Synthesis of RNA

Summary: Synthesis of RNA

Title: Synthesis of RNA

Claudia Höbartner

Research Group Nucleic Acid Chemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Address correspondence to: Claudia Höbartner, Am Fassberg 11, 37077 Göttingen

fax: 0049-551-201-1680, E-mail:

1. Abstract

The discovery of RNA interference and the therapeutic potential for modified RNA underline the growing importance of synthetic RNA in basic biomedical research. Synthetic RNA oligonucleotides are also indispensable tools for structural studies and biochemical analyses of RNA-RNA or RNA-protein interactions. The chemical synthesis of RNA offers the unique possibility to introduce site-specific modifications and attachment sites for biophysical labels. This chapter summarizes chemical modification strategies for RNA and gives a brief overview for incorporating modified oligonucleotides into larger RNA constructs by enzymatic ligation.

Keywords: solid-phase synthesis, phosphoramidite, post-synthetic modification, RNA ligation.

2. Theoretical background

2.1. RNA solid-phase synthesis

The automated chemical synthesis of RNA oligonucleotides consists of repeated coupling of ribonucleoside phosphoramidite building blocks on a solid support. The four steps of the synthesis cycle include: A) cleavage of the transient 5’-protecting group, B) activation of the phosphoramidite building block and coupling to the 5’-OH of the support bound nucleotide, C) capping of unreacted 5’-termini to prevent subsequent extension, and D) oxidation of the phosphite triester to a phosphate triester internucleotide bond. The four steps are repeated until the desired oligonucleotide length is assembled. The full-length RNA is then released from the solid support and the nucleobase and phosphate protecting groups are removed. Cleavage of the 2’-protecting groups affords the final oligoribonucleotide product.

Thisphosphoramidite-based RNA solid-phase synthesis cycle is highly similar to standard automated DNA solid-phase synthesis but the requirement for additional 2’-protecting groups makes RNA synthesis much more challenging. The key to successful solid-phase RNA synthesis is the choice of a suitable combination of orthogonal transient (R1) and permanent (R, R2, R3) protecting groups.It is of critical importance that the 2’-protecting groups remain completely intact until the final deprotection step and that they can be removed under conditions that do not affect the integrity of the target RNA. The increasing demand for synthetic RNA oligonculeotides has spurred renewed efforts in the development of new protecting group strategies with the goal to render RNA synthesis as efficient and reliable as DNA synthesis. The latest advances in chemical RNA synthesis have recently been reviewed[1-3]. Presently, the three most important families of phosphoramidite building blocks for RNA solid-phase synthesis (Figure 1A)belong to two classes of orthogonal protecting group strategies, the 5’-O-DMT-2’-O-silyl (1 and 2) and the 5’-O-silyl-2’-O-ACE (3) strategy.

The tert-butyldimethylsilyl (TBDMS) group of phosphoramidite 1has been the most commonly used 2’-alkylsilyl protecting group for RNA solid-phase synthesis since the 1980s[4]. A wide variety of building blocks is commercially available. However, the performance of TBDMS-based RNA synthesis has not reached the level of solid-phase DNA synthesis in terms of quality, yield and accessible oligonucleotide length.

The 2’-O-triisopropylsilyloxymethyl (TOM) groupof phosphoramidite building block 2was reported by Pitsch and coworkers in 1998 and represents a considerable advancement over conventional 2’-silyl protection[5]. The reduced steric demand of the TOM group compared to TBDMS during internucleotide bond formation allows for high coupling yields in short coupling times. Data for RNA synthesis in high yield and high quality have been reported for oligonucleotides up to 84 nucleotides in length.

