Discriminating Immunorecognition Pathways Activated by RNA Nanostructures

Abstract

Nucleic acid nanoparticles (NANPs) composed of therapeutic DNA, RNA, or a hybrid of both are increasingly investigated for their targeted and tunable immunomodulatory properties. By taking advantage of the NANPs’ unique and relatively straightforward self-assembling behavior, nucleic acid sequences can be designed from the bottom-up and specifically tailored to induce certain immune responses in mammalian cells (Johnson et al., Nucleic Acids Res 48:11785–11798, 2020). Although not yet used in the clinic, functionalized NANPs display promising advantages to be included in therapeutic applications. By adjusting the chemical composition of a limited selection of NANPs all sharing the same physicochemical properties, it is demonstrated how substituting RNA strands for different chemical analogs can increase the thermodynamic and enzymatic stability of NANPs. Altering the composition of NANPs also determines the cellular mechanisms which initiate immune responses, therefore impacting the subcellular targeting and delivery efficiency.

1. Introduction

The key roles of nucleic acids pertain to the regulation of gene expression and protein synthesis. Identification and targeting of specific genes or cellular pathways are achieved through the rational design of therapeutic nucleic acids (TNAs) [2]. High tunability for optimizable immunomodulatory properties and overall biocompatibility present TNAs as an advantageous therapeutic tool, yet clinical use is rare due to low serum stability, bioavailability of the negatively charged oligonucleotides, and likelihood of off-target inflammatory responses [3]. NANPs that trigger distinct immune responses have potential as antiviral treatments or vaccine adjuvants, and investigations are therefore focused on balancing the optimal therapeutic effect of NANPs through structural and physicochemical modifications [4]. The immunostimulatory properties of NANPs are strongly correlated with their chemical compositions, allowing for immunostimulatory activity to be optimized by the fine-tuning of NANP chemical properties [5]. As well as immunostimulatory properties, the thermostability and serum stability, often noted as disadvantages of nucleic acid nanotechnology, may be further optimized [6]. Previous work has produced and characterized a panel of NANPs composed of DNA, RNA, and 2′F-modified oligonucleotides [7, 8]. Construction of NANPs with the same sequences, connectivity, size, shape, and charge, with different chemical compositions, displays variation in physicochemical properties and, therefore, varies in immunostimulatory potential. For instance, the incorporation of 2′F-mofidied strands increases the melting temperature and the serum stability of both DNA and RNA NANPs [9]. While NANP bioavailability remains a challenge given the negative charge, complexing NANPs to lipid-based carriers enhances subcellular targeting and internalization. The precise design and chemical composition of NANPs can be applied to stimulate specific immune recognition by innate pattern recognition receptors in order to promote distinct and desirable immune responses [10].

2. Materials and Equipment

2.1. NANPs Synthesis

1. Synthetic oligonucleotides with 2′F-modified pyrimidines.

2. DNA strands with 5′-end Cy-3 labels.

3. Thermal cycler.

4. Freezer (−20 °C).

5. Heat block (100 °C).

6. RNA transcription kit.

7. Denaturing 8 M urea 18% polyacrylamide gel.

   1. Elution buffer: Tris-HCl, 89 mM pH 8.0, 0.3 M NaOAc, 0.1 mM EDTA.
   2. Ethanol precipitation: 2.5× volume of 100% EtOH and 1/10 volume of 3 M NaOAc.

8. Speed vacuum.

9. Double-deionized water.

10. Assembly buffer: Tris-borate buffer, 89 mM pH 8.3, 2 mM Mg (OAc)₂, 50 mM KCl.

11. Non-denaturing (native) polyacrylamide gel electrophoresis (native-PAGE).

12. Atomic force microscope.

2.2. UV-Melt

1. NANPs at 0.1–0.2 μM, volume of 100 μL in assembly buffer.

2. UV-melting cells: microcuvette cells, 10 mm path length.

3. PTFE stopper.

4. Spectrophotometer.

2.3. Dynamic Light Scattering (DLS)

1. DLS cells, microcuvette.

2. DLS instrument: Zetasizer nano-ZS.

2.4. Fetal Bovine Serum Stability Assay

1. NANPs at 1 μM in assembly buffer.

2. 20% FBS solution.

