Editing Capped mRNA (section)

== Radioactive capped mRNA ==

5′ Cap analog (10 mM)<br/>In addition to “conventional” cap analogs, which can be incorporated in two ways to produce mixtures of<br/>differently capped mRNAs, Ambion sells an antireverse cap analog (ARCA), which ensures that the cap is in the<br/>right orientation.<br/>[α-32P]GTP (800 Ci/mmol; 10 μCi/μL) or [α-32P]UTP (800 Ci/mmol; 10 μCi/μL)<br/>We recommend labeling capped transcripts with [α-32P]GTP because the concentration of GTP is already lowered for capping to be more efficient.<br/>ATP, GTP, UTP, and CTP stocks (10 mM each, pH 7.0)<br/>It is possible to initiate transcription with dinucleotides, i.e., UpG or CpG, or even mononucleosides (i.e., G).<br/>Transcripts initiated in this way can be 5′ -end labeled without prior phosphatase treatment.<br/>Dithiothreitol (DTT) (1 M)<br/>DNase I (10 units/μL)<br/>EDTA (0.5 M)<br/>Ethanol (100%)<br/>GlycoBlue (15 μg/μL)<br/>Phenol:chloroform:isoamyl alcohol (PCA; 25:24:1)<br/>RNasin (RNase inhibitor; 40 units/μL)<br/>SDS extraction buffer &lt;R&gt;<br/>Sodium acetate (3 M, pH 5.2)<br/>T3 or T7 RNA polymerase (depends on template promoter; 50 units/μL)<br/>Template DNA (linearized plasmid or gel-purified PCR product)<br/>Transcription buffer for RNA polymerase (5×) &lt;R&gt;<br/>Equipment<br/>Dry ice<br/>Gel-purification equipment<br/>Incubator preset to 37 ̊C<br/>Micro Bio-Spin Columns with Bio-Gel P-6 in Tris buffer (Bio-Rad)<br/>Microcentrifuge<br/>Microcentrifuge tubes (1.5 mL, polypropylene, capped)

&nbsp;H2O to 50 μL 

Template DNA (linearized plasmid or PCR product) 2 μg 

0.04 μg/μL ATP (10 mM) 2.5 μL 

0.5 mM CTP (10 mM) 2.5 μL 

0.5 mM UTP (10 mM) 2.5 μL 

0.5 mM GTP (10 mM) 2.5 μL 

0.5 mM Transcription buffer for RNA polymerase (5×) 10 μL 

1× RNasin (40 units/μL) 1 μL 

0.8 unit/μL DTT (1 M) 1 μL 

20 mM T3 or T7 RNA polymerase (50 units/μL) 1 μL 1 unit/μL&nbsp;

To monitor the nonradioactive transcription reaction, remove 10 μL of the reaction and transfer to a separate tube. Add 1 μL of [α- 32P]GTP or UTP (800 Ci/mmol; 10 μCi/μL) and process in parallel with unlabeled GTP. For trace labeled, add 0.2–0.5 μCi of GTP or UTP (800 Ci/mmol). Remove and set aside<br/>1 μL from any labeled reaction to use for input quantitation.<br/>This reaction should yield&nbsp; 10–20 μg of RNA at 100 cpm/ng

For high-specific-activity RNA, combine the following in the order listed:<br/><br/>&nbsp;H2O to 50 μL Template DNA (linearized plasmid or PCR product) 

2 μg 0.04 μg/μL ATP (10 mM) 2.5 μL 

0.5 mM CTP (10 mM) 2.5 μL 

0.5 mM UTP (10 mM) 2.5 μL 

0.5 mM GTP (0.5 mM) 2.5 μL 

25 μM Transcription buffer for RNA polymerase (5×) 10 μL 

1× RNasin (40 units/μL) 1 μL 

0.8 unit/μL DTT (1 M) 1 μL 

20 mM [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 10 μL 

2 μCi/μL T3 or T7 RNA polymerase (depends on template promoter) (50 units/μL) 1 μL 1 unit/μL&nbsp;

If labeling with [α- 32P]UTP, use 2.5 μL of 10 mM GTP (instead of 0.5 mM) and 2.5 μL of 0.5 mM UTP (instead of 10 mM) and add 10 μL of [α- 32P]UTP in the above reaction instead of [ 32P]GTP. Remove and set aside 1 μL to use for input quantitation. This reaction will yield 3 μg of RNA at 50,000–100,000 cpm/ng.

