagent‐DNA complex. Transfection complexes are generally delivered into the cells via endocytosis and translocate into the nucleus for mRNA transcription. Many commercial transfection reagents exist, with the most common composition classes being cationic lipids, cationic polymers, and complex blends. Representative examples of lipid‐based reagents include Lipofectamine, DharmaFECT kb, and ExpiFectamine [13, 14, 15]. Examples of cationic polymer‐based reagents include the polyethylenimine products PEIpro and PEI MAX [16]. Examples of complex blends include TransIT and FuGENE, both of which may contain DNA‐binding proteins [17], [18]. All transfection reagents come with manufacturer recommendations, but researchers must experimentally optimize DNA‐to‐reagent ratios for optimal use. Studies have shown that circular or linearized plasmid complex with transfection reagents differently, thus necessitating reagent and method optimization [19], [20]. Transfection methods exist for linearized RCA DNA (after cumbersome concatemer digestion and processing) [3], [8, 9, 10]; however, transfection optimization for large (nonlinearized) RCA DNA is not well established.

Here, we provide comparative RCA DNA transfection results for two of the most common mammalian cell types used in biopharmaceutical manufacturing (suspension Chinese hamster ovary [CHO] and adherent HEK293) using a panel of commercial transfection reagents. We demonstrate that the overall suitability of RCA DNA is highly dependent on the transfection reagent and cell line used, but expression patterns generally track with plasmid controls. Our study provides further context for effectively implementing RCA DNA in downstream transfection workflows and biopharmaceutical applications.

2. Materials and Methods

2.1. Plasmid DNA Preparation

Plasmid DNA encoding monomeric red fluorescent protein (mRFP) was constructed as follows: starting from pCAGG‐MCS (PVT19755, Life Science Market), a custom gBlock fragment (Integrated DNA Technologies) corresponding to a strong enhancer (woodchuck hepatitis virus posttranscriptional regulatory element [WPRE]) was cloned into the XhoI and BglII positions of the multiple cloning site (MCS), to create a pCAGG‐MCS‐WPRE expression vector. A custom gBlock fragment encoding mRFP was subsequently cloned into the AflII and XhoI sites of pCAGG‐MCS‐WPRE. Purified pCAGG‐mRFP‐WPRE plasmid was prepared from Escherichia coli using endotoxin‐free plasmid DNA purification kits (Qiagen #12362). Plasmid DNA for antibody expression experiments were generated internally at AstraZeneca and purified by DNA Maxiprep from E. coli. All plasmid concentrations were determined spectrophotometrically or by Quant‐iT PicoGreen dsDNA assay (Thermo Fisher Scientific) after aliquoting and digesting a sample to disrupt plasmid supercoiling, since PicoGreen measurements are more precise for linear DNA [21].

2.2. DNA Preparation by RCA

Random‐primed RCA reactions were performed analogous to previous reports [11] but at milliliter scales to generate milligram quantities of RCA DNA from plasmid inputs. RCA reactions contained GenomiPhi v2 reaction buffer (Cytiva) supplemented with 400 µM of each dNTP (Cytiva), purified phi29 enzyme (Cytiva), and exonuclease‐resistant random hexamer (40 µM final concentration). Prior to RCA, plasmids were treated with equal units of exonuclease I and III (New England Biolabs) to ensure that only circular templates could be templated [22]. After amplification, the generated RCA DNA product was purified by ethanol‐based precipitation or desalting columns using proprietary methods. DNA concentrations were determined directly by Quant‐iT PicoGreen dsDNA assay (Thermo Fisher Scientific). Unless otherwise stated, no other processing steps were performed on purified RCA DNA to facilitate transfection studies of large hyperbranched concatemeric DNA.

2.3. Agarose Gel Electrophoresis

Approximately 25 ng of RCA product was resolved (per lane) using 1xTAE, 0.8% agarose gel electrophoresis. A 1 Kb Plus DNA Ladder (Invitrogen) was loaded for size comparison. To confirm sequence identity by RCA processing, DNA was incubated with XhoI restriction enzyme (which cuts the pCAGG‐mRFP‐WPRE template plasmid at a single location) for 1 h at 37°C and activity was quenched using SDS‐containing purple gel loading dye (New England Biolabs). Following electrophoresis, the gel was stained with SYBR gold nucleic acid gel stain (Invitrogen) and imaged by Typhoon laser scanner (Cytiva).

