HighYield T7 Cap Analog Testkit

For in vitro use only!

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months after date of delivery

Description:
HighYield T7 Cap Analog Kit is designed to produce large amounts of Cap 0- or Cap 1-modified (m)RNA via in vitro transcription with T7 RNA polymerase. The resulting 5’-capped (m)RNA can subsequently be used for microinjection, transfection or in vitro translation experiments.

Anti-reverse cap analog (ARCA, m₂^7,3'-OGP₃G) co-transcriptionally introduces a 7-methylguanosine moiety (m⁷G, Cap 0 structure) required for efficient translation and increased stability of eukaryotic mRNA. Cap 1 AG (3‘-OMe) (m₂^7,3'-OGP₃(2'OMe)ApG) additionally introduces a 2’-ribose methylation of the first nucleotide downstream of m⁷G. The resulting Cap 1 structure is frequently found in higher eukaryotes and associated with a reduced immunogenicity of correspondingly modified mRNAs. 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 10 Cap 1 AG (3’-OMe)-capping reactions à 20 μl (5 mM Cap 1 AG (3’-OMe), 5 mM GTP, 5 mM CTP, 5 mM UTP, 5 mM ATP) and 5 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). 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 Cap 1 AG (3’-OMe)-capping reaction yields about 80-100 μg RNA after 30 min incubation (1 μg T7 control template
(A-initiating), 1.4 kb RNA transcript). 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)
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)

m₂^7,3'-OGP₃(2'OMe)ApG - Solution
1x 10 μl (100 mM)

m₂^7,3'-OGP₃G (ARCA Cap Analog) - Solution
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

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 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:

  Cap 1 AG (3‘-OMe)-capping: Linearized plasmid DNA or PCR products containing a double-stranded A-initiating T7 class II phi2.5 promotor region upstream of the target sequence.
  Minimum T7 promotor sequences:
  T7 class II phi2.5 promotor (A-initiating)
  5'-TAATACGACTCACTATTANN…-3’
  5'-TAATACGACTCACTATAANN…-3’
  Bold: First base incorporated into RNA, NN: ideally GG

  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 and Cap 1 AG (3'-OMe) (m₂^7,3'-OGP₃(2'OMe)ApG) concentration of 5 mM (Cap 1 AG (3'-OMe)-capping reaction) or a final NTP and ARCA concentration of 7.5 mM (ARCA-capping / No-capping reaction).

Cap 1 AG (3'-OMe)-capping reaction:
Depending on the RNA sequence and final application, individual reaction optimization may improve product yield and biological function (e.g. variation of template amount, variation of incubation time). An optimal balance between reaction and capping efficiency is usually achieved by a NTP and Cap 1 AG (3'-OMe)(m₂^7,3'-OGP₃(2'OMe)ApG) concentration of 5 mM (>90% capped RNA transcripts).

• 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).

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

• 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 UTP/UTP ratio, variation of template amount, variation of incubation time). 5'-capping can be achieved post-transcriptional capping with capping enzymes (alternativ approach to co-transcriptional ARCA- or Cap 1 AG (3'-OMe)-capping).

• 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^[8,9]. 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].

(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), #NU-855
• m₂^7,3'-OGP₃(2'OMe)ApG (Cap Analog), #NU-248

Selected References:
[1] Wienert et al. (2018) In vitro transcribed guide RNAs trigger an innate immune response via RIG-I pathway. PLoS Biol. 16 (7) :e2005840.
[2] Kim et al. (2018) CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 28 (3):367.