Are the Requirements for Using CleanCap mRNA Different Than ARCA mRNA?

The requirements for using CleanCap mRNA are no different than using ARCA mRNA. You can use your standard electroporation or transfection conditions. There are several advantages with using a CleanCap mRNA. The translational efficiency far exceeds that of a standard ARCA capped mRNA. You may want to re-purpose your solar eclipse glasses when viewing your cells after EGFP translation!

And just like with ARCA, you can submit your open reading frame to TriLink for custom mRNA transcription and we will do the rest!

I’ve identified the target sequence for a CRISPR Cas9 gene editing experiment. What components do I need?

To attain unmatched consistency between your CRISPR experimental replicates use TriLink’s CleanCap Cas9 mRNA and high quality sgRNA. sgRNA, also known as Single Guide RNA, a chimeric RNA composed of crRNA and tracrRNA, connected by a short RNA linker. The purpose of sgRNAs is to bind to Cas9 and direct the complex to a specific genomic location. Other technologies exists as a 2-part crRNA:tracrRNA guide RNAs.

TriLink’s sgRNA offers several advantages over 2-part guide RNAs. With a sgRNA there is no obligatory annealing step in vitro. This annealing step can be time consuming and inefficient. Especially when modified with 2’OMethyl and Phosphothioates, sgRNA is more stable to degradation by intracellular exonucleases (Porteus, Nature, June 2015). With enhanced stability, you will achieve better gene editing.

TriLink’s CleanCap Cas9 mRNA enables rapid gene expression and eliminates the risk of insertional mutagenesis that can be associated with DNA plasmid approach. The benefits of DNA-free, mRNA-based CRISPR over plasmid delivery include no danger of unintended DNA insertion, reduced toxicity, better on-target efficiency, and improved specificity. TriLink’s CleanCap Cas9 mRNA offers superior translational efficiency than conventional Cas9 mRNA. For maximal expression in cells or target organs, transfected mRNAs must avoid detection by pattern recognition receptors (PRRs) that evolved to sense improperly capped RNAs and double stranded RNA. PRR activation leads to cytokine production, translational arrest and cell toxicity or death. CleanCap mRNA is highly efficiently capped with a natural Cap1 structure and sequence engineered to improve activity.

Materials Required:

  • Synthetic guide RNA (sgRNA)
  • CleanCap Cas9 mRNA (modified L-7206 or wild type L-7606)
  • Tissue culture plates
  • Microcentrifuge Tubes
  • Cell Counter
  • Normal Growth Medium
  • Preferred Transfection Reagent (Lipofectamine, Nucleofection, TransIT, etc.)

What are your recommended conditions for running a gel for mRNA or long RNA transcripts?

The conditions we recommend for running a gel are:
We run a 1% agarose gel. Depending on the size of the gel/number of wells, we load 250 ng to 1 ug of mRNA. We dilute mRNA to 0.2 ug/uL, then add equal volume of NorthernMax Gly sample loading dye.

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For making a 1% agarose gel, we would use 50 mL of gel and 0.5 grams of agarose. Microwave the agarose until melted and allow to cool for ~10 min. Add 0.5 uL of ethidium bromide. Swirl gently to mix. Pour gel.

Which dyes are available and are there specific dyes that are most compatible with active translation? Is there one specific nucleotide that can accommodate the dye better than others with respect to translation?

Bulky dyes may block the ribosome during translation and we have seen lower translational efficiencies with more substitutions. We recommend not to fully substitute, but only substitute 10-50%. For our catalog products, we use a 25% substitution of the dye-labeled nucleotide. Please contact us for any additional information.

How much mRNA is needed to transfect cell cultures in 24-well plates?

There are typically two avenues for transfection of mRNA depending on cell type:

1. For adherent cells, use a transfection reagent such as MessengerMax (Invitrogen), mRNA TransIT (Mirus) or mRNA-In (MTI-GlobalStem).

a. For a 24-well plate format, use a ratio of 500 ng mRNA:1 ul transfection reagent in a total volume of 50 ul complexed mRNA/lipid per well. This is a good starting point and is described in this protocol. Diluting your working stock of mRNA down to 100 ng/ul works well to establish manageable volumes for mastermixes.

b. We’ve only used TransIT and mRNA-In in-house – from our experience these reagents give a dose response of FLuc activity from between 100 ng to 400 ng. Therefore, a minimum starting amount of 100 ng is sufficient. Use 100 ng mRNA: 1ul reagent in a complexed final volume of 50 ul per well.

c. Ratios for other plate formats need to be optimized according to the manufacturer’s instructions.