For bidirectional pulse‐field gel electrophoresis, RCA product (300 ng) was suspended in alkaline loading buffer (50 mM NaOH, 1 mM EDTA, 2.5% Ficoll 400, 0.025% bromocresol green) and electrophoresed on a 0.8% alkaline agarose gel at 2.5 v/cm in 25 mM NaOH, 1 mM EDTA running buffer, with an initial switch time of 1 and final switch time of 6, for 27 h. Denatured lambda DNA and 1 Kb Plus DNA Ladder (Invitrogen) were loaded for size comparison. The gel was neutralized in 1 M Tris pH 8 buffer prior to staining with SYBR Gold and imaging.

2.4. Transmission Electron Microscopy (TEM)

DNA substrate (pUC18 plasmid) and amplified product from TempliPhi reactions (1 ng/µL DNA in 0.25 M ammonium acetate containing 0.1% cytochrome‐C) were placed as 20 µL drops on parafilm and touched onto a 200‐mesh formvar/carbon grid, then rinsed with water twice. The grid with carbon film was then dipped in 30% ethanol containing 0.02% uranyl acetate for 5 s, then washed in 30% ethanol for 5 min, and dried. After drying, the grid was examined in dark‐field mode in an electron microscope, rotary‐shadowed at 10° angle. TEM images were then examined using imageJ to estimate minimum DNA length from visible sections (those protruding from the grid cannot be measured). In general, RCA DNA products were found in distinct clusters, with extended regions of uninterrupted double‐stranded DNA; however, for those connected to sets of nearby clusters, each measured section was summed to determine the minimum combined product length.

2.5. Cell Culture

Adherent HEK293T cells were purchased from ATCC (CRL‐3216). Cells were maintained at ATCC‐suggested conditions by culturing in Dulbecco's modified Eagle's medium (DMEM) with high glucose (Thermo Fisher Scientific, 11965092), 10% heat‐inactivated fetal bovine serum (ATCC, 30‐2020), and 2 mM L‐glutamine (ATCC 30‐2214). Cells were passaged in 75 cm² flasks with media replacement twice a week. Prior to plating for transfection, cells were enzymatically dissociated and collected. Briefly, flasks were washed with D‐PBS (ATCC, 30‐2200) to remove traces of serum prior to adding ∼ 2 mL Trypsin‐EDTA (Thermo Fisher Scientific, 25200056) and incubated at 37°C for 2 min. After cell detachment, excess complete media was added to neutralize trypsin. Cell suspensions were then transferred to a sterile centrifuge tube and concentrated at 250 × g for 5 min. Cell pellets were resuspended in fresh complete media and HEK293T were seeded into multi‐well plates for adherent culture.

ExpiCHO‐S cells (Thermo Fisher Scientific, A29127) were suspension‐cultured in ExpiCHO‐S expression medium (Thermo Fisher Scientific, A2910001) or CHOgro expression medium (Mirus Bio, MIR 6200), depending on specific recommendations from the transfection reagent manufacturer. CHOgro media were supplemented with poloxamer 188 solution (Mirus Bio, MIR6230) and L‐glutamine (Mirus Bio, 6240) to final concentrations of 0.3% and 4 mM, respectively. Prior to plating for transfection, ExpiCHO‐S were cultured in 250‐mL Erlenmeyer shaker flasks (Corning, 431144) at 37°C (7% CO₂) in 60 mL culture volume on a platform shaker (Thermo Fisher Scientific, 88881103) with a 19 mm orbital throw at 120 rpm. ExpiCHO‐S cells were subpassaged no more than two to three times to maintain culture densities < 6 ×10⁶ cells/mL with > 95% viability.

2.6. Transient Transfection of Adherent HEK293T Cells

Lipofectamine 3000 (Invitrogen, L3000008) transient transfections were performed in 12‐well tissue culture‐treated plates (Corning, CLS3513‐50EA). HEK293T cells were seeded at ∼1.6 × 10⁵ cells/mL in complete growth medium and incubated overnight to facilitate attachment and ∼75% confluency. DNA: Lipofectamine 3000 complexes were generated by first diluting stock DNA into Opti‐MEM I reduced serum medium (Thermo Fisher Scientific, 31985062) and then adding P3000 reagent. Separately, Lipofectamine 3000 (pre‐equilibrated to ambient temperature for ∼15 min) was diluted into Opti‐MEM I and then the DNA/P3000 mixture was added to this and incubated for 10–15 min at ambient temperature. Complexes were prepared in ∼100 µL volumes per transfection well and comprised 2 µL/well of P3000 reagent. Spent culture media were removed from each well before adding 900 µL of fresh media and ∼100 µL DNA:Lipofectamine 3000 complexes and incubating the plate for 3 days at 37°C (5% CO₂). To optimize complexation ratios, DNA mass was held constant (i.e., 1 µg per well) while the volume of Lipofectamine 3000 was tested at 1:1.5 and 1:3 ratios (µg DNA:µL reagent) per manufacturer instructions. Optimal DNA expression was observed at 1 µg DNA and 3 µL Lipofectamine 3000.

