RNA Technologies

In vitro synthesis of RNA (> 20 nt up to several thousand nt) is catalyzed by bacteriophage RNA polymerases using linear DNA as a template (in vitro transcription). T7 RNA polymerase is the most efficient and widely used RNA polymerase. A modified version (T7 P&L RNA polymerase) with proline 266 replaced by leucine (P266L) has been 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].

140 – 160 µg RNA are synthesized after 30 min incubation with our HighYield formulation (1 μg T7 control template, 1.4 kb RNA transcript).

Selected References

[1] Guillerez et al. (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance. Natl. Acad. Sci. U.S.A102:5958.
[2] Salvail-Lacoste et al. (2018) Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags. RNA19:1003.
[3] Lyon et al. (2018) A T7 RNA Polymerase Mutant Enhances the Yield of 5′-Thienoguanosine-Initiated RNAs. ChemBioChem19:142.

Synthetic mRNAs are widely used as alternative molecules to plasmid DNA for modulating protein levels in a therapeutic and research context. Their biological functionality depends on both a 5’ Cap structure and nucleotide modifications that ensure efficient translation and reduced immunogenicity [1]-[16]. The classic combination of modifications consists of ARCA (Cap 0), 5-methylcytidine and pseudouridine which are conveniently introduced by correspondingly modified nucleotides via in vitro transcription.

Table 1: Overview on available (m)RNA synthesis kit configurations

Selected References

[1] Karikó et al.(2005) Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity23:165.
[2] Karikó et al.(2008) Incorporation of Pseudouridine into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability. Mol. Ther.16(11):1833.
[3] Kormann et al.(2011) Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nature Biotechnology29(2):154.
[4] Warren et al.(2011) Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. Cell Stem Cell7:618.
[5] Svitkin et al.(2017) N1-methyl-pseudouridine in mRNA enhances translation through eIF2alpha-dependent and independent mechanisms by increasing ribosome density. Nucleic Acid Res45(10):6023.
[6] Andies et al.(2015) N1-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J. Control. Release217:337.
[7] Li et al.(2016) Effects of Chemically Modified Messenger RNA on Protein Expression. Bioconjugate Chem.27:849.
[8] Arango et al.(2018) Acetylation of Cytidine in mRNA Promotes Translation Efficiency. Cell175(7):1872.
[9] Sinclair et al.(2017) Profiling Cytidine Acetylation with Specific Affinity and Reactivity. ACS Chem. Neurosci.12(12):2922.
[10] Dominissini et al.(2016) The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA. Nature530:441.
[11] Wienert et al. (2018) In vitro transcribed guide RNAs trigger an innate immune response via RIG-I pathway. PLoS Biol. 16 (7) :e2005840.
[12] Kim et al. (2018) CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 28 (3):367.
[13] Badieyan et al. (2019) Concise Review: Application of Chemically Modified mRNA in Cell Fate Conversion and Tissue Engineering. Stem Cells Translational Medicine8:833.
[14] Hadas et al. (2019) Optimizing Modified mRNA In Vitro Synthesis Protocol for Heart Gene Therapy. Molecular Therapy: Methods & Clinical Development 14:300.
[15] Shatkinet al. (1976) Capping of eukaryotic mRNAs. Cell 9(4):645.
[16] Gallowayet al.(2019) mRNA cap regulation in mammalian cell function and fate. Biochimica et Biophysica Acta 1862(3):270.

In vitro synthesis of RNA is catalyzed by bacteriophage (SP6, T7 or T3) RNA polymerases using linear DNA as a template. Ribonucleoside triphosphates (NTPs) are the building blocks of these in vitro transcription reactions.

Some naturally occuring RNAs such as messenger RNAs (mRNAs) or long non-coding RNAs (lncRNAs) contain a variety of modifications e.g. 5‘-end guanosinemethylation (5‘-Cap), 3‘-end adenosine (poly(A)) tail or several internal base modifications which markedly influence their biological function (e.g. translation efficiency, stability or immunogenicity).

These structural features can be introduced during in vitro transcription via correspondingly modified NTPs and cap analoga to obtain biologically functional synthetic RNAs.

Kits for enzymatic RNA Synthesis and poly(A) tailing are available as well.

