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In vitro transcription of self-assembling DNA nanoparticles – Scientific Reports

Republished By Plato
Republished By Plato

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Gene-bearing DNA origami scaffolds were prepared as previously described24, summarized as follows.

Standard PCR

Primers for amplification of the Green Fluorescent Protein (GFP) gene were designed using SnapGene and purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA). The sequence of each primer is listed in Supplementary Table 1. Phusion® polymerase for the PCR reaction was purchased from New England Biolabs (NEB, Ipswich, MA, USA).

For the generation of a duplex gene containing all components for transcription, each PCR reaction mixture was prepared in 50 µL final volume, composed of 1 × Phusion® HF buffer (NEB), 200 nM dNTP mix (NEB), 500 nM sense primer (T7EGFP sense = undesired sense strand), 500 nM antisense primer (T7EGFP anti = desired antisense strand), 10 ng plasmid template (pCMV-T7-EGFP; Addgene, Watertown, MA, USA), 0.5 µL Phusion® DNA polymerase, and nuclease-free water to volume. Each PCR was performed using the following thermocycler steps: 30 s at 98 °C, 30 s at 58 °C, and 1 min at 72 °C for 30 cycles24.

For the generation of duplex gene missing promoters, each PCR reaction mixture was prepared in 50 µL final volume, composed of 1 × Phusion® HF buffer (NEB), 200 nM dNTP mix (NEB), 500 nM sense primer (RT-sense), 500 nM antisense primer (T7EGFP anti), 10 ng pCMV-T7-EGFP (Addgene), 0.5 µL Phusion® DNA polymerase, and nuclease-free water to volume. Each PCR was performed using the following thermocycler steps: 30 s at 98 °C, 30 s at 59 °C, and 1 min at 72 °C for 30 cycles24.

The reaction products were mixed with 6 × loading dye (15% Ficoll®-400, 60 mM EDTA, 19.8 mM Tris–HCl, 0.48% SDS, 0.12% Dye 1, 0.006% Dye 2, pH 8 at 25 °C; NEB) and then loaded onto a 1% agarose gel pre-stained with SYBR safe DNA dye (Invitrogen, Waltham, MA, USA). Electrophoresis was carried out at 8 V/cm for 1 h. The SYBR Safe-containing DNA was visualized using a 490 nm wavelength (blue) transilluminator and an amber filter24.

Asymmetric PCR (aPCR)

Primers used in aPCR were identical to those used in standard PCR. Along with sense and antisense primers, a 3’ terminal modified primer (3’ T7EGFP blocker) was used. The 3’ T7EGFP blocker was designed using SnapGene and purchased from IDT. Its sequence is listed in Supplementary Table 1.

For the generation of a scaffold containing all components for transcription, each aPCR reaction was carried out in 50 µL total volume, composed of 1 × LongAmp® Taq buffer (60 mM Tris-SO4, 20 mM (NH4)2SO4, 2 mM MgSO4, 3% glycerol, 0.06% IGEPAL® CA-630, 0.05% Tween® 20, pH 9.1 at 25 °C) from NEB, 500 nM T7EGFP anti, 25 nM T7EGFP sense, 475 nM 3’ T7EGFP blocker, 300 nM dNTP mix from NEB, 10 ng double-stranded GFP gene (dsT7EGFP; generated by standard PCR), 2 µL LongAmp® Taq DNA polymerase (NEB), and nuclease-free water to final volume. Each aPCR was performed using the following thermocycler steps: 30 s at 94 °C, 30 s at 58 °C, and 2 min at 65 °C for 25 cycles24.

For the generation of a scaffold containing all components for transcription except the promoter element, each PCR reaction was carried out in 50 µL total volume, composed of 1 × LongAmp® Taq buffer (60 mM Tris-SO4, 20 mM (NH4)2SO4, 2 mM MgSO4, 3% glycerol, 0.06% IGEPAL® CA-630, 0.05% Tween® 20, pH 9.1 at 25 °C) from NEB, 1 µM T7EGFP anti, 20 nM RT sense, 300 nM dNTP mix from NEB, 10 ng double-stranded GFP gene devoid of promoters (dsT7EGFP -T7; generated by standard PCR), 2 µL LongAmp® Taq DNA polymerase (NEB), and nuclease-free water to final volume Each aPCR was performed using the following thermocycler steps: 30 s at 94 °C, 30 s at 59 °C, and 2 min at 65 °C for 25 cycles24.

The reaction product was loaded onto a 1% agarose gel pre-stained with 1 × SYBR Safe (Invitrogen), electrophoresed, and visualized as above.