TransIT‐LT1 (Mirus, MIR 2304) transient transfections were performed in 12‐well tissue culture‐treated plates. HEK293T cells were seeded at ∼1.6 × 10⁵ cells/mL in complete growth medium and incubated overnight to facilitate attachment and ∼75% confluency. DNA: TransIT‐LT1 complexes were generated by diluting stock DNA into Opti‐MEM I and then adding TransIT‐LT1 (pre‐equilibrated to ambient temperature) into the diluted DNA with gentle mixing and incubating for 15–20 min. Complexes were prepared in 100 µL volumes per transfection well and administered dropwise into the media. Plates were gently rocked to evenly mix prior to incubating the cells for 3 days at 37°C (5% CO₂). To optimize complexation ratios, DNA mass was held constant (i.e., 1 µg per well) while the volume of TransIT‐LT1 was increased proportionally to establish 1:2–1:6 ratios (µg DNA:µL reagent). Optimal DNA expression was observed at 1 µg DNA and ∼5 µL TransIT‐LT1.

PEIpro (Polyplus, 101000017) transient transfections were performed in 12‐well tissue culture‐treated plates. HEK293T cells were seeded at ∼1.6 × 10⁵ cells/mL in complete growth medium and incubated overnight to facilitate attachment and ∼75% confluency. DNA:PEIpro complexes were generated by separately diluting stock DNA and PEIPro reagent in Opti‐MEM I as instructed by the manufacturer, prior to combining and incubating for 15 min at ambient temperature. Complexes were prepared in 100 µL volumes per transfection well and administered dropwise into the media. Plates were gently rocked to evenly mix prior to incubating the cells for 3 days at 37°C (5% CO₂). To optimize complexation ratios, DNA mass was held constant (e.g., 0.5–2 µg per well) while the volume of PEIpro was increased proportionally to establish 1:1–1:3 ratios (DNA:reagent). Optimal DNA expression was observed at ∼0.5 µg DNA and ∼1.5 µL PEIpro.

DharmaFECT kb (Horizon, T‐2006‐01) transient transfections were performed in 12‐well tissue culture‐treated plates. HEK293T cells were seeded at ∼1.6 × 10⁵ cells/mL in complete growth medium and incubated overnight to facilitate attachment and ∼75% confluency. DNA: DharmaFECT kb complexes were generated by separately diluting stock DNA and DharmaFECT kb reagent in Opti‐MEM I as instructed by the manufacturer, prior to adding the diluted DharmaFECT kb reagent to the diluted DNA and incubating for 10 min at ambient temperature. Complexes were prepared in 200 µL volumes per transfection well. Spent culture media were removed from each well before adding 800 µL of fresh media and ∼200 µL DNA: DharmaFECT kb complexes and incubating the plate for 3 days at 37°C (5% CO₂). To optimize complexation ratios, DNA mass was held constant (i.e., 2 µg per well) while the volume of DharmaFECT kb was increased proportionally to establish 2:4–2:9 ratios (µg DNA:µL reagent) per the range recommended by the manufacturer. Optimal DNA expression was observed at 2 µg DNA and 8 µL DharmaFECT kb.

2.7. Transient Transfection of Suspension CHO Cells

PEI MAX transient transfections were performed in 6‐well ultra‐low attachment tissue culture plates (Millipore Sigma, CLS3471‐24EA). ExpiCHO‐S cells were pre‐cultured in media and approximately 12 × 10⁶ cells were plated per well (cell density ∼4 × 10⁶ cells/mL × 3 mL). PEI MAX powder (Polysciences Inc., 24765‐1) was dissolved in cell culture‐grade sterile water, adjusted to pH 7.2, and sterile‐filtered and aliquoted at a final stock solution of 1 mg/mL. DNA: PEI MAX complexes were generated by first diluting DNA stocks in serum free DMEM and adding PEI MAX to the diluted DNA, allowing complexation to take place at ambient temperature for 15 min. Complexes were prepared in 300 µL volumes per transfection well. DNA: PEI MAX complexes were added to cell suspensions and mixed by orbital shaking (120 rpm at a 19 mm orbital throw) for 3 days at 37°C (7% CO₂). To optimize complexation ratios, DNA mass was held constant (e.g., 3 µg, 6 µg, or 12 µg per well) while the volume of PEI MAX was increased proportionally to establish 1:1–1:4 ratios (DNA:reagent). Optimal DNA expression was observed at 12 µg DNA and 24 µL PEI MAX.