2. For cells in suspension, such as CD34+ cells, electroporation has traditionally been the mode of mRNA delivery. However, transfection reagents have advanced and some are able to transfect cells in suspension reliably, such as the transfection reagent mRNA-In (MTI-GlobalStem).

a. For electroporation, an instrument from Lonza is recommended based on advice from a trusted collaborator.

b. Use 1-15 ug of mRNA per million cells in a 100 ul volume.

What protocol should I use for labeling Aminoallyl FLuc mRNA?

Suggested protocol for labeling Aminoallyl FLuc mRNA

This protocol is compatible for use with most water-soluble NHS esters*. If using a non-water soluble NHS-ester, different processing will be required. Precipitation in alcohol, such as isopropanol or ethanol, should be considered to remove excess NHS ester and any hydrolysis products. Multiple precipitations may be required.

*This protocol has been tested at small scales (100 ug) using Aminoallyl FLuc mRNA and Cyanine 5. Scales larger than 10 mg may require modifications to the protocol. The protocol has been designed to minimize mRNA degradation during the labeling process.

Salt exchange of Aminoallyl FLuc mRNA
1. Concentrate desired amount of Aminoallyl FLuc mRNA with 3K centrifugal filter. Reduce volume to ~0.25X of original.
2. Add 100 mM NaHCO3 to achieve total volume of 1.25X of original.
3. Repeat steps 1-2 at least three times. Add enough 100 mM NaHCO3 at final step to achieve 1X original volume of product. Concentration should be at ~ 1 mg/mL.

Labeling Reaction
4. Add 1X volume 100 mM NaHCO3 in H20. Mix gently by inverting several times.
5. Add 2X volume 2 mM NHS ester solution in DMSO. Mix gently by inverting several times.
6. Incubate 90 min at room temperature. If label is fluorescent, make sure to shield from light.
7. Add ~0. 35X volume 4M hydroxylamine in H20. Mix gently by inverting several times.
8. Incubate in dark for 15 min at room temperature. If label is fluorescent, make sure to shield from light.

Processing
9. Concentrate ~3X with 10K centrifugal filter device.
10. Add back 3X volume H2O.
11. Repeat steps 9-10 at least 6 times.

We recently purchased eGFP mRNAs (L-6101 and L-6402) as controls for mRNA delivery experiments. I have transfected HEK293 and DU145 cells using lipofectamine 3000 and PEI (5:1 weight ratio) and am unable to see GFP expression (by FACS) after 7 or 24 hours. By FACS (looking at cy5) the delivery looks good. I have tried both the labeled (L-6402) and unlabeled (L-6101) side by side and transfected 100 or 200 ng/well (96-well plate) into cells at about 50% confluency. Control transfections using these cell lines with a CMV-eGFP plasmid produce almost off-scale GFP expression when measured by FACS. Is there something wrong with my protocol and what is the timing and level of GFP expression I should expect in comparison to a plasmid transfection?

Dear Scott,

Thank you for your inquiry. I would suggest checking for mRNA degradation. Make sure you are using serum free reagents (ie Optimem) that are rigorously RNAse free. We use special pipets for mRNA and for example do not do minipreps or maxipreps with these pipets. RNasezap can be used to clean work surfaces and pipets. FACS signal from the Cy labeled RNA does not ensure that there was good delivery since the RNA could simply be trapped in an endosome. This experiment should work well in the 293 cells, we have no experience with the other cell line. Additionally, you make want to experiment with the timing. Though I would expect to see EGFP expression at your indicated time points, many factors influence expression and half-life. We and others typically see peak expression between 12-18 hours.

Regards,
Brea

What concentrations of mRNA are considered high,100ng/ul? Which buffer should I use to store mRNA? My mRNA activity still drops after 3 months at -80C and stored at 100ng/ul. I’ll appreciate any help-Marcia

Dear Marcia,

In general, 100 ng/uL is not considered a high concentration. We supply our mRNA at 1 mg/mL in 10 mM Tris-HCl, pH 7.5. That said, the mRNA sequence and structure can dictate its solubility. We recommend heating the mRNA for 15 min at 37°C to improve solubility. Long incubations at elevated temperatures should be avoided.