Double-stranded DNA (dsDNA) purification

A Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA) was used to extract dsDNA from agarose gels. Gel bands containing target dsDNA were removed using a clean razor blade. Three times the gel slice volume of the provided agarose dissolving/binding buffer was added to each gel fragment and incubated at 55 °C on a heat block for 15 min. Each dissolved gel solution was transferred to a provided silica-based spin column and centrifuged at 10,000 relative centrifugal force (rcf) for 60 s in a table-top centrifuge. 200 µL of ethanol-based DNA wash buffer was added to each spin column and centrifuged at 10,000 rcf for 30 s. A washing step was repeated before centrifuging at 10,000 rcf for 60 s for the complete removal of ethanol. Flow-through from all steps was discarded. After transferring each spin column to a clean microcentrifuge tube, 6–20 µL of the provided elution buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.5) was added directly to the matrix of each spin column followed by centrifugation at 10,000 rcf for 60 s for DNA collection. A fraction of each purified dsDNA was mixed with 6 × loading dye (NEB) and loaded onto 1% agarose gel pre-stained with 1 × SYBR safe (Invitrogen). The gel was run at 8 V/cm for 1 h. The yield of the purified dsDNA sample was evaluated by measuring band intensities relative to a known control using GelAnalyzer 19.1 available at www.gelanalyzer.com (accessed on 19 August 2021)24.

Single-stranded (ssDNA) purification

A Zymoclean Gel RNA Recovery Kit from Zymo Research was used to purify ssDNA from agarose gels. The gel bands containing target ssDNA were excised with a clean razor blade. Three times the gel slice volume of the provided agarose dissolving/binding buffer was added to each excised gel band and melted at 55 °C on a heat block for 15 min. Each dissolved gel solution was transferred to a provided silica-based spin column and centrifuged at 12,000 rcf for 2 min. 400 µL RNA Prep buffer was added to each spin column followed by centrifugation at 12,000 rcf for 1 min. Washing was carried out by the addition of 800 µL ethanol-based wash buffer followed by centrifugation at 12,000 rcf for 30 s. After repeating the washing step with 400 µL ethanol-based wash buffer, each spin column was centrifuged at 12,000 rcf for 2 min to remove residual ethanol. Flow-through in all steps was discarded. After transferring each spin column to clean microcentrifuge tubes, 6–20 µL of provided nuclease-free water was added directly to the column matrix, and the spin columns were centrifuged at 10,000 rcf for 1 min for retentate collection. A fraction of each purified ssDNA was mixed with 6 × loading dye (NEB) and the yield was estimated by gel electrophoresis as described above24.

DNA nanoparticle construction

DNA nanoparticles were designed using caDNAno (www.cadnano.org) and staples were purchased from IDT (Supplementary Tables 2 and 3). DNA nanoparticles were prepared by mixing single-stranded GFP gene (ssT7EGFP or ssT7EGFP -T7; generated by aPCR) to a final concentration of 91.4 nM and each staple to a final concentration of 457 nM in 1 × TAE buffer supplemented with 12.5 mM Mg(OAc)2 (TAEM) in a final volume of 50 µL. The staple set for each DNA nanoparticle is listed in Supplementary Table 4. The mixture was incubated at 90 °C for 10 min in a water bath followed by gradual cooling to room temperature. Products of this reaction were mixed with 6 × loading dye (NEB) and then loaded onto 1% agarose gel containing 12.5 mM Mg(OAc)2 pre-stained with 1 × SYBR Safe DNA dye (Invitrogen). Electrophoresis was carried out in TAEM buffer at 6 V/cm for 90 min. The gel was visualized as above. DNA origami was purified using a Freeze ‘N Squeeze™ DNA Gel Extraction Spin Column (Bio-Rad, Hercules, CA, USA). Gel bands containing target DNA origami were sliced and removed using a clean razor blade, then transferred to Freeze ‘N Squeeze™ DNA Gel Extraction Spin columns. Spin columns containing target DNA origami gel slices were incubated at − 20 °C for 5 min followed by centrifugation at 13,000 rcf in a table-top centrifuge for 3 min at room temperature. The concentration of the purified DNA origami samples was measured using a NanoDrop™ instrument.

In vitro transcription (IVT)

All IVT reactions were carried out in 20 µL total volume using a HiScribe® T7 Quick High Yield RNA Synthesis Kit (NEB). Each reaction contained 10 µL NTP buffer mix (10 mM each NTP; NEB), 10 ng DNA template (linearized GFP plasmid (LpCMV-T7-EGFP), dsT7EGFP, ssT7EGFP, each DNA nanoparticle (designated as described in Table 1 below and in the legend for Fig. 2b), 2 µL T7 RNA polymerase mix, and nuclease-free water to volume. Each reaction was carried out at 37 °C for 2 h. For T7GHL FS, T7GHL PO, and T7GHL HS, a reaction time course of 30, 60, 90, and 120 min was carried out for a preliminary comparison of the relative transcription rates of these constructs and substrate longevity.