Table 1: Raw materials for RNA Synthesis

Random labeled single-stranded RNA probes can be synthesized by in vitro transcription. The reaction is catalyzed by bacteriophage T7 RNA polymerase that incorporates labeled NTPs (mostly UTP) as substitute for their natural counterpart using linear, RNA probe-encoding DNA as template.

The optimal ratio of labeled NTP/NTP in terms of product yield and labeling efficiency depends on both the type of labeled NTP and final application. Individual optimization of labeled NTP/NTP ratio can easily be achieved with the single nucleotide format of our HighYield T7 RNA Labeling Kits. They offer maximum optimization flexibility in contrast to fixed NTP labeling mixes combined with high product yields. The combination of (poly)-HRP-conjugated Biotin/Digoxigenin detection reagents with AF 488 (Alexa Fluor® 488)-, AF546 (Alexa Fluor® 546)- or AF594 (Alexa Fluor® 594)-labeled tyramide further increases detection sensitivity of up to 100-fold.

One-step labeling of short RNA is achieved via T4 RNA Ligase 1-mediated incorporation of labeled pCps.

AF (Alexa Fluor®)- and ATTO® dyes are hydrophilic fluorescent dyes with remarkable photostability and thus ideally suited for fluorescence imaging applications. Biotin- and Desthiobiotin labeling allows efficient RNA pull-down.

Table 1: Spectroscopic properties of aptamer/dye complexes

Selected References

[1] Neubacher et al. (2019) RNA Structure and Cellular Applications of Fluorescent Light-Up Aptamers. Angew. Chem. Int. Ed. 58:1266.
[2] Ouellet (2016) RNA Fluorescence with Ligth-Up Aptamers. Front. Chem 4:29.
[3] George et al. (2018) Intracelluar RNA-tracking methods. Open Biol 8:180104.
[4] Dologosheina et al. (2016) Fluorophore Binding RNA aptamers and their application. Wires RNA 7(6):843.
[5] Paige et al. (2011) RNA mimics of green fluorescent protein. Science 333(6042):642.
[6] Song et al. (2014) Plug-and-Play fluorophores extend the Spectral Properties of Spinach. J. Am. Chem. Soc. 136:1198.
[7] Filonov et al. (2014) Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution. J. Am. Chem. Soc. 136:16299.
[8] Song et al. (2017) Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nat. Chem. Biol. 13(11):1187.

Northern Blot analysis is a reliable hybridization technique frequently used for the detection of a specific RNA transcript (e.g. mRNA) within a complex mixture. Historically, mainly radioactively labeled hybridization probes have been used however, non-radioactive labels such as Digoxigenin, Biotin or near-infrared fluorescent dyes are attractive alternatives in terms of probe stability, handling convenience and improved safety profiles.^[1-3]

Using RNA probes (“riboprobes”) generated by in vitro transcription instead of double-stranded DNA probes results in significantly higher assay sensitivity due to increased riboprobe affinity to RNA target and a higher thermodynamic stability of RNA:RNA complex.^[4-6] Riboprobes thus allow a reduced exposure time and more stringent washing conditions resulting in a lower background signal. The combination of (poly)-HRP-conjugated detection reagents with AF 488 (Alexa Fluor® 488)-, AF546 (Alexa Fluor® 546)- or AF594 (Alexa Fluor® 594)-labeled tyramide further increases detection sensitivity of up to 100-fold.

Selected References

[1] Miller et al. (2018) Near-infrared fluorescent northern blot. RNA 24(12):1871.
[2] O’Neill JW et al. (1994) Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. Biotechniques 17(5):870.
[3] Brauburger et al. (2017) Nonradioactive Northern Blot Analysis to Detect Ebola Virus Minigenomic mRNA. Methods in Molecular Biology 1628: DOI 10.1007/978-1-4939-7116-9_11.
[4] Melton et al. (1984) Efficient in vitro synthesis of biologicaly active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promotor. Nucleic Acids Research 12(18):7035.
[5] Srivastava et al. (2000) Analysis of RNA by Northern Blotting Using Riboprobes. The Nucleic Acid Protocols Handbook 38:249.
[6] Srivastava et al. (1991) Use of riboprobes for Northern blotting analysis. Biotechniques 11:584.