TransIT‐PRO (Mirus, MIR 5740) transient transfections were performed in six‐well ultra‐low attachment tissue culture plates. ExpiCHO‐S cells were pre‐cultured in CHOgro media according to manufacturer instructions and approximately 12 × 10⁶ cells were plated per well (cell density∼4 × 10⁶ cells/mL × 3 mL). DNA:TransIT‐PRO complexes were generated in CHOgro complex formation solution (300 µL per transfection well) by first adding stock DNA and subsequently adding TransIT‐PRO, incubating this mixture for not longer than 5 min at ambient temperature. Complexes were added to cell suspensions, immediately followed by CHOgro titer enhancer (Mirus, MIR 6220, 60 µL per well) and mixed by orbital shaking (120 rpm at a 19 mm orbital throw) for 3 days at 37°C (7% CO₂). To optimize complexation ratios, DNA mass was held constant (e.g., 3 µg per well) while the volume of TransIT‐PRO was increased proportionally to establish 1:1–1:4 ratios (µg DNA:µL reagent). Optimal DNA expression was observed at 3 µg DNA and 9 µL TransIT‐PRO.

FuGENE 4K (FuGENE, 4K‐1000) transient transfections were performed in 24‐well deep well plates (Qiagen, 19583). ExpiCHO‐S cells were pre‐cultured in CHOgro media and approximately 12 ×10⁶ cells were plated per well (cell density ∼4 × 10⁶ cells/mL × 3 mL). Stock DNA was diluted in DMEM and FuGENE 4K reagent (pre‐equilibrated to ambient temperature) was added to the diluted DNA, incubating for ∼13 min at ambient temperature. Complexes were prepared in 300 µL volumes per transfection well. DNA:FuGENE 4K complexes were added to cell suspensions and mixed by orbital shaking (275 rpm at a 19 mm orbital throw) for 3 days at 37°C (7% CO₂). To optimize complexation ratios, DNA mass was held constant (i.e., 3 µg per well) while the volume of FuGENE 4K was increased proportionally to establish 1:3–1:5 ratios (µg DNA:µL reagent) according to manufacturer recommendations. Optimal DNA expression was observed at 3 µg DNA and 9 µL FuGENE 4K.

ExpiFectamine (Thermo Fisher Scientific, A29129) transient transfections were performed in 96‐well deep well plates (Axygen, PDW20CS) and 24‐well deep well Plates (Qiagen, 19583). ExpiCHO‐S cells were pre‐cultured in ExpiCHO media according to manufacturer instructions. On the day prior to transfection, the cells were split to a final density of 3–4 × 10⁶ cells/mL and cultured overnight. On the day of transfection, the cells were diluted to ∼4–6 × 10⁶ cells/mL with fresh ExpiCHO media and 0.8 mL or 3 mL aliquots were transferred into 96‐well or 24‐well deep well plates, respectively. DNA stocks were diluted in Gibco OptiPRO SFM (Thermo Fisher Scientific, 12309050) and mixed prior to adding ExpiFectamine reagent and mixing well. Complexes were prepared in 300 µL volumes per transfection well (24‐well) or 65 µL volumes per transfection well (96 well). For the latter, a master mix was prepared for n + 1 = 4 wells and aliquoted into three transfection wells for triplicate analysis. Complexes were mixed with cells by gentle pipetting and plates were covered with a gas‐permeable seal (AeraSeal adhesive microplate seals, E&K Scientific, T896100‐S). Plates were cultured at 37°C with 7% CO₂ with shaking (225 rpm for 24‐well plate at a 19 mm orbital throw or 900 rpm for 96‐well plate at a 3 mm orbital throw). The following day, CHO Enhancer (Thermo Fisher Scientific, A29129) and ExpiCHO Feed reagents were added following manufacturer instructions and culture temperature was optionally reduced to 32°C (5% CO₂) for 2 more days of culture. To optimize complexation ratios, DNA:reagent ratios (µg:µL) were explored around the manufacturer's recommended instructions, but we ultimately observed that the highest mean expression was achieved at a ratio of 3 (i.e., 3 µg DNA and 9 µL ExpiFectamine), consistent with prior reports [23].

