Most eukaryotic mRNAs require a 7-methyl guanosine (m7G) cap structure at the 5´-end and a poly(A) tail at the 3′-end for efficient translation. The HiScribe T7 mRNA Kit with CleanCap Reagent AG utilizes an optimized RNA synthesis formulation and trinucleotide cap analog technology for co-transcriptionally capping mRNAs that contain a natural Cap-1 structure in a single simplified reaction without compromising RNA yield. By using a DNA template with a T7 promoter sequence followed by an AG initiation sequence and an encoded poly(A) tail, mRNAs can be transcribed with a 5´-m7G Cap-1 structure that is polyadenylated, translationally competent and able to evade the cellular innate immune response.
The HiScribe T7 mRNA Kit with CleanCap Reagent AG is formatted with individual vials of NTPs and CleanCap Reagent AG to allow for partial or complete substitution of modified NTPs, with a total kit yield of 1.8 mg of mRNA. Cap-1 mRNA synthesized from this kit is suitable for many applications, including transfections, microinjections, in vitro translation, preclinical mRNA therapeutic mRNA studies as well as RNA structure and function analysis.
This kit contains sufficient reagents for 20 reactions (20 µl each). Each standard reaction yields ≥ 90 µg of RNA from 1 µg CLuc AG Control Template DNA. Each kit can yield ≥ 1.8 mg RNA.
Figure: 1: CleanCap Reagent AG
FIgure 2: Schematic of CleanCap Reagent AG Promoter and Initiation Sequence
Figure 3: CleanCap Reagent AG results in higher mRNA synthesis yield than the ARCA analog
All reactions were performed with 5 mM CTP, 5 mM UTP and 6 mM ATP. Standard IVT reactions included 5 mM GTP and no cap analog. ARCA reactions contained a 4:1 ratio of ARCA:GTP (4mM:1mM). IVT with CleanCap Reagent AG contained 5 mM GTP and 4 mM CleanCap Reagent AG and was performed according to recommended protocol (Standard mRNA Synthesis, HiScribe T7 mRNA Kit with CleanCap Reagent AG). Reactions were incubated for 2 hours at 37°C, purified and quantified by NanoDrop®.
This product is related to the following categories:
DNA Template:
The DNA template must be linear and contain the T7 RNA Polymerase Promoter with the correct orientation in relation to the target sequence to be transcribed, followed by an AG initiation sequence.
Modified NTPs:
Biotin-, Fluorescein-, Digoxigenin-, Aminoallyl-, Pseudouridine-5—Triphosphate, etc.
General:
Thermocycler, microcentrifuge, nuclease-free water, nuclease-free tubes and tips
Purification:
Phenol, chloroform, ethanol, 3 M Sodium Acetate, pH 5.2 or Ammonium Acetate, Monarch® RNA Cleanup Kit (500 µg; T2050), equipment and reagents for RNA quantitation
Gel Analysis:
Gels, running buffers, loading dye, nucleic acid ladders, gel apparatus, power supply
Verify that the sequence following the T7 promoter contains the AG initiating sequence.
Control Reaction
The CLuc AG control template is a linearized plasmid containing the Cypridina luciferase gene under the transcriptional control of the T7 promoter. The initiating sequence has been changed to an AG by site-directed mutagenesis to be compatible with CleanCap Reagent AG. The size of the run-off transcript is ~1.76 kb. The control reaction, following the standard reaction protocol, should yield > 90 µg of RNA in 2 hours at 37°C.
If the control reaction is not working, there may be technical issues with the reaction set up. Repeat the reaction following the protocol exactly (thawing specified reagents to room temperature, setting the reaction up at room temperature, and adding the components in the exact order listed in the manual. Take every precaution to avoid RNase contamination.
The control plasmid sequence can be found within the DNA Sequences and Maps Tool under the name “pCMV-CLuc2 AG Control Plasmid”. The CLuc AG control template is generated by linearizing the plasmid with the restriction enzyme XbaI.
Low Yield of Full-length RNA
If the transcription reaction generates full-length RNA but yields are significantly lower than expected, it is possible that contaminants in the DNA template are inhibiting the RNA Polymerase or the DNA template concentration may be incorrect or too low. Additional purification of the DNA template may be required. Phenol:chloroform extraction is recommended (see template DNA preparation section).
Low yield of Short Transcript
High yields of short transcripts (< 300 nts) are achieved by extending incubation time and increasing the amount of template. Incubation of reactions up to 16 hours (overnight) or using up to 2 μg of template DNA can help achieve maximum yield.
RNA Transcript Smearing on Denaturing Gel
If the RNA appears degraded (smeared) on a denaturing agarose or polyacrylamide gel, the DNA template may be contaminated with RNase. DNA templates contaminated with RNase can affect the length and yield of RNA synthesized (smear below the expected RNA length). If the DNA template is contaminated with RNase, we recommend performing phenol:chloroform extraction followed by ethanol precipitation (see template DNA preparation section) and dissolving the DNA in nuclease-free water.
RNA Transcript of Larger Size than Expected
If the RNA transcript appears larger than the expected size on a denaturing gel when compared to a single-stranded RNA ladder, the plasmid DNA that is used for the template may not be completely digested. Even if small amounts of undigested circular plasmid DNA is present, T7 RNA Polymerase can produce large amounts of long transcripts. Check the digestion of the plasmid for complete digestion compared to a sample of undigested plasmid. If undigested plasmid is present repeat the restriction digest.
Alternatively, larger sized bands may be observed when the RNA is not completely denatured due to the presence of strong secondary structure.
RNA Transcript of Smaller Size than Expected
If denaturing gel analysis indicates the presence of smaller bands than expected it is most likely due to premature termination by T7 RNA Polymerase. Sequences that resemble T7 RNA Polymerase termination signals will cause premature termination. For GC-rich templates, or templates with known strong secondary structure, incubation at 42°C may improve the yield of full-length transcript.
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