The 5’-O-silyl-2’-O-ACE phosphoramidites3were described in 1998 by Caruthers and coworkers and followed a complete redesign of earlier protecting group strategies, now using fluoride-labile 5’-silyl groups and acid labile 2’-orthoester protecting groups[6].This innovative ACE chemistryhas advanced to a highly powerful and commercially offered RNA synthesis method although it preserves only a few aspects of the traditional DNA synthesis concept, requires alterations to commonly applied reagents and procedures and involves changes to standard instrumentation. The ACE methodology enables RNA synthesis in excess of 70 bases in length, is highly scaleable and is applicable to high throughput RNA production.Custom RNA synthesis by Dharmacon is based on the ACE chemistry. Most other companies that offer custom RNA synthesis service apply 5’-O-DMT-2’-O-silyl chemistry. Representative companies are listed in the Table.

2.2. RNA modifications

2.2.1. RNA modification during solid-phase synthesis

The most importantprerequisite for successful incorporation of nucleoside analogs via solid-phase phosphoramidite chemistry is the chemical compatibility of the desired modification with all conditions encountered during chain assembly and deprotection.Various modified phosphoramidites compatible with the5’-O-DMT-2’-O-silyl protection schemeare commercially available from different sources (e.g., GlenResearch, ChemGenes, Berry Associates, Link Technologies, etc.). A subset of these modifications is also available via custom RNA synthesis services offered by various companies as listed in the Table.

Representative classes of nucleobase modifications in Figure 1Binclude:alkylated nucleobases(4-9) that are mainly used to mimic natural modifications[7]; nucleobases with altered patterns of exocyclic amino groups and ring nitrogen atoms (10-20) for structural and mechanistic studies of RNA folding and catalysis[8];thio-substituted nucleobases (21,22);halogenated nucleosides (23-28);fluorescent nucleoside analogs (29-31);amino-tethered (32) and convertible nucleosides (33-36) for post-synthetic RNA modification[9].

Ribose modifications include 2’-O-methyl RNA40, 2’-amino-2’-deoxyRNA 41(only pyrimidine nucleosides commercially available; purine analogs have been described[10-11]), 2’-deoxy-2’-fluoro RNA42, LNA 43, and2’-methylseleno RNA 44(only uridine derivative commercially accessible; other three nucleoside 2’-SeMe phosphoramidites for 2’-O-silyl- and 2’-O-ACE strategies have been reported [12-13]).

In addition to nucleobase- and ribose-modified nucleoside analogs, a large variety of non-nucleoside phosphoramidites is commercially available and can be used to incorporate amino or thiol groups via alkyl linkers at the 5’- or 3’-terminus or at internal positions. Internucleotide spacers (mono-, tri-, or hexaethylene glycol units) or abasic site analogs can be introduced, as well as fluorophores (e.g., fluorescein and its derivatives, tetramethylrhodamine, cyanine dyes, etc.), quenchers, biotin, acridine, psoralen, and cholesterol. Most internal modifications are supplied as DMT-protected phosphoramidites, but several are also offered for combination with ACE chemistry.

Instead of replacing standard phosphoramidites with modified nucleoside building blocks during solid-phase synthesis, the standard oxidation solutions can be replaced by alternative reagents, which results in the synthesis of backbone-modified RNA (e.g. phosphorothioate or phosphoroselenoate RNA).

2.2.2. Post-synthetic RNA modification

The post-synthetic modification of RNA oligonucleotides relies on introduction of nucleoside analogs containing reactive functionalities by solid-phase synthesis and enables the site-specific attachment of various reporter groups and chemical devices (Figure 2). Useful types of nucleoside derivatization reactions include: nucleophilic aromatic substitution of appropriate leaving groups on nucleobases (the convertible nucleoside approach using 33-36);formation of thioether or disulfide bonds (Figure 2A) at phosphorothioates 44, thiouridine 21 or thiol-containing alkyl linkers;palladium-catalyzed cross-coupling reactions to halogenated nucleotides (Figure 2B); and functionalization of various amino groups via formation of amide bonds or ureido groups (Figure 2C).

