Yields of chemically-synthesized RNA decrease exponentially with increasing length because coupling efficiencies at each step are 90-99% (depending on the type of incorporated base). Thus it is not feasible to synthesize very long RNA chemically. In contrast, bacteriophage polymerases, such as T7 RNA polymerase, are quite processive and it is therefore possible to obtain RNA by transcription that are thousands of bases long and migrate as a single band on a denaturing gel.

Types of long RNA transcripts

Unmodified: Unmodified RNA transcripts are useful in biochemical assays, RNA structure studies and as probes for RNA protection experiments.

Base Modified: TriLink offers an extensive catalog of modified nucleoside triphosphates (NTPs) that impart desirable characteristics to in vitro transcribed RNA such as increased nuclease stability, increased translation or altered interaction with innate immune receptors. For example, incorporation of 5-methylcytidine-5'-triphosphate and pseudouridine-5'-triphosphate has been shown to reduce innate immune stimulation in culture and in vivo while enhancing translation. RNA can be functionalized using aminoallyl NTPs for later conjugation to other molecules, such as dyes. Biotin groups can also be incorporated at the transcription step.

Capped & Polyadenylated (mRNA or messenger RNA): In cells, the ribosome translates mRNA into proteins. mRNA in eukaryotic cells has a 5’ cap [m7G(5')ppp(5')G] which stabilizes the mRNA and enhances translation. A cap can be incorporated in a transcription by including a mixture of cap analog and GTP (usually at a 4:1 ratio). Approximately 80% of synthesized mRNA will possess a 5’ cap, while the remaining 20% will possess a 5’ triphosphate. In some cases it is desirable to remove the 5’ triphosphate with a phosphatase treatment.

The first cap analog to be introduced was mCAP [m7G(5')ppp(5')G]. 50% of the time, mCAP is inserted in the correct orientation to enhance translation. The other 50% of molecules are not substrates for efficient translation, reducing the specific activity of the transcript. More recently, Anti-Reverse Cap Analog (ARCA) was introduced [3’ O-Me-m7G(5')ppp(5')G](1). ARCA can only insert in the proper orientation, resulting in mRNAs that are translated twice as efficiently as those initiated with mCAP. Efficient translation of the mRNA into protein also requires a poly(A) tail. This can be introduced by including a poly(dT) stretch at the end of the transcription template. Often this is accomplished by a PCR step that utilizes a primer containing the poly(dT) stretch.

Templates

The customer can provide their own transcription template containing a bacteriophage promoter or they can provide a plasmid or PCR product containing the sequence of interest. TriLink will amplify the plasmid or PCR product and add the required elements (promoter and poly(A) tail, if required) to the PCR primers.

Scales and Yields

Synthesis scales yielding µgrams to hundreds of milligrams are available. We quote based on starting synthesis scale due to the fact that transcription yields vary from template to template. However, based on our experience, we will make a conservative estimate of the scale necessary to obtain the quantity our customers require. We will ship all material produced.

Purification Options

Lithium Chloride (LiCl) Precipitation: LiCl preferentially precipitates RNA relative to DNA. LiCl precipitation, followed by a 70% ethanol wash, concentrates the RNA and removes most protein, DNA and NTPs. For applications that are very sensitive to DNA contamination, a DNase treatment or additional purification is recommended.

Acidic Guanidinium Thiocynate Phenol Extraction: Extraction with acidic guanidinium thiocynate phenol preferentially extracts RNA while removing most proteins and DNA. A subsequent isopropanol precipitation and 70% ethanol wash concentrates the RNA and removes most protein, DNA and NTPs. For applications that are very sensitive to DNA contamination, a DNase treatment or additional purification is recommended.

Silica-gel Membrane Spin Column Purification: RNA can be bound to silica-gel membranes, washed and eluted in water or TE pH 7. This process removes most proteins, DNA and NTPs.

Additional Enzymatic Treatments

DNase Treatment: For many applications, it is desirable to remove the DNA transcription template following transcription. DNase treatment degrades the DNA template into smaller fragments that can then be removed in subsequent purification steps.

Phosphatase Treatment: Transcription leaves a 5’ triphosphate at the end of the RNA. RNA with a terminal triphosphate induces innate immune responses in mammalian cells by activating RIG-I. In other applications, it may be desirable to 5’ end label the RNA using T4 polynucleotide kinase. This requires removal of the 5’ triphosphate. Therefore, we offer RNA dephosphorylation by phosphatase treatment. Prior purification is required due to competition for the phosphatase by free NTPs in the transcription reaction.

Analysis

Spectrophotometric Scan: A wavelength scan from 220-320 nm allows calculation of concentration and provides an estimate of contaminating protein, salt and phenol.

Gel Analysis: An analytical gel sample is treated with glyoxal to disrupt secondary structure and run on an agarose gel.


Documentation

All long RNA transcripts have fully traceable documentation and are supplied with a certificate of analysis.

Calculating RNA Concentrations

For long RNA with even base distributions, an common rule of thumb is that 1 OD₂₆₀ is approximately 40 µg. For sequences with highly skewed base distributions, this approximation does not hold. Modified NTPs often have different extinction coefficients. In these cases, exact extinction coefficient should be calculated and used to determine concentration using Beer’s law. Beer’s law states that Absorbance= (Extinction coefficient) x (path length in cm) x (concentration).