3. 3% agarose gel.

4. Image software.

2.5. Source and Propagation of Cell Lines

1. Immortalized primary human microglia (hμglia) cells (gift from Dr. Jonathan Karn, Case Western Reserve University).

2. THP monocyte-like cell line THP-1-Dual cells.

3. Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine solution (FBS) and penicillin/streptomycin (100 U/mL–100 g/mL).

4. Cell media: RPMI 1640, 2 mM glutamine, 25 mM HEPES, 10% heat-inactivated FBS, 100 g/mL Normocin, penicillin/streptomycin (100 U/mL and 100 g/mL).

5. QUANTI-Blue; secreted embryonic alkaline phosphatase (SEAP) detection reagent.

6. QUANT-Luc; luciferase detection reagent.

7. HEK hTLR reporter cell lines: HEK-Blue^™ hTLR cells overexpress the human TLR3, 7, or 9 genes and inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene.

8. Cell culture medium HEK-Blue detection.

2.6. Reporter Cell Lines

1. THP1-Dual reporter cell line.

2. HEK-Blue reporter cell line (HEK-Lucia RIG-I cells).

3. 96-well plate.

4. Lipofectamine 2000 (L2K).

2.7. Transfection of Microglia

1. L2K.

2. DOTAP (MilliporeSigma).

3. DMEM supplemented with 5% FBS.

4. Cell media supplemented with 100 U/mL penicillin-100 g/mL streptomycin.

2.8. siRNA Knockdown

1. Control siRNA (silencer select negative control number 1 siRNA).

2. siRNA targeting RIG-I (RIGI) (Thermo Fisher Scientific assay identification number s223615).

3. siRNA targeting RNA polymerase III subunit A (Thermo Fisher Scientific assay identification number s21945).

4. RNAiMAX.

2.9. Quantification of Cytokines in Cell Supernatants

1. Rat anti-human IL-6 capture antibody.

2. Biotinylated rat anti-human IL-6 detection antibody.

3. Polyclonal rabbit antihuman IFN-β capture antibody.

4. Biotinylated polyclonal rabbit anti-human IFN-β detection antibody.

5. Streptavidin–horseradish peroxidase (HRP).

6. Tetramethylbenzidine substrate.

7. H₂SO₄.

8. Recombinant cytokines for IL-6 (BD Pharmingen).

9. Recombinant cytokines for IFN-β (Abcam).

2.10. Immunoblot Analysis

1. Rabbit polyclonal antibody against RIG-I.

2. Rabbit monoclonal antibody against RNA polymerase III subunit A.

3. Rabbit monoclonal antibody against GAPDH.

4. Rabbit monoclonal antibody against histone 3.

5. Rabbit monoclonal antibody against COX IV.

6. HRP-conjugated secondary anti-rabbit antibody.

7. WesternBright ECL kit.

8. Mouse monoclonal antibody against β-actin.

3. Methods

3.1. Synthesis of NANPs

1. DNA template strands are amplified via PCR. From these DNA strands, the 2′F-modified RNA strands to be prepared using 2′F in vitro T7 RNA transcription kit, followed by purification with an 8 M urea 15% PAGE.

2. Use short-wavelength UV shadowing to visualize the RNA bands and excise the individual bands using a scalpel. Place each individual gel piece in elution buffer for 4 h at 37 °C.

3. Allow for ethanol precipitation overnight using 2.5× volume of 100% EtOH and 1/10 volume of 3 M NaOAc.

4. Centrifuge at 16,500× g for 30 min to pellet the precipitate, followed by a wash with 80% EtOH and drying in a speed vacuum.

5. Use double-deionized water to rehydrate the 2′F-RNA pellet and store at −20 °C.

6. The RNA, DNA, and fluorinated NANPs are assembled in assembly buffer by mixing corresponding oligonucleotide strands at 1 μM and heating to 100 °C followed by slow cooling at a rate of 1 °C/min from 90 °C to 4 °C in a PCR thermal controller.

7. Evaluate the purity of each synthesized batch of NANPs by means of AFM imaging and native-PAGE.

3.2. UV-Melt Experiments

1. The assembled NANPs, at concentration of 0.1–0.2 μM and a volume of 100 μL in assembly buffer, are degassed in a speed vacuum for 5 min.

