HighYield T7 mRNA Modification Testkit (ARCA)

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 Modification Testkit (ARCA) is designed to produce large amounts of differentially uridine-, cytidine- or adenosine-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 e.g. (pseudo)uridine methylation or thiolation as well as 5'-capping have been shown to increase translation efficiency and to reduce immunogenicity [1]-[10] 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, m^7,3'-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.

The kit contains sufficient reagents for 50 ARCA-capping reactions à 20 μl (6 mM ARCA, 1.5 mM GTP, 7.5 mM CTP, 7.5 mM UTP, 7.5 mM ATP). Amounts of each modified NTP are sufficient for 6 reactions à 20 μl each. An individual optimization of Cap Analog concentration or incorporation of modified nucleotides (e.g. Pseudo-UTP or N⁴-Acetyl-CTP) up to 100% substitution can easily be achieved with the single nucleotide format.

A 20 μl of ARCA-capping reaction yields about 30-50 μg RNA after 30 min incubation (1 μg T7 control template (G-initiating), 1.4 kb RNA transcript). Yields may however vary depending on the template (promotor design, sequence length, secondary structure formation)).

Content:
HighYield T7 RNA Polymerase Mix
incl. RNase inhibitor and 50 % glycerol (v/v)
3x 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
6x 10 μl (100 mM)

Pseudo-UTP
1x 10 μl (100 mM)

N¹-Methylpseudo-UTP
1x 10 μl (100 mM)

5-Methoxy-UTP
1x 10 μl (100 mM)

2-Thio-UTP
1x 10 μl (100 mM)

5-Methyl-CTP
1x 10 μl (100 mM)

N⁴-Acetyl-CTP
1x 10 μl (100 mM)

N⁶-Methyl-ATP
1x 10 μl (100 mM)

N¹-Methyl-ATP
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
2x 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

• to perform all reactions in sterile, RNAse-free tubes using sterile pipette tips.
• to wear gloves when handling samples containing RNA.
• to keep all components tightly sealed both during storage and reaction procedure.

Template requirements

• Template type:
  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
  
• Template quality: DNA template quality directly influences yield and quality of transcription reaction. Linearized plasmid DNA needs to be fully digested and to be free of contaminating RNase, protein and salts. We recommend selecting restriction enzymes that generate blunt ends or 5´-overhangs and purification by phenol/chloro¬form extraction. A PCR mixture can be used directly however, better yields will usually be obtained with purified PCR products (e.g. via silica-membrane based purification columns).
  
• mRNA production: For the production of functional mRNA, the DNA template needs to encode the following structural features e.g. 3’-UTR, 5’-UTR, correctly orientated target sequence and poly(A)-tail. Alternatively, poly (A)-tailing can post-transcriptionally be performed with Poly(A) polymerase.

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 a NTP (e.g. UTP) by a modified NTP (e.g. Pseudo-UTP). Please note: Substitution of ATP by N¹-Methyl-ATP is only feasible up to 50%.

ARCA-capping reaction:
Depending on the RNA sequence and final application, individual reaction optimization may improve product yield and biological function (e.g. modified NTP/NTP 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.

Exemplary reaction for modified UTPs (adjust accordingly for modified CTPs or ATPs):

• Place HighYield T7 RNA Polymerase Mix on ice.
• Thaw all remaining components at room temperature (RT), mix by voretexing and spin down briefly.
• Assemble all components at RT to a nuclease-free microtube (sterile pipette tips) in the following order:
• Mix PCR-grade water, HighYield T7 Reaction Buffer and DTT by voretexing and spin down briefly.
• Add nucleotide solutions and template DNA, vortex and spin down briefly.
• Add HighYield T7 RNA Polymerase Mix vortex and spin down briefly.
• Incubate for 2h at 37°C in the dark (e.g. PCR cycler). Individual optimization may increase product yield (0.5h–4h at 37°C).

No-capping reaction:
Depending on the RNA sequence and final application, individual reaction optimization may improve product yield and biological function (e.g. modified NTP/NTP ratio, variation of template amount, variation of incubation time). 5'-capping can be achieved post-transcriptionally with capping enzymes (alternativ approach to co-transcriptional capping with cap analogs).

Exemplary reaction for modified UTPs (adjust accordingly for modified CTPs or ATPs):

• Place HighYield T7 RNA Polymerase Mix on ice.
• Thaw all remaining components at room temperature (RT), mix by voretexing and spin down briefly.
• Assemble all components at RT to a nuclease-free microtube (sterile pipette tips) in the following order:
• Mix PCR-grade water, HighYield T7 Reaction Buffer and DTT by voretexing and spin down briefly.
• Add nucleotide solutions and template DNA, vortex and spin down briefly.
• Add HighYield T7 RNA Polymerase Mix vortex and spin down briefly.
• Incubate for 2h at 37°C in the dark (e.g. PCR cycler). Individual optimization may increase product yield (0.5h–4h at 37°C).

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^[1,2]. 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: [1],[2].

(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).

Related products:

• m₂^7,3'-OGP₃G (ARCA Cap Analog) - Solution, #NU-855
• Pseudo-UTP, #NU-1139
• N¹-Methylpseudo-UTP, #NU-890
• 5-Methoxy-UTP, #NU-972
• 2-Thio-UTP, #NU-1151
• 5-Methyl-CTP, #NU-1138
• N⁴-Acetyl-CTP, #NU-988
• N⁶-Methyl-ATP, #NU-1101
• N¹-Methyl-ATP, #NU-987

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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] Svitkin et al.(2017) N1-methyl-pseudouridine in mRNA enhances translation through eIF2alpha-dependent and independent mechanisms by increasing ribosome density. Nucleic Acid Res45(10):6023.
[6] Andies et al.(2015) N1-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J. Control. Release217:337.
[7] Li et al.(2016) Effects of Chemically Modified Messenger RNA on Protein Expression. Bioconjugate Chem.27:849.
[8] Arango et al.(2018) Acetylation of Cytidine in mRNA Promotes Translation Efficiency. Cell175(7):1872.
[9] Sinclair et al.(2017) Profiling Cytidine Acetylation with Specific Affinity and Reactivity. ACS Chem. Neurosci.12(12):2922.
[10] Dominissini et al.(2016) The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA. Nature530:441.
[11] Wienert et al. (2018) In vitro transcribed guide RNAs trigger an innate immune response via RIG-I pathway. PLoS Biol. 16 (7) :e2005840.
[12] Kim et al. (2018) CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 28 (3):367.