2.3. Combined chemical and enzymatic strategies

To generate longer RNAs than routinely achievable by direct chemical synthesis, modified and non-modified RNA fragments can be covalently joined by enzymatic ligation. The protein enzymes T4 DNA ligase or T4 RNA ligase are commonly used to activate the 5’-terminal phosphate of the donor RNA by adenylation and join it to the 3’-hydroxyl group of the acceptor fragment. T4 DNA ligase catalyzes the ligation of two RNA substrates that are precisely aligned in a fully base-paired RNA-DNA heteroduplex, whereas T4 RNA ligase is used to join two single-stranded RNAs in the absence of a splint oligonucleotide. The required RNA segments can either all be prepared by chemical synthesis or larger fragments can be generated enzymatically by in vitro transcription using T7 RNA polymerase. Although T7 RNA polymerase accepts certain modified NTPs as substrates it is usually not possible to introduce modified nucleotides site-specifically using standard DNA templates.

Numerous examples have been reported for the successful combination of chemical synthesis of modified RNA and enzymatic ligation methods. Recent examples involve the convertible nucleoside approach and thiol-specific RNA labelling together with enzymatic ligation for engineering of pre-mRNA and snRNA constructs[14-15]. In these studies, a site-specifically attached hydroxyl radical probe (Fe-BABE) was used to investigate the architecture of early spliceosomal complexes.

3. Brief representative protocols

Protocol 1: Incorporation of modified phosphoramidites during solid-phase synthesis

The coupling conditions for modified phosphoramidite building blocks may require alterations of the standard RNA synthesis protocols, as usually specified in the accompanying product sheets. In general, the modified phosphoramidites are applied as 100 mM solutions in dry acetonitrile. At least 10 equivalents of modified amidites are used and coupling times are set for up to 12 minutes. It is advisable to use phosphoramidites with nucleobase protecting groups that can be cleaved under ultramild conditions, since these will be compatible with most nucleobase modifications. Certain RNA modifications require additional steps during the synthesis cycle to guarantee integrity of the final RNA after deprotection. An example is the synthesis of 2’-SeMe RNA that requires treatment of the growing oligonucleotide chain with a reducing agent after each oxidation step.

Protocol 2: Coupling of biophysical probes to aliphatic amino groups on RNA

Primary amino groups are incorporated into RNA by phosphoramidite reagents or amino-modified RNA fragments can be purchased from commercial sources. The iso(thio)cyanate, NHS or STP ester derivatized labelling reagents are dissolved in DMF or DMSO, and aliquots corresponding to 10-1000 fold excess over amino-RNA (1-100µM in sodium borate or sodium bicarbonate buffer pH 8.5 - 9.0) are used for the labelling reaction. The final content of organic solvent in the labelling mixture should not exceed 50%. Reactions with active esters are usually carried out at 25°C for 12 hours, whereas labelling with more reactive isothiocyanate derivates is performed at 4°C. Excess labelling reagent is removed by precipitation, extraction, or gel filtration. The labelled RNAs are purified by denaturing PAGE, RPHPLC or anion exchange HPLC.

Protocol 3: Enzymatic ligation of RNA fragments using T4 RNA or T4 DNA ligase

RNA fragments and a splint oligonucleotide (when appropriate) are annealed in 5 mM Tris or HEPES buffer at pH 7.5 at a RNA concentration of 10-40 µM by heating the sample to 95°C for 2 min and slow cooling to room temperature over 15-60 min. Ligase buffer containing appropriate amounts of MgCl2 and ATP are added, as well as ligase enzyme, and the ligation reaction is incubated at 20 or 37°C for 1-5 hours. After phenol-chloroform extraction, the ligated RNA productsare purified by denaturing PAGE or anion exchange HPLC. Critical parameters include concentrations of MgCl2 and ATP, incubation temperature and reaction time. For optimal ligation yields, especially for large scale reactions, the conditions should be carefully optimized.