2.8. Flow Cytometry

For CHO suspension culture, ∼200 µL cell suspension samples were harvested 2–3 days after transfection. For adherent HEK293T samples, cells were trypsinized to detach and quenched with complete media before analyzing suspensions. Red fluorescent protein (RFP)‐positive cells were measured and recorded in the PE channel using CytoFLEX LX flow cytometer (Beckman Coulter Life Sciences). Events were analyzed using Cytexpert 2.3 software. A forward scatter dot plot (FSC‐A) was plotted against a side scatter dot plot area (SSC‐A) to distinguish cell debris from live cells and a gate was applied around the live cells. Then, a forward scatter plot‐area (FSC‐A) on the x‐axis was drawn against forward scatter height (FSC‐H) on the y‐axis to distinguish “doublets” from singlet events. PE fluorescence intensity (y‐axis) was plotted on a log scale against the fluorescence intensity (x‐axis) of FITC to exclude any spectral spillover and apply a gate for the PE‐positive population. Finally, the mean fluorescence intensity (MFI) was recorded among all singlet live cells.

2.9. Comparative Testing of Antibody Production

AstraZeneca proprietary G22 CHO cells [24] were seeded at 2 × 10⁶ viable cells/mL. After 24 h, 3 mL aliquots of cells at 4 × 10⁶ viable cells/mL were decanted into wells of a 24‐well block (Whatman 24‐well, round well bottom, WHA77015110, Merck). For constructs transfected with PEI, cells were treated with 1% DMSO 1 h pre‐transfection (Sigma, D2650) and then transfected with 1 µg DNA/mL of cells using PEI‐MAX (PolySciences, 24765) at a DNA:PEI ratio of 1:5 (a previously established optimum ratio for antibody production in this CHO line). Both the PEI‐MAX and DNA were each diluted in 150 mM NaCl before combining to form a DNA‐PEI complex and added directly to the cells. For TransIT‐PRO transfection, cells were transfected with 1 µg DNA per mL of cells (3 µg total per well) using 9 µL of TransIT‐PRO at a 1:3 ratio (TransIT‐PRO transfection reagent, Mirus Bio, MIR5740) according to the down‐selected conditions for ExpiCHO‐S (see Figure S3). The DNA was diluted in CHOgro complex formation solution, the TransIT‐PRO reagent was added directly to this, incubated for 5 min then added directly to the cells. Post‐transfection, all cultures were maintained at 37°C for 4 h, then the temperature was reduced to 34°C as the optimal induction temperature for this proprietary CHO system (determined by extensive internal testing). The cultures were maintained at 34°C, 5% CO₂, 80% humidity for 7 days and supplemented with AstraZeneca proprietary feeds. Viable cell density, total cell density, and cell viability were measured using a Vi‐Cell XR cell counter (Beckman Coulter). Titer quantitation was performed by BLI assay using Octet Protein A Biosensors (Sartorius, 185010).

3. Results and Discussion

3.1. Confirmation of Unique RCA DNA Structure Compared to Plasmid

We conducted electron microscopy (TEM) studies of representative RCA DNA molecules to contextualize reports from previous AFM studies [5]. Our TEM images presented in Figure 1 (and Figure S1B–D) confirm that RCA DNA is structurally unique compared to plasmid, with extended regions of uninterrupted double‐stranded DNA and loops of hyperbranched DNA reminiscent of the pinwheel or butterfly illustrations of random‐primed RCA polymerization [4], [5]. Using a conversion factor of three base pairs per nanometer, the apparent length of several RCA DNA molecules was estimated computationally and found to range from > 50,000 bp to > 300,000 bp (Figure S1B–D), with a numerical average of > 188,000 bp. By comparison, the observed length of supercoiled plasmid DNA (Figure 1A insert; Figure S1A) was found to be on average 2622 bp, in good agreement with its expected length of 2680 bp.

FIGURE 1.

Characterization of rolling‐circle amplified DNA. (A) Representative TEM image of RCA DNA relative to plasmid (insert). Scale bars are 200 nm. (B) Agarose gel electrophoresis of RCA DNA in either unprocessed concatemeric state (Lane 1) or after enzymatic digestion to form non‐concatemeric monomers of the original plasmid template (Lane 3). Lane 2 is a DNA size ladder as marked. (C) Denaturing pulse‐field gel electrophoresis of the unprocessed RCA DNA from panel B after alkaline strand separation (Lane 2). Lane 1 is a DNA size ladder as marked.

