Synthesis of differentially cytidine-modified (m)RNA with Cap 0 structure
Cat. No. | Amount | Price (EUR) | Buy / Note |
---|---|---|---|
RNT-138 | 1 kit | 345,00 | Add to Basket/Quote Add to Notepad |
For general laboratory use.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months after date of delivery
Description:
HighYield T7 mRNA Cytidine Modification Testkit is designed to produce large amounts of differentially cytidine-modified (m)RNA via in vitro transcription with T7 RNA polymerase. Included Cap analog ARCA allows additional co-transcriptional introduction of a Cap 0 moiety. The resulting 5’-capped (m)RNA can subsequently be used for microinjection, transfection or in vitro translation experiments.
Epigenetic internal mRNA modifications such as cytidine methylation or acetylation as well as 5'-capping have been shown to increase translation efficiency and to reduce immunogenicity [1]-[7] however, the optimal combination needs to be individually termined for each mRNA target.
Internal modifications are introduced by correspondingly labeled nucleotides. Anti-reverse Cap analog (ARCA, m7,3'-OGP3G) co-transcriptionally introduces a 7-methylguanosine moiety (m7G, Cap 0 structure) required for efficient translation and increased stability of eukaryotic mRNA. 3'-O-methylation of the m7G moiety allows incorporation in the correct (“anti-reverse”) orientation only resulting in a 100 % translatable capped (m)RNA population.
Nukleotide | Reference |
5-Methyl-CTP | [1]-[4] |
N4-Acetyl-CTP | [5],[6] |
ARCA | [3],[4] |
A 20 μl of ARCA-capping reaction without modified cytidines yields about 30-50 μg RNA after 30 min incubation (1 μg T7 G-initiating control template, 1.4 kb RNA transcript). Yields may however vary depending on the template (promotor design, sequence length, secondary structure formation))as well as on the combination and substitution rate of modified cytidines.
Content:
HighYield T7 RNA Polymerase Mix
incl. RNase inhibitor and 50 % glycerol (v/v)
2x 40 μl
HighYield T7 Reaction Buffer
1x 200 μl (10x), HEPES-based
ATP - Solution
1x 100 μl (100 mM)
GTP - Solution
1x 100 μl (100 mM)
CTP - Solution
1x 100 μl (100 mM)
UTP - Solution
1x 100 μl (100 mM)
ARCA - Solution
2x 10 μl (100 mM)
5-Methyl-CTP
1x 10 μl (100 mM)
N4-Acetyl-CTP
1x 10 μl (100 mM)
T7 G-initiating control template (1.4 kbp)
1x 10 μl (200 ng/μl), 1.4 kbp PCR fragment plus T7 class III phi6.5 promotor (G-initiating) resulting in ~1400 nt RNA transcript
PCR-grade water
1x 1.2 ml
DTT
1x 100 μl (100 mM)
To be provided by user
T7 Promotor-containing DNA template
RNA purification tools
RNAse-free DNAse I
Important Notes (Read before starting)
Prevention of RNAse contamination
Although a potent RNase Inhibitor is included, creating a RNAse-free work environment and maintaining RNAse-free solutions is critical for performing successful in vitro transcription reactions. We therefore recommend
Template requirements
ARCA-capping: Linearized plasmid DNA or PCR products containing a double-stranded G-initiating T7 class III phi6.5 promotor region upstream of the target sequence.
Minimum T7 promotor sequences:
T7 class III phi6.5 promotor (G-initiating)
5'-TAATACGACTCACTATAGNN…-3’
Bold: First base incorporated into RNA, NN: ideally CG
In vitro Transcription protocol
The general protocols are set up for 1 μg DNA template (refer to "Important Notes" regarding template requirements), a final NTP concentration of 7.5 mM, a final ARCA concentration of 6 mM and 100% substitution of CTP by a modified CTP (ARCA-capping / No-capping reaction).