For moderate-specific-activity RNA, combine the following in the order listed: 

&nbsp;H2O to 50 μL 

Template DNA (linearized plasmid or PCR product) 

2–4 μg 0/04–0.08 μg/μL ATP (10 mM) 2.5 μL 

0.5 mM CTP (10 mM) 2.5 μL 

0.5 mM UTP (10 mM) 2.5 μL 

0.5 mM GTP (0.5 mM) 5 μL

0.1 mM Transcription buffer for RNA polymerase (5×) 10 μL 

1× RNasin (40 units/μL) 1 μL 0.8 unit/μL DTT (1 M) 

1 μL 20 mM [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 1–4 μL 

1 unit/μL T3 or T7 RNA polymerase (depends on template promoter) (50 units/μL) 5 μL 1 μCi/μL

Adjust the amount of [α- P]GTP to get the desired specific activity. This reaction will yield RNA at 4000–8000 cpm/ng.

2. Incubate the reaction for 1–2 h at 37 ̊C. 3. Add 1 μL of 10 units/μL (for each 50 μL transcription reaction) DNase I and incubate for 15 min at 37 ̊C. 4. Add EDTA to 10 mM. 5. For labeled reactions, remove unincorporated nucleotides with a Micro Bio-Spin Column and then dilute to 200 μL with SDS extraction buffer. For unlabeled or trace-labeled reactions, dilute with an equal volume of SDS extraction buffer. Add 1/10 volume of 3 M sodium acetate (pH 5.2) to both labeled and unlabled reactions. 6. Extract with PCA (see Purification of RNA by SDS Solubilization and Phenol Extraction [Rio et al. 2010]). 7. Add 2 μL of 15 μg/μL GlycoBlue or glycogen to serve as carrier or to aid in resuspending the pellet. 8. Add 2.5 volumes of ethanol. Freeze on dry ice or in dry ice/ethanol bath ( 15 min). Thaw. Centrifuge at maximum speed in a microcentrifuge for 10 min. Carefully pipette off the supernatant and dispose off by an appropriate waste method.

9. Resuspend the pellets in 2 mM EDTA (2–5 μL for each 50 μL of transcription reaction for gel purification). If RNA is to be used directly, resuspend in H2O.

10. Gel-purify the RNA.

Check a small aliquot of longer transcripts (>600–700 nucleotides) on the gel for intactness. If the RNA is a single band with no trailing of radioactivity below, the transcripts can be used without gel purification.

Capping of In Vitro-Synthesized Transcripts For capping, mix the following reagents in the order listed: 

Template DNA (linearized plasmid or PCR product) 2–4 μg

0.04–0.08 μg/μL ATP (10 mM) 2.5 μL 

0.5 mM CTP (10 mM) 2.5 μL 

0.5 mM UTP (10 mM) 2.5 μL 

0.5 mM GTP (1 mM) 5 μL 

0.1 mM Transcription buffer for RNA polymerase (5×) 10 μL 

1× RNasin (40 units/mL) 1 μL 

0.8 unit/μL DTT (1 M) 

1 μL 20 mM Cap analog (see above) (40 mM) 

1.25 μL 1 mM T3 or T7 polymerase (50 units/μL) 

1 μL 1 unit/μL [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 5 μL 

1 μCi/μL H2O to 50 μL 



Substitute any cap analog, dinucleotide (NpG), or GMP for 7mGpppG at a final concentration of 1 mM. 2. Perform Steps 2–10 above.

Random Incorporation of Modified Nucleotides during In Vitro Transcription The following is a “typical” in vitro transcription reaction designed to obtain a capped, moderate-specific-activity, biotin- or 4-thio-U-substituted RNA; the modified uridine is incorporated at approximately every fourth uridine residue. See Discussion.

1. For substitution, mix the following reagents in the order listed: 

Template DNA (linearized plasmid or PCR product) 2–4 μg 

0.04–0.08 μg/μL ATP (10 mM) 2.5 μL 0.5 mM CTP (10 mM) 2.5 μL 

0.5 mM UTP (10 mM) 3.75 μL 

0.375 mM Biotin-UTP or 4-thio-UTP (1 mM) 

6.25 μL 0.125 mM GTP (1 mM) 5 μL 

0.1 mM Transcription buffer for RNA polymerase (5×) 10 μL 

1× RNasin (40 units/mL) 1 μL 

0.8 unit/μL DTT (1 M) 1 μL 

20 mM Cap analog (see above) (40 mM) 1.25 μL 

1 mM T3 or T7 polymerase (50 units/μL) 1 μL 

1 unit/μL [α-32P]GTP (800 Ci/mmol; 10 μCi/μL) 5 μL 

1 μCi/μL H2O to 50 μL 



2. Perform Steps 2–10 above.

Problem (Step 9): The RNA does not completely resuspend. Solution: Resuspend the RNA in a larger volume or dilute it and reprecipitate with ethanol. Very efficient transcriptions may produce a PPi precipitate in large amounts and interfere with solubility. This problem can be overcome by addition of pyrophosphatase.