These structural findings contextualize the migration patterns of unprocessed RCA DNA within traditional agarose gel electrophoresis assays (Figure 1B). To illustrate, we amplified a 6038 bp plasmid encoding an RFP (pCAGG‐mRFP‐WPRE) and analyzed the RCA product in either native (unprocessed) or enzymatically digested (processed) states. (Figure 1B). Unprocessed RCA DNA shows a smeared size distribution by gel with the bulk species migrating beyond the size resolution limit of the gel (> 15 kb) with additional species trapped (or “deposited” [5]) in the loading well, unable to enter the gel (Lane 1, Figure 1B). This trapped RCA DNA likely contains DNA strands with internal replication forks, as DNA strands with three or more ends cannot migrate in gel systems [25]. Despite such unique structural properties, it is well known that RCA DNA is directly compatible with DNA sequencing [4], [26]. Next, we confirmed that unprocessed RCA DNA is a double‐stranded tandem‐repeat of authentic circular template. We digested RCA DNA with restriction enzyme (to cut the original template plasmid at a single location) and observed RCA DNA species were processed into ∼6 kb monomeric size (Lane 3, Figure 1B) with virtually no residual DNA trapped in the loading well. Lastly, we denatured unprocessed (hyperbranched) RCA DNA into single strands and analyzed by alkaline pulse‐field gel electrophoresis (Figure 1C). The bulk of the ssDNA appeared greater than 15 kb with approximately 50% of the densitometry signal > 50 kb, in line with TEM estimations.

3.2. Optimization and Comparison of RCA DNA Transfection in Adherent HEK293 Cells

We systematically investigated protocols for transfecting large unprocessed RCA DNA (derived from pCAGG mRFP‐WPRE plasmid) into adherent HEK293T cells. These transfection protocols were tested using TransIT‐LT1, Lipofectamine 3000, DharmaFECT kb, and PEIpro reagents (as representative examples of mixed‐, lipid‐, and polymer‐based reagents, respectively). Of note, TransIT and PEIpro reagents are available in cGMP grades for viral vector manufacturing (commonly performed in HEK293‐derived cell lines). We included plasmid transfection controls to benchmark protein expression against RCA DNA. Per manufacturer‐provided transfection ranges, we evaluated several reagent‐DNA ratios and ranked cytoplasmic intensity of expressed RFP by flow cytometry analyses of MFI at 2–3 days post‐transfection (see Figure S2). Optimal ratios were down‐selected according to the combined MFI responses of both plasmid and RCA DNA, and these optimums were serially repeated in technical replicate experiments. Figure 2 summarizes our optimized results. Transfection of RCA DNA (or plasmid) with TransIT‐LT1 or Lipofectamine 3000 provided the highest RFP intensity, which is consistent with prior RCA DNA transfection reports using different grades of these reagents [5], [11]. PEIpro also worked well for transfecting RCA DNA (or plasmid), especially when compared against DharmaFECT kb, which generated lower RFP expression for both plasmid and RCA DNA. We observed a bell‐shaped expression pattern for PEIPro:plasmid DNA complexes after transfection (Figure S2), as was been reported by others [27]; however, RCA DNA expression was nearly constant across the same conditions (Figure S2). Consequently, RCA DNA tended to work better than plasmid at the 1:1 “recommended condition” provided by the PEIPro manufacturer, due to a dramatic decline in comparative plasmid transfection fidelity. Across all tested reagents, the mean and median fluorescence intensities from RCA DNA transfections were ∼threefold lower than plasmid (at equivalent DNA mass) using this adherent HEK293T model system (Figure 2). However, plate‐based adherent cell culture can be a poor predictor of suspension‐culture performance and commercial‐scale production [28]. In total, our results identify TransIT‐LT1, PEIpro, and Lipofectamine 3000 as suitable transfection reagents for RCA DNA delivery into adherent HEK293T cells.

FIGURE 2.

Comparative transfection of adherent HEK293T cells. (A) Mean fluorescence intensity of red fluorescent protein (RFP) after transfection of RCA DNA (left) or plasmid (right) into adherent HEK293T cells. Pre‐optimized transfection conditions (DNA:reagent) are listed. Asterisks denote statistical significance relative to TransIT‐LT1 and Lipofectamine 3000 (*, p‐value < 0.05; ***, p‐value < 0.001). (B) Representative flow cytometry data showing singlet live‐cell counts as a function of RFP fluorescence after transfection of RCA DNA (left) or plasmid (right) in panel A. Saturated fluorescence > 10⁷ RFU (relative fluorescence unit) intensity is stacked onto the perimeter line and not shown.

