Long RNA Synthesis: Leading the Way in mRNA™
TriLink has supplied the market with high quality RNA oligos since 1996. The addition of RNA synthesis by transcription rounds out TriLink's offering to include all lengths of RNA. Any length of RNA may be requested from modified monomers to longmer RNA, in the kilobases. Visit OligoBuilder® to price and order RNA oligos <100 bases. For long RNA, 100+ bases, request a quote:
Chemical Synthesis vs Synthesis by Transcription
RNA synthesis by transcription is an alternative to chemical RNA oligo synthesis and is useful when a long RNA is needed, typically 100 bases and above. Yields of chemically-synthesized RNA decrease dramatically with increasing length due to the compound affect of coupling efficiencies of less than 100%. (View Understanding Oligonucleotide Synthetic Yields to learn more.) Thus with current chemistries, it is not cost effective or efficient to synthesize high quality longmer RNA chemically. In contrast, RNA synthesis by transcription enables lengths up to thousands of bases due to the extremely high processivity and fidelity of bacteriophage polymerases used in the process.
Synthesis by Transcription
In enzymatic synthesis a DNA template is first synthesized, and then used as a template for transcription. Several factors should be considered when requesting a long RNA synthesis by transcription:
Initiating Nucleotide: Bacteriophage polymerases such as T7, SP6 and T3 RNA polymerase preferentially initiate with a guanine residue. Therefore, TriLink recommends that the sequence begin with a 5′ G. If it is acceptable to initiate with 5′ GCG 3′, this is preferred, as will be discussed in the section on 5′ and 3′ End Heterogeneity below. Initiation with an adenosine residue is available, but approximately 10 fold less efficient, which will dramatically reduce the final yield.
5′ and 3′ End Heterogeneity: Long RNA synthesis by transcription may have some heterogeneity at the 5′ and 3′ end. However, 5′ end heterogeneity can be significantly reduced by initiating with the sequence 5′ GCG 3′ (1). At the 3′ end, T7 RNA polymerase can add an untemplated nucleotide (or sometimes several) to the transcribed RNA (2, 3). This untemplated nucleotide is usually an A or C residue.
5′ Triphosphate: In vitro transcribed RNA transcripts have a 5′ pppG. Both double stranded and single stranded 5′ pppG RNA can stimulate the cytosolic innate immune sensor RIG-I (retinoic acid-inducible gene-I) (4, 5). It has been reported that in vitro transcripts can be powerful activators of RIG-I (6). In cases where innate immune stimulation would be problematic, the 5′ triphosphate can be removed by an optional phosphatase treatment.
Transcriptional Termination Signals: In bacteria, bacteriophage polymerases terminate at transcription termination signals. These are strong hairpins followed by a string of U residues (7). During in vitro transcription, certain sequences can cause premature transcription termination, resulting in a truncated product. Transcription termination or polymerase slippage can also occur at polyuridine stretches such as 5′ UUUUCUU 3′ (7). While potentially problematic sequences can be identified using RNA folding programs, these are not completely predictive. Thus, the ability of a new transcription template to produce a full length transcript must be experimentally validated.