4. Troubleshooting

Problem / Reason+Solution
Poor coupling yield of modified phosphoramidite / -water (>30 ppm) in acetonitrile used for dissolving the phosphoramidite; use molecular sieves, keep solutions under Argon
-too low amidite concentration
-too short coupling time
-lines not purged
Poor yield of amino group derivatization / -amine-containing buffers used
-amino groups on RNA not fully deprotected
-isocyanate or active ester reagents hydrolyzed; use high quality (amine-free) DMF for the stock solution
Poor ligation yield with T4 DNA ligase / -check for RNA secondary structure in ligation fragments; use disruptor oligos or design new ligation site
-wrong ATP or MgCl2 concentration
Severe RNA degradation during ligation / -RNase contamination of T4 DNA ligase; change supplier
Poor ligation yield with T4 RNA ligase / -competing circularization of RNA donor substrate; use 3’-phosphor-ylated donor substrates, ensure proper annealing

Figure legends

Figure 1. A. Ribonucleoside phosphoramidite building blocks for commercialized RNA synthesis methods using either 5’-O-DMT-2’-O-silyl (1 and 2) or 5’-O-silyl-2’-O-ACE chemistry (3). B. A collection of commercially available modified nucleosides for solid-phase synthesis of RNA by phosphoramidite chemistry. All building blocks are compatible with the 5’-O-DMT-2’-O-silyl protection scheme. Base-modified nucleosides 4-36 are available with the 2’-O-TBDMS protecting group. The asterisk* indicates additional availability as 2’-O-TOM protected phosphoramidite; # denotes availability via custom synthesis service using 2’-O-ACE chemistry (Dharmacon).

Figure 2. Selected examples of postsynthetic RNA modification stategies. A. Thioether formation with -haloacetamides and disulfide formation with methanethiosulfonate reagents. B. Sonogashira cross-coupling of terminal alkynes to 5I-U on the solid support. C. Amino groups reacting with isocyanate or isothiocyanate form urea or thiourea bonds, reactions with NHS or STP esters give amide bonds. R groups are biophysical labels or reporter groups such as fluorophores, ion complexation reagents, photocrosslinking reagents or spin labels.

References

[1]Beaucage, S. L., and Reese, C. B. (2009). Recent advances in the chemical synthesis of RNA. Curr. Protoc. Nucleic Acid Chem., 2.16.11-12.16.31.

[2]Höbartner, C., and Wachowius, F. (2009) Chemical synthesis of modified RNA, In The chemical biology of RNA (Mayer, G., Ed.), Weinheim, Germany: Wiley-VCH Verlag GmbH, in press.

[3]Chow, C. S., Mahto, S. K., and Lamichhane, T. N. (2008). Combined Approaches to Site-Specific Modification of RNA. ACS Chem Biol 3, 30-37.

[4]Usman, N., Ogilvie, K. K., Jiang, M. Y., and Cedergren, R. J. (1987). The automated chemical synthesis of long oligoribuncleotides using 2'-O-silylated ribonucleoside 3'-O-phosphoramidites on a controlled-pore glass support: synthesis of a 43-nucleotide sequence similar to the 3'-half molecule of an Escherichia coli formylmethionine tRNA. J. Am. Chem. Soc. 109, 7845-7854.

[5]Pitsch, S., Weiss, P. A., Jenny, L., Stutz, A., and Wu, X. (2001). Reliable Chemical Synthesis of Oligoribonucleotides (RNA) with 2'-O-[(Triisopropylsilyl)oxy]methyl(2'-O-tom)-Protected Phosphoramidites. Helv. Chim. Acta 84, 3773-3795.

[6]Scaringe, S. A., Wincott, F. E., and Caruthers, M. H. (1998). Novel RNA Synthesis Method Using 5‘-O-Silyl-2‘-O-orthoester Protecting Groups. J. Am. Chem. Soc. 120, 11820-11821.

[7]Helm, M. (2006). Post-transcriptional nucleotide modification and alternative folding of RNA. Nucleic Acids Res. 34, 721-733.

[8]Das, S. R., Fong, R., and Piccirilli, J. A. (2005). Nucleotide analogues to investigate RNA structure and function. Curr. Opin. Chem. Biol. 9, 585-593.

[9]Edwards, T. E., and Sigurdsson, S. T. (2005) Modified RNAs as Tools in RNA Biochemistry, In Handbook of RNA Biochemistry (Hartmann, R. K., Bindereif, A., Schön, A., and Westhof, E., Eds.), Weinheim, Germany: Wiley-VCH Verlag GmbH, pp 112-129.