3.3. Optimization and Comparison of RCA DNA Transfection in Suspension CHO Cells

Fluorescent assays are known to have good correlative potential to large‐scale antibody production in CHO cells [28]; therefore, we evaluated RCA DNA transfection of suspension ExpiCHO‐S cells using the same cytoplasmic RFP assay applied to HEK293T cells. Transfection protocols were optimized for suspension delivery using TransIT‐PRO, FuGENE 4K, ExpiFectamine, and PEI MAX reagents as representative examples of mixed‐, lipid‐, and polymer‐based reagents, respectively. Following manufacturer‐provided guidelines, we evaluated several DNA:reagent ratios and quantified cytoplasmic expression of RFP by flow cytometry at approximately 2–3 days post‐transfection (see Figure S3). Optimal ratios were down‐selected according to the combined MFI responses of both plasmid and RCA DNA, and these optimums were serially repeated in technical replicate experiments. Figure 3 summarizes our optimized results. TransIT‐PRO and FuGENE 4K provided the highest mean expression of RCA DNA, followed very closely by ExpiFectamine, and this pattern mirrored the results from plasmid controls. Transfection with PEI MAX required ∼4x more total DNA (including plasmid) to achieve comparable RFP expression thresholds to other transfection reagents (Figure S3). Despite requiring more input DNA, lower average MFI (∼twofold) was observed for RCA DNA:PEI MAX complexes compared to plasmid. Interestingly, for all other tested reagents, the mean and median fluorescence intensities from RCA DNA transfections were largely comparable to plasmid at equivalent DNA mass (i.e., less than a 1.5‐fold difference in MFI between RCA DNA and plasmid). Our results identify TransIT‐Pro, FuGENE 4K, and ExpiFectamine as preferred transfection reagents for RCA DNA delivery into suspension ExpiCHO‐S cells.

FIGURE 3.

Comparative transfection of suspension ExpiCHO‐S cells. (A) Mean fluorescence intensity of red fluorescent protein (RFP) after transfection of RCA DNA (left) or plasmid (right) into suspension ExpiCHO‐S cells. Pre‐optimized transfection conditions (DNA:reagent) are listed. Asterisks denote statistical significance relative to TransIT‐Pro and FuGENE 4k (*, p‐value < 0.05; **, p‐value < 0.01). (B) Representative flow cytometry data showing singlet live‐cell counts as a function of RFP fluorescence after transfection of RCA DNA (left) or plasmid (right) in panel A. Saturated fluorescence > 10⁷ RFU (relative fluorescence unit) intensity is stacked onto the perimeter line and not shown.

3.4. Validation of RCA DNA in a Biopharma‐Relevant Workflow for Antibody Production

The mainstay of biopharmaceutical antibody development utilizes transient transfection of CHO suspensions for high throughput screening of IgG binding activity and production titers during preclinical development. The Biologics Engineering team at AstraZeneca uses repositories of high‐quality plasmids to facilitate antibody screening in a proprietary G22 CHO cell lineage [24]. Transfection protocols have been carefully developed using PEI MAX to facilitate iterative screening of plasmid assets at low reagent costs. The targeted antibody secretion titers are ∼200 mg/L (on average) using this transfection screening assay (represented by the dashed lines in Figure 4). We evaluated the utility of RCA DNA substitution into this workflow by taking the same stock plasmid and amplifying by RCA prior to testing transfection of unprocessed RCA DNA into G22 CHO cells. In parallel, we evaluated double‐transfection formats in which two RCA DNA products (separately encoding IgG heavy and light chains, respectively) were co‐transfected into G22 cells. Figure 4A reveals very low antibody expression for all RCA DNA formats delivered by PEI MAX, with titers at the lower‐limit of assay quantification (∼tenfold lower than plasmid). Based on previous mRFP screening results (Figure 3), we next substituted TransIT‐Pro in for PEI MAX and re‐tested transfections at the same mass of RCA DNA (and plasmid) in G22 CHO. Figure 4B shows that TransIT‐Pro enabled RCA DNA to restore antibody titers to ∼200 mg/L expectations, thereby confirming the trends predicted by Figure 3 for RFP. Moreover, our data show that two different RCA DNA products can be co‐transfected into CHO cells to achieve sufficient antibody titers for biopharmaceutical screening. Interestingly, antibody titers from plasmids were also elevated using TransIT‐Pro (∼twofold higher than PEI MAX and RCA DNA titers), albeit using a transfection protocol that was only pre‐optimized for ExpiCHO‐S cells (Figure S3). Since transfection parameters can differ substantially among CHO host lines [23], it is possible that even higher titers may be obtained from G22 CHO cells after optimizing reagent‐RCA DNA ratios for these cells. Nevertheless, using the current ExpiCHO‐optimized protocol (3 µg DNA and 9 µL TransIT‐PRO), RCA DNA can be swapped for plasmid and achieve sufficient yields in this biopharma‐relevant CHO workflow.

FIGURE 4.

Comparison of antibody expression titers following transfection of RCA DNA or plasmid into CHO cells. Secreted antibody titers were obtained from media 7 days after transfection using (A) PEI MAX or (B) TransIT‐PRO. Cells were transfected with one of three different sets of DNA—a single dual‐expression cassette vector for antibody 1 (purple bars), a two‐vector set for antibody 1 (light blue bars), or a two‐vector set for antibody 2 (green bars). DNA was prepared for transfection either by synthetic RCA or plasmid fermentation, as indicated.