RNA structure determination can be performed by liquid state nuclear magnetic resonance (NMR) spectroscopy that requires millimolar amounts of isotopically labeled RNA to obtain well-resolved signals.

A successful approach to generate sufficient isotopically labeled RNA amounts is the in vitro transcription-mediated synthesis of 19F-labeled RNA using fluorinated NTPs[1]. 2F-ATP or 5F-UTP are incorporated instead of their natural counterparts in a non-perturbing way (intact base-pairing properties) and simultaneously function as a sensitive NMR reporter group (larger chemical shift dispersion than 1H)[1].

Selected References

[1] Graber et al. (2008) 19F NMR Spectroscopy for the Analysis of RNA Secondary Structure Populations. J. Am. Chem. Soc. 130 (51):17230.
[2] Sochor et al. (2016) S(19)F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy. J. Biomol. NMR 64 (1):63.
[3] Scott et al. (2004) Enzymatic synthesis and 19F NMR studies of 2-fluoroadenine-substituted RNA. J. Am. Chem. Soc. 126 (38):11776.

Table 1: Overview on available dsRNA detection products

Selected References

[1] Schönborn et al. (1991) Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res.19: 2993.
[2] Lukacs (1994) Detection of virus infection in plants and differentiation between coexisting viruses by monoclonal antibodies to double-stranded RNA. J. Virol. Methods47: 255.
[3] Lukacs (1997) Detection of sense:antisense duplexes by structure-specific anti-RNA antibodies. In: Antisense Technology. A Practical Approach, C. Lichtenstein and W. Nellen (eds), pp. 281-295. IRL Press, Oxford
[4] Weber et al. (2006) Double-Stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. Journal of Virology 80(10): 5059.
[5] Knoops et al. (2008) SARS-Coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLOS Biology 6(9): e226.
[6] Richardson et al. (2010) Use of antisera directed against dsRNA to detect viral infections in formalin-fixed paraffin-embedded tissue. Journal of Clinical Virology 49: 180.
[7] 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 Research 39(21): e142.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are the hallmark of a bacterial defense system that forms the basis for CRISPR/Cas sequence-specific gene targeting technology ^[1-5].

CRISPR/Cas systems typically consists of two components:

1.

 RNA-guided CRISPR-associated (Cas) endonuclease(e.g. class II endonuclease Cas9)

2.

Sequence-specific guide RNA

 (e.g. sgRNA)

There are several ways to introduce Cas endonucleases and sequence-specific guide RNA into cells however, delivery as ribonucleoprotein (RNP) complex or RNA molecules shows the highest efficiency in most cases.^[6-7]

Selected References

[1] Jinek et al. (2012) A programmable dual-RNA-guided, DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816.
[2] Wang et al. (2016) CRISPR/Cas9 in Genome Editing and Beyond. Annu. Rev. Biochem. 85:227.
[3] Gaj et al. (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31(7):397.
[4] Aldi et al. (2018) The CRISPR tool kit for genome editing and beyond. Nature Communications 9:1911.
[5] Pickar-Oliver et al. (2018) The next generation of CRISPR–Cas technologies and applications. Nature Reviews Molecular Cell Biology 20:507.
[6] DeWitt et al. (2017) Genome editing via delivery of Cas9 ribonucleoprotein. Methods 121:9.
[7] Kim et al. (2014) Highly efficient RNA-guided genome editing in humancells via delivery of purified Cas9 ribonucleoproteins. Genome Research 24:1012.

T7 RNA Polymerase-mediated in vitro transcription is an easy, fast and currently the most cost-efficient way of guide RNAs synthesis.

HighYield T7 sgRNA Synthesis Kit (SpCas9) allows the cloning-free preparation of SpCas9-specific single-guide RNA (sgRNA).^[1,2] Other (s)gRNA-encoding T7 DNA templates (e.g with a different SpCas9 scaffold or for different Cas endonucleases) can efficiently be in vitro transcribed with the HighYield T7 RNA Synthesis Kit.

Selected References

[1] Jinek et al. (2012) A programmable dual-RNA guided DNA Endonuclease in adaptive bacterial immunity. Science 337:816.
[2] Modzelewski et al. (2018) Efficient mouse genome engineering by CRISPR-EZ technology. Nature Protocols 13 (6) :1253.
[3] Hendel et al. (2015) Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nature Biotechnology 33 (9):985.