In regards to the diminishing activity of your mRNA, have you checked for degradation?  In addition to the solubility, your issues with mRNA activity may also result from RNA degradation over time. To combat degradation you should use RNase-free reagents and materials and use proper technique. Additionally, we suggest that you aliquot your RNA to limit freeze/thaw cycles.  A higher concentration may also improve stability however  could exacerbate your issues with solubility.

Please let us know if we can help you further.

Regards,

Brea

What are the benefits of using mRNA over plasmid or viral-based approaches for vaccine and immunotherapy development?

mRNA offers several advantages over traditional plasmid and viral-based approaches:

  1. mRNA boasts a superior safety profile. As a transient carrier of genetic information, it is metabolized naturally and poses little to no risk of genomic integration. Additionally, no inactivated viruses or pathogens are needed.
  2. mRNA serves the dual purpose of expressing the desired antigen as well as acting as an adjuvant.
  3. mRNA triggers a more diverse immune response. Because the mRNA encoded epitopes are intracellular, they are recognized by the immune system in an MHC class-independent manner.
  4. mRNA can more readily transfect difficult-to-transfect cell types because it functions in the cytoplasm. DNA vaccines can be limited by lack of access to the nucleus.
  5. mRNA manufacturing is easily scalable. Because mRNA transcription is carried out completely in vitro, to hundreds of millions of vaccine doses with a lead time of as little as a few weeks. This allows for rapid deployment of a new antigen during pandemics.
  6. mRNA is easily customizable. The ease of manufacturing makes it a viable option for personalized treatments.

How do I concentrate mRNA?

mRNA can be concentrated using an Amicon® Ultra 30 kDa or 100 kDa size exclusion filter. These are available from EMD Millipore in various sizes. Simply add the mRNA to the filter and spin for a short time (~3-5 min) at the recommended speed. mRNA will be retained in the top chamber. Spin in brief rounds of centrifugation until the desired concentration is reached.  Note that over-concentrating the mRNA could lead to precipitation. Carefully remove the mRNA from the top chamber using a pipette. Calculate the actual concentration of the mRNA using a spectrophotometer or a Nanodrop™ device.

 

What base modifications can be incorporated into my custom mRNA or long RNA?

TriLink offers a variety of modified rNTPs suitable for in vitro transcription. We have assessed transcription efficiency based on final product formation of a 1.9 kb transcript with the following triphosphates at 100% substitution using T7 polymerase.

NTP Transcription Efficiency
5-Aminoallyl-CTP (N-1065) +++
2-Amino-ATP (N-1001) +++
5-Br-UTP (N-1054) +
5-Carboxy-CTP (N-1084) ++
5-Carboxy-UTP (N-1091) n/a
5-Carboxymethylester-UTP (N-1096) ++
7-Deaza-ATP (N-1061) ++
5-Formyl-CTP (N-1085) +++
5-Formyl-UTP (N-1090) +
5-Hydroxy-CTP (N-1089) ++
5-Hydroxy-UTP (N-1092) ++
5-Hydroxymethyl-CTP (N-1087) ++
5-Hydroxymethyl-UTP (N-1086) +++
5-Iodo-UTP (N-1012) +
5-Methoxy-CTP (N-1094) ++
5-Methoxy-UTP (N-1093) ++
N6-Methyl-Amino-ATP (N-1083) ++
N6-Methyl-ATP (N-1013) +
5-Methyl-CTP (N-1014) +++
Pseudo-UTP (N-1019) +++
Thieno-CTP (N-1097) n/a
Thieno-GTP (N-1088) ++
1-Thio-ATP (N-8005) +
2-Thio-UTP (N-1032) +++

+++ = Greater than 75% efficiency compared to unmodified NTP
++ = Between 25-75% efficiency compared to unmodified NTP
+ = Less than 25% efficiency compared to unmodified NTP
n/a = No significant product formed with 100% substitution

In all cases, transcription was carried out at 37°C.

I don’t see any expression after transfection. What should I do?

We suggest validating the mRNA in an easily transfected cell line, such as HEK-293 cells. You may also want to include a GFP plasmid as a positive control. Note that while the GFP plasmid will provide confirmation of transfection, is does not verify delivery to the correct cellular compartment. The target compartment for the plasmid is the nucleus and the target compartment for the mRNA is the cytoplasm.