4. Conclusions

Our data highlight that delivery and expression of RCA DNA is highly influenced by the transfection reagent and cell type used. While certain combinations of transfection reagents and RCA DNA may be suboptimal to plasmid controls, we identified conditions where RCA DNA achieves comparable performance (or otherwise meets acceptable thresholds for high throughput screening). The unique ability to produce up to gram quantities of RCA DNA rapidly (in days compared to several weeks for scaling plasmid DNA in fermenters) provides tangible benefits for exploiting large synthetic DNA in transfection workflows, including faster time‐to‐result and ease of scale‐up potential for cell seed trains. Furthermore, RCA is one of the few solutions for propagating minimal DNA constructs (i.e., lacking prokaryotic elements) [29], so new synthetic DNA workflows might be enabled in bioprocessing cell lines using RCA.

PEI‐based transfection reagents including PEI MAX are recognized as cost‐effective solutions for screening antibody titers in suspension CHO cells, albeit one requiring higher amounts of DNA (per transfection) than other types of reagents [23]. In our tests of ExpiCHO cells, we also observed that fourfold higher DNA input (including plasmid) must be used with PEI MAX to obtain comparable RFP fluorescence intensities as non‐PEI reagents. However, RCA DNA expression using PEI MAX was unusually lower than other tested CHO reagents, thereby magnifying comparative expression differences between plasmid and RCA DNA (for both IgG and RFP). We did not evaluate if higher RCA DNA mass might have improved antibody production using PEI MAX. Instead, we show that TransIT‐PRO improved RCA DNA transfection efficiency for both IgG and RFP, albeit further method optimization is warranted for G22 cells since we applied a protocol customized for ExpiCHO cells (i.e., publications have shown transfection parameters can differ substantially among CHO lineages) [23]. TransIT‐Pro also improved the performance of plasmid DNA in CHO cells relative to PEI MAX, consistent with prior reports [30].

Protein‐containing transfection reagents have demonstrated superior abilities for transfecting CHO and HEK293 cell lines relative to cationic lipids and cationic polymers [28]. In this regard, it is interesting that patent literature for TransIT and FuGENE (i.e., two of the highest‐performing reagents in our study) suggest these compositions comprise DNA‐binding proteins (i.e., histone H1) [17], [18] Thus, we hypothesize the presence of DNA‐binding proteins may enhance transfection of RCA DNA into mammalian cells (versus reagents that lack protein components, like PEI MAX). That being said, mixed composition reagents like TransIT are also highly sensitive to culture media formulations [30], and indeed we observed better protein expression from Mirus CHOgro expression media (using TransIT‐PRO or FuGENE 4K) compared to Gibco ExpiCHO expression media (data not shown).

This current study is not without certain experimental limitations. Our HEK293 experiments were limited to adherent cells and future studies will evaluate RCA DNA transfection using suspension‐adapted lineages, for which specific transfection reagents have been developed to produce viral vector‐based gene therapies (for example, Gibco AAV‐MAX and FectoVIR reagents). Our observed trends for better RCA DNA performance in suspension CHO versus adherent HEK293 mirror what has been reported by comparative plasmid transfection of suspension versus adherent lines [28]. Contributing reasons for this include the fact that certain reagents have been optimized for suspension cells, and suspension lines achieve much higher cell densities than surface area–limited adherent cells, which when combined with fluidic agitation may augment random diffusion interactions between DNA‐reagent complexes and suspension cells. Our survey of commercial reagents was intentionally limited since new product versions are released continually, often with re‐branding that makes it difficult to track product composition changes. The molecular mechanism for how cells accomplish effective expression from large RCA DNA concatemers is still unclear and warrants further intracellular study. It is also unclear how many expression‐competent gene copies are present in RCA concatemers (and further transcribed by cells), so a molar comparison to plasmid DNA was not possible and we utilized DNA mass as a transfection comparator. Lastly, we have not explored whether biochemical modification of RCA reactions might modulate transfection and/or expression parameters. In total, further work may improve upon the baseline performance reported in our current study.

Conflicts of Interest

Evelina Loghin, Erik Kvam, Brian Davis, Lisa Lowery, Christine Surrette, and John Nelson are employees of GE HealthCare (GEHC) and have filed intellectual property on behalf of GEHC that pertain to aspects of this work. Nathan Hudson, Jenny Keen, and Roger B. Dodd are employees of AstraZeneca.

Supporting information

Supplementary Figures: bab70100‐sup‐0001‐figuresS1‐S3.pdf

Data Availability Statement

The data used to support the findings of this study have been included in this article as Supporting Information.