[10]Karpeisky, A., Sweedler, D., Haeberli, P., Read, J., Jarvis, K., and Beigelman, L. (2002). Scaleable and efficient synthesis of 2'-deoxy-2'-N-phthaloyl nucleoside phosphoramidites for oligonucleotide synthesis. Bioorg. Med. Chem. Lett. 12, 3345-3347.

[11]Dai, Q., Deb, S. K., Hougland, J. L., and Piccirilli, J. A. (2006). Improved synthesis of 2'-amino-2'-deoxyguanosine and its phosphoramidite. Bioorg. Med. Chem. 14, 705-713.

[12]Micura, R., Höbartner, C., Rieder, R., Kreutz, C., Puffer, B., Lang, K., and Moroder, H. (2007). Preparation of 2'-deoxy-2'-methylseleno-modified phosphoramidites and RNA. Curr. Protoc. Nucleic Acid Chem., 1.15.11-11.15.34.

[13]Puffer, B., Moroder, H., Aigner, M., and Micura, R. (2008). 2'-Methylseleno-modified oligoribonucleotides for X-ray crystallography synthesized by the ACE RNA solid-phase approach. Nucleic Acids Res. 36, 970-983.

[14]Dönmez, G., Hartmuth, K., Kastner, B., Will, C. L., and Lührmann, R. (2007). The 5' end of U2 snRNA is in close proximity to U1 and functional sites of the pre-mRNA in early spliceosomal complexes. Mol. Cell 25, 399-411.

[15]Kent, O. A., and MacMillan, A. M. (2002). Early organization of pre-mRNA during spliceosome assembly. Nat. Struct. Biol. 9, 576-581.

Abbreviations

ACE bis(2-acetoxyethoxy)methyl orthoester; DMF dimethylformamide, DMSO dimethylsulfoxide, DMT 4,4,’-dimethoxytrityl, NHS N-hydroxysuccinimid, RP reversed phase, STP 4-sulfonyl-tetrafluorophenyl, TBDMS tert-butyldimethylsilyl, TOM triisoproylsilyloxymethyl.

Table. Selection of companies currently offering custom synthesis of modified and unmodified RNA oligonucleotides a

Company
website / synthesis scales offered / µmol / available RNA length / nt
(for guaranteed yield)
Biomers.net
/ 0.2, 1.0 / 3-80
Biosynthesis Inc.
/ 0.1
0.25, 1.0, 5, 10 / 10-50
10-70
Dharmacon RNAi technologies
/ 0.05
0.2
0.4
1.0 / 10-40
2-67
2-100
2-35
Eurogentec
/ 0.04
0.2, 1.0 ,2.5, 5, 10 / 15-30
8-50
Eurofins MWG Operon
/ 0.05, 0.2, 1.0 / 10-40
IBA GmbH
/ 0.2, 1.0, 15 / up to 50-70
(depending on scale and sequence)
Integrated DNA technologies
/ 0.1
0.25, 1.0, 5, 10 / 10-50
5-50
Metabion
/ 0.2, 1.0 / 2-80
Microsynth
/ 0.04
0.2, 1.0
15 / 10-30
5-65
15-40
Midland Certified Reagent Company Inc.
/ 0.2, 1.0, 10 / up to 50
Primm Biotech Inc.
/ 0.2, 1.0, 10 / up to 50
Sigma Genosys
/ 0.05, 0.2, 1.0 / 7-45
Trilink Biotechnolgies
/ 0.2, 1.0, 15 / no info on website

a Please note that commercial sources are subject to change. This list contains representative examples as of September 2009. Several companies that specialize mainly on siRNA synthesis and don’t offer custom RNA synthesis of longer oligonucleotides are not listed. Other companies specialized on DNA synthesis may also offer custom RNA synthesis service.

Summary

Figure 1

Figure 2

Höbartner- 1 -