Preparation of Circular RNA In Vitro Protocol

Experiment Summary

This protocol describes a simple and straightforward way to obtain single-stranded circular RNA sequences in vitro. Linear RNA that is phosphorylated at the 5' end is first prepared by a chemical or enzymatic method, then circularized using ligase. The function of the prepared circular RNA molecule, such as an ability to induce translation, can then be investigated.

Fig. 1 Schematic of the in vitro preparation of single-stranded (ss) circular RNA using ligase.

Materials and Reagents

A. Denaturing Polyacrylamide Gel Electrophoresis (PAGE) of RNA

Equipment for electrophoresis
0.5 M EDTA (pH 8.0)
10× TBE buffer
2× Formamide gel loading solution [80 (v/v) % formamide, 10 mM EDTA (pH 8.0), 0.02 (w/v) % xylene cyanole FF, 0.02 (w/v) % bromophenol blue]
2 × Formamide gel loading solution without dye [80 (v/v) % formamide, 10 mM EDTA (pH 8.0)]
40% acrylamide solution (acrylamide: bisacrylamide = 19: 1)
10% denaturing polyacrylamide gel solution [10% acrylamide (acrylamide: bisacrylamide = 19: 1), 7.5 M urea, 25 (v/v) % formamide, 1 × TBE]
Ammonium persulfate (APS), 30 (w/v) % solution in water
N, N, N', N'-Tetramethylethylenediamine (TEMED)
Stains-All gel staining solution [0.02 (w/v) % Stains-All in 50% N, N-dimethylformamide]
SYBR Green II Nucleic Acid Gel Stain
Low Range ssRNA Ladder
Millex LH filter
Amicon Ultra-4 Centrifugal Filters
Amicon

B. Chemical Synthesis of linear 5'-Phosphorylated RNA

Standard RNA synthesis reagents
Chemical Phosphorylation Reagent
Reagents required for automated DNA/RNA synthesis
33% methylamine in ethanol
40% aqueous methylamine
Millex LH filter
Tetrabutylammonium fluoride (TBAF), 1 M solution in tetrahydrofuran (THF)
1 M Tris-HCl (pH 7.4)
NAP-25 column
3 M NaOAc (pH 5.2)
20 mg/mL glycogen: add 1 μL of this solution per 1.5/2 mL
tube of alcohol-precipitation
Isopropyl alcohol
T4 DNA ligase
60 (w/v) % PEG6000
DNA oligomer(s) of 20-nt as a template for the ligation reaction

C. In Vitro Transcription of Linear 5'-Phosphory- Lated RNA

Plasmid DNA containing the RNA sequence
A set of PCR primers: substitute two 2'-deoxynucleotides at the 5'-end of the reverse primer with 2'-O-methylated nucleotides
PrimeSTAR HS DNA Polymerase
Equipment and reagents for agarose gel electrophoresis
Gel Extraction Kit
MEGAscript T7 Transcription Kit
750 mM guanosine 5'-monophosphate

D. Circularization of 5'-Phosphorylated Linear RNA Using Ligase

5'-phosphorylated linear RNA.
DNA oligomer of 20-nt as a template for the ligation reaction
T4 DNA ligase
60 (w/v) % PEG6000
RNase R

Procedure

A. Preparation of Denaturing Polyacrylamide Gel

To 40 mL of 10% denaturing polyacrylamide gel solution, add 120 μL of 30% APS, 40 μL of TEMED, and mix well. Cast the solution between the glass plates immediately. Place a comb between the plates and let the gel polymerize for at least 1 h.

B. Analysis and Purification of RNA by Denaturing Polyacrylamide Gelelectrophoresis (PAGE)

Add an equal volume of 2 × formamide gel loading solution to the RNA solution.
Heat the solution at 90 °C for 3 min. Load the solution onto a 5-10% denaturing polyacrylamide gel.
Run the gel at a constant 10-20 W for 1-2 h.
Stain the gel for analysis with Stains-All or SYBR Green II dye. If staining with SYBR Green II, visualize the RNA using a gel imaging system and UV excitation.
When purifying the RNA, wrap the gel in plastic film, place it on a TLC plate, and detect the RNA using a UV lamp. Mark the band with a marking pen, excise it using a razor blade, and transfer it into a 15 mL tube. Add 4 mL of water to the gel and crush it with a disposable serological pipet. Extract RNA by incubation at room temperature with shaking for 4 h to overnight. After a brief centrifugation, remove the supernatant and store it at 4 °C. Add another 4 mL of water to the crushed gel, and incubate it for several hours. Remove the supernatant and combine it with the first supernatant. Filter the solution with a Millex LH filter to remove small pieces of gel. Desalt and concentrate the solution using an Amicon Ultra-4 centrifugal device, according to the procedure provided by the manufacturer. Finally, precipitate the RNA from the concentrated/desalted solution using 3 M NaOAc (pH 5.2) and isopropyl alcohol.

C. Chemical Synthesis of linear 5'-Phosphorylated RNA

Divide the sequence of linear RNA into several, synthesizable lengths.
Synthesize the RNA lengths on an automated DNA/RNA synthesizer.
Deprotect the oligoribonucleotide.
Check the purity of synthesized RNA oligomers by denaturing PAGE.
Purify the RNA by preparative denaturing PAGE.
Ligate synthesized RNA fragments with ligase to a full-length sequence.

D. Synthesis of Precursor linear 5'phosphorylated RNA by In Vitro Transcription

Construct the transcription template DNA by PCR.
Carry out in vitro transcription.

E. Synthesis of Circular RNA by Ligation

Ligate 5'-phosphorylated linear RNA to form a circle.
Isolate/purify the circular RNA from the mixture described above by preparative denaturing PAGE.

F. Circularity Check of RNA Using RNase R.

Treat the possible circular RNA and linear precursor RNA with RNase R, a 3'→5' exoribonuclease, and compare its reactivity by denaturing PAGE analysis. Only linear RNA should be digested. The composition of a typical reaction mixture is: 40 ng/μL RNA, 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM MgCl₂, 2 U/μL RNase R. Incubate the mixture at 37 °C for 5-30 min.

* For research use only. Not intended for any clinical use.

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Preparation of Circular RNA In Vitro Protocol

Experiment Summary

Materials and Reagents

Procedure

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