HighYield T7 Enzyme Testkit

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 Enzyme Testkit is designed to produce large amounts of RNA via in vitro transcription. It contains three different T7 RNA Polymerase variants to search for the optimal match for a specific application:

1. HighYield T7 RNA Polymerase Mix:

• based on wildtype T7 RNA Polymerase (T7)
• commonly used variant

• based on a modified T7 RNA polymerase (T7 P&L)
• associated with decreased abortive transcription [1], increased 5' homogeneity of transcripts synthesized from A-initiating phi2.5 promoter [2], increased 5' incorporation efficiency of GTP analogs [3]

• based on a modified T7 RNA Polymerase (T7 PURE)
• reduced double-stranded RNA (dsRNA) formation and thus less RNA immunogenicity and reduced down-stream purification efforts for dsRNA contamination removal [4-7].

One kit contains sufficient reagents for 15 reactions with each HighYield RNA Polymerase Mix (20 μl reaction each, 7.5 mM each NTP).

Yields of a 20 μl reaction (1 μg T7 control template, 1.4 kb RNA transcript) which may however, vary depending on the template (promoter design, sequence length, secondary structure formation):

Content:
HighYield T7 RNA Polymerase Mix
1x 40 μl incl. RNase inhibitor and 50 % glycerol (v/v)

HighYield T7 P&L RNA Polymerase Mix
1x 40 μl incl. RNase inhibitor and 50 % glycerol (v/v)

HighYield T7 PURE RNA Polymerase Mix
1x 40 μl incl. RNase inhibitor and 50 % glycerol (v/v)

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)

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 resulting in ~1400 nt RNA transcript

T7 A-initiating control template (1.4 kbp)
1x 10 μl (200 ng/μl), 1.4 kbp PCR fragment plus T7 class II phi2.5 promotor (A-initiating) resulting in ~1400 nt RNA transcript

PCR-grade water
1x 1.2 ml

DTT
1x 150 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: Linearized plasmid DNA or PCR products containing a double-stranded T7 class II phi2.5 or class III phi6.5 promotor region upstream of the target sequence.

  Minimum T7 promotor sequences:

  T7 class III phi6.5 promotor
  5'-TAATACGACTCACTATAGNN…-3’
  Bold: First base incorporated into RNA, NN: ideally CG

  or

  T7 class II phi2.5 promotor
  5'-TAATACGACTCACTATTANN…-3'
  Bold: First base incorporated into RNA, NN: ideally GG
  

• 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/chloroform 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 please ensure that the DNA template encodes the required structural features e.g. 3’-UTR, 5’-UTR, correctly orientated target sequence and poly A-tail. Alternatively, polyA-tailing can post-transcriptionally be performed with Poly A polymerase.

In vitro Transcription protocol
The protocol is optimized for 0.5 μg - 1 μg DNA template (refer to "Important Notes" regarding template requirements).

• Place HighYield T7 or HighYield T7 P&L or HighYield T7 PURE 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 or HighYield T7 P&L or HighYield T7 PURE RNA Polymerase Mix, vortex and spin down briefly.
• Incubate for 2h at 37°C in the dark (e.g. PCR cycler). Depending on the RNA sequence 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: [8],[9].

RNA purification
Purification of RNA is required for certain applications such as RNA concentration mesurement. 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.

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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Selected References:
[1] Guillerez et al. (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance. Natl. Acad. Sci. U.S.A 102:5958.
[2] Salvail-Lacoste et al. (2018) Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags. RNA 19:1003.
[3] Lyon et al. (2018) A mT7 RNA Polymerase Mutant Enhances the Yield of 5'-Thienoguanosine-Initiated RNAs. ChemBioChem 19:142.
[4] Nelson et al. (2020) Impact of mRNA chemistry and manufacturing process on innate immune activation. J.L. Sci.Adv. 6:eaaz6893.
[5] Mu et al. (2018) An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res. 46 (10):5239.
[6] Karikó et al. (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res. 39 (21):e142.
[7] Baiersdörfer et al. (2019) A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA. Ther. Nucleic Acids 15:26.
[8] Wienert et al. (2018) In vitro transcribed guide RNAs trigger an innate immune response via RIG-I pathway. PLoS Biol. 16 (7): e2005840.
[9] Kim et al. (2018) CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 28 (3) :367.