Problem (Step 10): The RNA yield is low. Solution: Rerun the reaction, making sure that the mixture remains in solution while assembling the reaction. The reactions are assembled at room temperature in Step 1 to avoid precipitation. If the reactions containing high concentrations of cap and nucleotides form a precipitate, heat them for 5 min at 37 ̊C and briefly vortex before adding the enzyme.

Problem (Step 10): The RNA is degraded. Solution: Make sure that the template was nuclease-free. If necessary, after plasmid linearization, dilute 100 μL of digestion with 200 μL of SDS extraction buffer. Add proteinase K (100 μg/mL), digest for 15 min at 37 ̊C, add 1/10 volume 3 M sodium acetate (pH 5.2) and proteinase K, phenol-extract, and ethanol-precipitate. Make sure that all solutions are RNase-free.

These methods are designed to prepare RNAs by in vitro transcription. Before setting up an in vitro transcription reaction, it is necessary to have a clear idea of how much RNA is needed and at what specific activity. In addition, is the RNA to be capped or substituted? Capping is usually performed in conjunction with radiolabeling and/or with other internal modifications. The presence of a cap at the 5′ end of in vitro-synthesized transcripts can be useful in many situations, e.g., by preventing 5′ to 3′ nuclease activity or by promoting translation. “Caps” come in several forms: 7mGpppG, ApppG, and GpppG. 7mGpppG serves as a recognition factor for the translation initiation factor eIF-4E and thus is important when making RNAs that will be used for translation. The other analogs are primarily used to prevent nucleolytic attack. Capping during transcription involves the initiation of RNA chains with the cap (obviously, cap cannot be incorporated internally). The extent of capping is determined by competition between cap and GTP;

therefore, cap must be present in excess of GTP. We recommend that the concentration of cap be 10-fold that of GTP; under such conditions, 90% of the RNAs will be capped. Because of constraints on the concentrations of cap that can be achieved (1 mM is practical), it is necessary to reduce the concentration of GTP to 100 μM to achieve efficient capping.

Many modified nucleotides can be incorporated into in vitro-synthesized RNA, including, but not limited to, biotinylated UTP, 4-thio-UTP, bromo-UTP, iodo-UTP, and many adenosine analogs (with α-thiophosphates). Biotinylated RNAs are used in affinity selection experiments; 4-thio-, 5-bromo-, and 5-iodo-U-substituted RNAs are useful for cross-linking studies; and α-thio-based analogs are primarily used for modification–interference experiments. When designing in vitro transcription reactions that incorporate modified nucleotides, it is important to consider the level of incorporation desired. For example, in modification–interference approaches, transcripts should contain one or even fewer modifications per transcript, whereas in biotin applications, heavier substitution (several per transcript) is desirable. The degree of substitution is determined by competition between the unmodified and modified nucleotide just as in capping. Because some modified nucleotides are incorporated less efficiently than their unmodified counterparts, the degree of substitution may not correspond exactly to the ratio of modified to unmodified nucleotides present in the transcription reaction. Thus, it is sometimes necessary to titrate the level of the modified nucleotide to achieve the desired level of substitution. Oversubstitution with biotin can sometimes cause RNA to partition with the organic phase because of the hydrophobic spacer. Transcripts heavily substituted with biotinylated nucleotides should not be extracted with phenol.

SDS Extraction Buffer 

Reagent Quantity (for 500 mL) 

Final concentration Tris–HCl (1 M, pH 7.5) 

10 mL 20 mM EDTA (0.5 M, pH 8.0) 

1 mL 1 mM SDS (10% w/v) 

25 mL 0.5% (w/v) H2O 464 mL 



Transcription Buffer for RNA Polymerase (5×) 

Reagent Quantity (for 10 mL) Final concentration Tris–HCl (1 M, pH 8.0) 2 mL 200 mM MgCl2 (1 M) 400 μL 40 mM NaCl (5 M) 0.5 mL 250 mM Spermidine (100 mM) 1 mL 10 mM H2O 6.1 mL&nbsp;