Table 1 Names of DNA scaffolds and DNA nanoparticles used in this study.
Full size table

Purification of IVT products

Following IVT, 30 µL of nuclease-free water was added to each IVT reaction product to increase the reaction volume. Each IVT product was then purified using a Monarch® RNA Cleanup Kit (NEB). 100 µL of RNA binding buffer was added to each 50 µL IVT product followed by the addition of 150 µL of absolute ethanol. Each mixture was then transferred to a provided silica-based spin column and centrifuged in a table-top centrifuge. 500 µL of ethanol-based DNA wash buffer was added to each spin column and centrifuged as above. The washing step was repeated once more and the flow-through from all steps was discarded. Each spin column was transferred to a clean microcentrifuge tube and 10 µL of the provided nuclease-free water was added directly to the matrix of each spin column followed by centrifugation for DNA collection. All centrifugation steps were carried out in 16,000 rcf for 60 s.

Evaluation of IVT products

IVT products from all DNA samples were analyzed by gel electrophoresis. Purified GFP PCR and linearized GFP plasmid IVT products were diluted 100-fold to avoid over-staining. Purified RNA products were mixed with an equal volume of 2 × RNA loading dye (95% formamide, 0.02% SDS, 0.02% bromophenol blue, 0.01% Xylene Cyanol, 1 mM EDTA; NEB). Samples were heated to 70 °C for 10 min prior to gel loading. Electrophoresis was carried out at 8 V/cm for 1 h. The gels were post-stained with 1 × TAE solution containing 1 × SYBR gold (Invitrogen) for 2 h. The stained gels were visualized using a 490 nm wavelength transilluminator and an amber filter.

Reverse transcription PCR (RT-PCR)

RNA templates for RT-PCR were prepared by carrying out an IVT reaction using each DNA template (LpCMV-T7-EGFP, dsT7EGFP, ssT7EGFP, T7GHL PO, T7GHL HS, T7GHL FS, and T7GHL BP) as described above. To minimize the possible background signal caused by the presence of residual DNA template, 1 ng of each DNA template (rather than 10 ng) was used for these reactions, and the incubation time was extended to overnight to maximize RNA production under these conditions. Following IVT, DNA was hydrolyzed by treatment with DNase. A 50 µL DNase mixture was prepared by mixing 20 µL IVT product with 2 µL RNase-free DNase-I and nuclease-free water to volume. All DNase reactions were carried out at 37 °C for 30 min. DNase-treated RNA products were purified using a Monarch® RNA Cleanup Kit (NEB) as described above. Primers for amplification of the GFP gene were designed using SnapGene and purchased from IDT. The sequence of each primer is listed in Supplementary Table 1. RT-PCR was performed using a OneTaq® One-step RT-PCR kit (NEB). Each RT-PCR mixture was carried out in 50 µL final volume, containing 1 × Quick-Load® OneTaq One-step reaction mix (1.6 mM MgCl2, 250 nM dNTP mixture), 400 nM sense primer (RT-sense), 400 nM antisense primer (RT-anti), 1 µL of each purified RNA product, 1 × OneTaq® One-step enzyme mix (ProtoScript® II reverse transcriptase, OneTaq® Hot Start DNA polymerase, Murine RNase inhibitor, and stabilizer), and nuclease-free water to volume. To assess any contribution to PCR signal from residual DNA resulting from incomplete DNase hydrolysis, negative controls were prepared in parallel. These reactions contained the same components as RT-PCR mixtures but substituted OneTaq® Hot Start DNA polymerase for the OneTaq® One-step enzyme mix (i.e., lacking reverse transcriptase altogether). In this case, any signal represented the amplification of residual DNA remaining after DNase treatment. Reactions were carried out by treating each RT-PCR mixture at 48 °C for 30 min followed by PCR. Each PCR was performed using the following thermocycling steps: 30 s at 94 °C, 30 s at 60 °C, and 1 min at 68 °C for 15 cycles. Each product was loaded onto a 1% agarose gel pre-stained with SYBR-safe DNA dye (Invitrogen). The gel was electrophoresed and visualized as above.

Transmission electron microscopy (TEM)

TEM was performed as previously described25. Briefly, samples for TEM imaging were prepared in a concentration range of 0.5 nM to 5 nM. 12 μL of the sample was placed on glow-discharged carbon-coated 400 mesh copper TEM grids. After two minutes of incubation, the sample solution was removed using filter paper and replaced with 12 μL of freshly prepared uranyl formate negative staining solution. The stain was removed after 30 s, and the grids were air-dried. TEM images were acquired at 25,000 × magnification using a JEOL 1230 TEM (Peabody, Ma, USA) equipped with a Gatan Inc. 2 k × 2 k Ultrascan camera (Pleasanton, CA, USA)25.

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