Which transfection reagent should I use?

Several companies offer transfection reagents. Our collaborators have used TransIT®-mRNA Transfection Kit (Mirus),  Stemfect™ (Stemgent), mRNA-In™ (MTI-GlobalStem), RmesFect™ Transfection Reagent (Oz Biosciences), and Lipofectamine™ RNAi Max (Life Technologies) with success. We suggest testing a matrix of transfection reagents and varying ratios of RNA to transfection reagent. Just as with plasmids and oligonucleotides, the optimal transfection procedure will need to be determined empirically. Some cell types are intrinsically easy to transfect (e.g. HEK-293 cells ~97%). Efficient delivery to other cell types, such as some primary cells can be very challenging.

My experimental system contains RNases. How do I combat degradation?

For many applications it is desirable to increase the nuclease stability of the RNA. The most common approach is to substitute canonical bases with 2’-fluoro modified NTPs. Bacteriophage polymerases do not efficiently incorporate 2’ modified NTPs. However, selection strategies have been utilized to evolve polymerases that can incorporate 2’ modified NTPs. Researchers commonly substitute pyrimidine bases with 2’ fluoro modified bases when making RNAs for biological applications, such as aptamers.

What precautions should I take when working with mRNA and long RNA?

We recommend that surfaces are wiped down with RNase Zap® and disposable plasticware is used for all supplies and reagents that will contact RNA. Use RNase-free reagents and a fresh bottle of serum-free media for diluting RNA and lipids. Water can be made RNase free by treating with DEPC and autoclaving. Alternatively, you can purchase RNase-free reagents. If possible, dedicate a set of pipettes for RNA work and use barrier tips. Note that serum contains Rnases and will likely degrade your RNA very quickly.

I would like to supply my own transcription template. What elements are required?

 mRNA Transcription Template:

  1. Bacteriophage promoter, preferably the T7 promoter
  2. 5′ untranslated region with a strong Kozak sequence
  3. ORF beginning with a start codon and ending with a stop codon
  4. 3′ untranslated region
  5. Poly(A) track top strand (this is not the same as a poly(A) signal)*
  6. Unique restriction site at the end of the cassette that is suitable for linearization

*Most plasmids designed for expression in a eukaryotic cell contain a poly(A) signal rather than a poly (A) stretch. If you do not have a poly(A) track in your plasmid we can add a poly(A) tail through a poly(A) polymerase reaction.

If your plasmid does not contain one or more of these elements, please contact us.

Long RNA Transcription Template:

  1. Bacteriophage promoter, preferably the T7 promoter
  2. Sequence that starts with G or GCG
  3. Unique restriction site at the 3′ end of the sequence to prevent runoff transcription*

*Please note that your transcript may contain a few untemplated nucleotides on the 3′ end.

If your plasmid does not contain one or more of these elements, please contact us.

What information is required for a custom mRNA or long RNA synthesis quote?

Step 1: Identify the Source of Transcription Template:   There are two primary options for the template source. We can synthesize and clone your sequence into our specially designed plasmid or you may submit your own template (PCR product or plasmid). The template must contain the elements as described in the ‘Transcription Templates’ section.

Step 2: Provide the Sequence: If you choose to have us synthesize and clone into our specialized plasmid, we need to know the sequence you would like to express.

Step 3: Identify a Transcription Scale:  Yield estimations below are based on a 1-2 kb transcript.

Transcription Scale Uncapped RNA Expected Yield Capped RNA Expected Yield
0.5 mL 0.5 – 1.5 mg 0.25 – 0.75 mg
1.0 mL 1 – 3 mg 0.5 – 1.5 mg
2.0 mL 2 – 6 mg 1 – 3 mg
4.0 mL 4 – 12 mg 2 – 6 mg
6.0 mL 6 – 18 mg 3 – 9 mg
10 mL 10 – 30 mg 5 – 15 mg

*Please inquire for smaller scales. TriLink also has the capability to do high throughput, small-scale RNA transcript synthesis.

Step 4: Select Modifications:  Tell us if you would like modified bases in your mRNA such as a 7-methylguanosine cap, 2-thiouridine, pseudouridine and 5-methylcytidine.

Step 5: Determine Number of Constructs:  It is much more economical to order the simultaneous synthesis of several RNAs.