ARCA-capping reaction:
Depending on the RNA sequence and final application, individual reaction optimization may improve product yield and biological function (e.g. modified CTP/CTP ratio, variation of ARCA:GTP ratio, variation of template amount, variation of incubation time). An optimal balance between reaction and capping efficiency is usually achieved by an ARCA:GTP ratio of 4:1 (approx. 80% capped RNA transcripts). The synthesis of RNA transcripts >/= 5000 nt may require higher GTP concentrations. Lowering the ARCA:GTP ratio (e.g. 2:1) lowers the capping efficiency but may significantly improve the yield of full-length transcripts.
Component | Volume | Final conc. |
PCR-grade water | X μl | |
HighYield T7 Reaction Buffer (10x) | 2 μl | 1x |
DTT (100 mM) | 2 μl | 10 mM |
ARCA (100 mM) | 1.2 μl | 6 mM |
GTP (100 mM) | 0.3 μl | 1.5 mM |
modified CTP (100 mM) | 1.5 μl | 7.5 mM |
UTP (100 mM) | 1.5 μl | 7.5 mM |
ATP (100 mM) | 1.5 μl | 7.5 mM |
Template DNA | X μl | 1 μg |
HighYield T7 RNA Polymerase Mix | 2 μl | |
Total volume | 20 μl |
No-capping reaction:
Depending on the RNA sequence and final application, individual reaction optimization may improve product yield and biological function (e.g. modified UTP/UTP ratio, variation of template amount, variation of incubation time). 5'-capping can be achieved posttranscriptionally with capping enzymes (alternativ approach to co-transcriptional with cap analogs).
Component | Volume | Final conc. |
PCR-grade water | X μl | |
HighYield T7 Reaction Buffer (10x) | 2 μl | 1x |
DTT (100 mM) | 2 μl | 10 mM |
GTP (100 mM) | 1.5 μl | 7.5 mM |
modified CTP (100 mM) | 1.5 μl | 7.5 mM |
UTP (100 mM) | 1.5 μl | 7.5 mM |
ATP (100 mM) | 1.5 μl | 7.5 mM |
Template DNA | X μl | 1 μg |
HighYield T7 RNA Polymerase Mix | 2 μl | |
Total volume | 20 μl |
Please note: Reagents for the following steps are not provided within this kit.
DNA template removal
Depending on the down-stream application, removal of template DNA might be required. We recommend a salt-resistant, high efficiency DNAase such as Turbo™DNAse (ThermoFisher). Follow the manufacturer instructions.
Removal of 5'-triphosphate groups
5'-ends of in vitro phosphorylated RNAs carry a triphosphate group that is known to trigger RIG-1 mediated innate immune response in mammalian cells[7,8]. Removal with phosphatases (e.g. CIP) before final purification is therefore recommended for RNA probes intended for transfection experiments. Please refer to the following references for more detailed information: [7],[8].
(m)RNA purification
Purification of (m)RNA is required prior to transfection or (m)RNA quantitation by absorbance measurement. Spin column purification will remove proteins, salts and unincorporated nucleotides. Please follow the manufacturer instructions and ensure that the columns match with product size and possess a sufficient binding capacity (e.g. RNA Clean & Concentrator™ columns (Zymo Research) or Monarch® RNA Cleanup kit (NEB)). Other RNA purification methods such as LiCl precipitation may work but have not been tested.
(m)RNA quantitation
RNA concentration can be determined by absorbance measurement at 260 nm (A260) according to the Law-of-Lambert-Beer (A260 = 1 corresponds to 40 μg/ml ssRNA).
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BIOZ Product Citations:
Selected References:
[1] Karikó et al.(2005) Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity23:165.
[2] Karikó et al.(2008) Incorporation of Pseudouridine into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability. Mol. Ther.16(11):1833.
[3] Kormann et al.(2011) Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nature Biotechnology29(2):154.
[4] Warren et al.(2011) Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. Cell Stem Cell7:618.
[5] Arango et al.(2018) Acetylation of Cytidine in mRNA Promotes Translation Efficiency. Cell175(7):1872.
[6] Sinclair et al.(2017) Profiling Cytidine Acetylation with Specific Affinity and Reactivity. ACS Chem. Neurosci.12(12):2922.
[7] Wienert et al. (2018) In vitro transcribed guide RNAs trigger an innate immune response via RIG-I pathway. PLoS Biol.16(7):e2005840.
[8] Kim et al. (2018) CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 28(3):367.