2. Place the 100 μL into UV-melting cells equipped with a PTFE stopper. Use an Agilent spectrophotometer at 260 nm to measure melting profiles over a temperature range of 20–100 °C at a ramp rate of 0.1 °C/min. Each UV-melt assay is repeated at least three times.

3. Fit the absorbance data using the nonlinear dose response function of Origin Pro.

3.3. DLS

1. The assembled NANPs, at concentration of 1 μM and a volume of 100 μL in assembly buffer, are filtered through 50 kDa Ultracel-50 regenerated cellulose membrane at 12,000× g for 2.5 min.

2. Transfer the 100 μL to DLS micro-cuvette cells, and analyze using a Zetasizer nano-ZS instrument at 25°.

3.4. FBS Stability Assay

1. Incubate the assembled NANPs, at a concentration of 1 μM in assembly buffer, at pH 8.0 in 20% FBS solution.

2. At time intervals of 1 min to 12 h, take aliquots, freeze them, and later analyze the retention of NANP structures by means of a 3% agarose gel.

3. Incubate corresponding NANPs in buffer for 12 h at 37 °C without FBS as a control.

4. Quantify the remaining fraction of NANPs from the gel image using ImageJ software.

5. Use Origin Pro software to plot the fraction percentage as a function of incubation time.

3.5. Cell Line Propagation

1. Primary hμglia cells were transformed with lentiviral vectors expressing hTERT and SV40 T antigen. This cell line was a generous gift from Dr. Jonathan Karn, at Case Western Reserve University (see Note 1).

2. Maintain the hμglia cell line in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS and penicillin/streptomycin (100 U/mL–100 μg/mL) to maintain the cells.

3. Maintain the THP-1-Dual reporter cells according to supplier guidelines in RPMI 1640, 2 mM glutamine, 25 mM HEPES, 10% heat-inactivated FBS, 100 μg/mL Normocin, penicillin/streptomycin (100 U/mL and 100 μg/mL) (see Note 2).

4. Use QUANTI-Blue to measure reporter proteins in the cells, along with QUANTI-Luc reagent to detect luciferase activity.

3.6. Reporter Cell Lines

1. Seed HEK-Blue and THP1-Dual reporter cell lines 4 × 10⁵ cells per well in a 96-well plate. Allow cells to adhere overnight or use at once.

2. Mix and incubate NANPs and L2K for 30 min.

3. Transfect the reporter cells with 5 nM NANP/L2K complexes.

4. Incubate cells along with the transfection reaction for 24 h.

5. Add 20 μL of supernatant from the THP1-Dual HEK-Blue hTLR3,7,9 cells to 180 μL of QUANTI-Blue in a 96-well plate.

6. Incubate mixtures at 37 °C for 3 h and read resulting absorbance at 620 nm.

7. Mix 20 μL of the supernatant from the THP1-Dual cells and HEK-Lucia RIG-I cells with 50 μL of QUANTI-Luc reagent in a black-walled 96-well plate, followed by an immediate luminescence measurement (see Figs. 1 and 2).

Fig. 1.

All RNA triangular NANPs and triangular NANPs with RNA centers and DNA sides stimulate hTLR7. (Reproduced from Ref. [1])

Fig. 2.

All RNA triangles, triangles with RNA center and DNA sides, triangles with RNA center and 2′F U/C modified RNA sides, and triangles with DNA center and 2′F U/C modified RNA sides stimulate RIG-I. (Reproduced from Ref. [1])

3.7. Microglia Transfection

1. Use L2K or DOTAP to transfect the hμglia cell line.

2. Incubate NANPs for 30 min with either L2K or DOTAP followed by transfection of hμglia using NANPs at final concentration of 5 nM for 4 h in DMEM with 5% FBS.

3. Replace cell culture media with media supplemented with 100 U/mL penicillin-100 μg/mL streptomycin.

4. Collect the cell supernatants for analysis at indicated time points.

3.8. siRNA Knockdown

1. Transfect hμglia cells with 5 nM of either negative control siRNA, siRNA targeting RIG-I, or siRNA targeting RNA polymerase III subunit A, using RNAiMAX according to the manufacturer’s guidelines.

2. Incubate the cells for 48 h prior to transfection with NANPs, and collect cell lysates and supernatants to evaluate at indicated time points.

3.9. Quantification of Cytokines in Cell Supernatants

1. Perform specific capture ELISA to quantify human IL-6 and IFN-β production.

2. Use rat anti-human IL-6 capture antibody (2 μg/mL in 0.1 M Sodium Bicarbonate Buffer) or polyclonal rabbit anti-human IFN-β capture antibody (0.25 μg/mL in 33.5 mM Sodium Carbonate 0.1 M Sodium Bicarbonate Buffer) to coat high binding 96-well ELISA plates overnight at 4 °C.

3. Wash wells three times with 1X Phosphate Buffered Saline-Tween 20.

4. Block all wells in 1% BSA 1X Phosphate Buffered Saline for 1 h at room temperature.

5. Wash wells three times with 1X Phosphate Buffered Saline-Tween 20.

6. Load either 100 μL of experimental samples or recombinant cytokines to generate a standard curve for IL-6 and/or IFN-β, and incubate for 2 h at room temperature or overnight at 4 °C.

7. Wash wells three times with 1X Phosphate Buffered Saline-Tween 20.

8. Incubate with either biotinylated rat anti-human IL-6 detection antibody (2 μg/mL in 1% BSA 1X Phosphate Buffered Saline) or a biotinylated polyclonal rabbit anti-human IFN-β detection antibody for IFN-β capture ELISAs (0.25 μg/mL in 1% BSA 1X Phosphate Buffered Saline) for 2 h at room temperature.

9. Wash wells three times with 1X Phosphate Buffered Saline-Tween 20.

10. Incubate 20 min at room temperature in the dark with Streptavidin–horseradish peroxidase (HRP) (dilute according to manufacture guidelines in 1% BSA 1X Phosphate Buffered Saline).

11. Detect bound antibody with tetramethylbenzidine substrate.

12. Stop the reaction with H₂SO₄.

13. Measure the absorbance at 450 nm.

14. Determine the cytokine concentration in cell supernatants by extrapolation of absorbance to the standard curve (see Note 3 and Fig. 3).

Fig. 3.

NANPs modified with 2′F stimulate cytokine production. (Reproduced from Ref. [1])

3.10. Immunoblot Analysis

1. Evaluate cell lysates for the expression of RIG-I and RNA polymerase III subunit A by immunoblot analyses.

2. Samples were run on either 10% or 12% PAGE.

3. Following transfer block blots in either a 5% milk or 5% BSA 1X Tris Buffered Saline-Tween 20 for 1 h at room temperature according to the antibody recommendations.

4. Incubate blots overnight at 4 °C with rabbit polyclonal antibody against RIG-I, a rabbit monoclonal antibody against RNA polymerase III subunit A, a rabbit monoclonal antibody against GAPDH, a rabbit monoclonal antibody against histone, or a rabbit monoclonal antibody against COX.

5. Wash blots with 1X Tris Buffered Saline-Tween 20, and then incubate blots with HRP-conjugated secondary antibody (select appropriate secondary antibody for each given primary antibody).

6. Detect bound antibody with the WesternBright ECL kit.

7. To assess total protein loading, deactivate or use a stripping buffer prior to reprobing immunoblots with a mouse monoclonal antibody against β-actin.

8. Western blots should be conducted using samples from a minimum of three biological repeats (see Fig. 4).

Fig. 4.

NANPs with DNA center and 2′F U/C modified RNA sides stimulate RNA polymerase III. (Reproduced from Ref. [1])

4. Notes

1. Immortalized primary human microglia and the THP monocyte-like cell line were selected as they are models for tissue-specific and circulatory immune cells, respectively. These cells are understood to be crucial for inflammation in response to pathogen-related molecular patterns, often recognizable with nucleic acids.

2. Two inducible reporter constructs were integrated into the THP-1 monocyte cell line to allow observations of IRF and NF-κB pathways.

3. Dilute the recombinant cytokines for IL-6 or IFN-β to acquire standard curves.