Download Synthesis of RNA - Stamm revision

Summary: Synthesis of RNA
in the summary picture : can you please highlight the changes in each step by putting a
colored box underneath the group ?
question asked : what nucleotide interacts with a protein ?
what is the secondary structure of RNA ?
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Title: Chemical 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: [email protected]
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. In this chemistry, is the 3’ nucleotide always linked
to the column, is this common in all chemistries?, what lengths can typically be done?
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This phosphoramidite-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
not clear what the orthogonal means, can you explain and put in glossary (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. Can you explain here DMT and ACE
The tert-butyldimethylsilyl (TBDMS) group of phosphoramidite 1 has 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) group of phosphoramidite building
block 2 was 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 phosphoramidites 3 were 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 chemistry has 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
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service apply 5’-O-DMT-2’-O-silyl chemistry. Representative companies are listed in the
Table.1
In the introduction, can you briefly talk about the instruments used for
synthesis? Is there are ‘standard (ABI?) vendor, how much is the approximate
setup cost? What is the advantage of doing this type of chemistry yourself, vs
going to a company?
In this or the next chapter, can you talk about mixing DNA and RNA bases in oligos, we
do this to for example block the 5” or 3’ ends of oligos used for library construction
2.2. RNA modifications
2.2.1. RNA modification during solid-phase synthesis
The most important prerequisite 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 the 5’-O-DMT-2’-Osilyl protection scheme are 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 1B include:
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]; thiosubstituted 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 RNA 40, 2’-amino-2’-deoxy RNA 41
(only pyrimidine nucleosides commercially available; purine analogs have been
described [10-11]), 2’-deoxy-2’-fluoro RNA 42, LNA (locked nucleic acids) 43, and 2’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
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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; palladiumcatalyzed cross-coupling reactions to halogenated nucleotides (Figure 2B); and
functionalization of various amino groups via formation of amide bonds or ureido groups
(Figure 2C). can any of these modifications be done as long as the RNA is bound to the
column? I am asking, as we try to link a chemical to an oligo, ideally, we would have a
derivatized oligo, couple our compound, wash the non-reacted stuff away and elute the
whole coupled compound
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. Could you make a figure for this? 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.
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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.
Regarding the ligation of two RNA molecules, which generally are difficult. One
possibility is to use 5’rAppCTGTAGGCACCATCAAT/3ddC type oligos, where you
have the oligo activated and ligate without ATP. Typical, the yields are fairly high. MiRNA
people use this method and we had good success with it in tailing short RNAs for libaries
first described here, is now in IDT kits
Lau NC, Lim LP, Weinstein EG, and Bartel DP 2001 An abundant class of tiny
RNAs with probable regulatory roles in Caenorhabditis elegans. Science
294:858-862.
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, what are these 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 (1-100
µM 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
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labelling reagent is removed by precipitation, extraction, or gel filtration. The labelled
RNAs are purified by denaturing PAGE, RP HPLC 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 phenolchloroform extraction, the ligated RNA products are 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. How do you get rid of the
RNA splinter oligo, DNAse treatment? Can you make a picture for this?
4. Troubleshooting
Problem
Poor coupling yield of modified
phosphoramidite
Poor yield of amino group derivatization
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-
Poor ligation yield with T4 DNA ligase
-
Severe RNA degradation during ligation
-
Poor ligation yield with T4 RNA ligase
-
Reason + Solution
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
amine-containing buffers used
amino groups on RNA not fully
deprotected
isocyanate or active ester reagents
hydrolyzed; use high quality (aminefree) DMF for the stock solution
check for RNA secondary structure in
ligation fragments; use disruptor oligos
or design new ligation site
wrong ATP or MgCl2 concentration
RNase contamination of T4 DNA
ligase; change supplier
competing circularization of RNA
donor substrate; use 3’-phosphorylated 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
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chemistry (3). B. A collection of commercially available modified nucleosides for solidphase 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 not clear what this is 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]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
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.
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.
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.
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.
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.
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.
Helm, M. (2006). Post-transcriptional nucleotide modification and alternative
folding of RNA. Nucleic Acids Res. 34, 721-733.
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.
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.
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.
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[11]
[12]
[13]
[14]
[15]
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.
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.
Puffer, B., Moroder, H., Aigner, M., and Micura, R. (2008). 2'-Methylselenomodified oligoribonucleotides for X-ray crystallography synthesized by the ACE
RNA solid-phase approach. Nucleic Acids Res. 36, 970-983.
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 premRNA in early spliceosomal complexes. Mol. Cell 25, 399-411.
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 dimethyl formamide, DMSO dimethyl
sulfoxide, 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
synthesis scales
available RNA length / nt
website
offered / µmol
(for guaranteed yield)
Biomers.net
0.2, 1.0
3-80
www.biomers.net
Biosynthesis Inc.
0.1
10-50
www.biosyn.com
0.25, 1.0, 5, 10
10-70
Dharmacon RNAi technologies 0.05
10-40
www.dharmacon.com
0.2
2-67
0.4
2-100
1.0
2-35
Eurogentec
0.04
15-30
www.eurogentec.com
0.2, 1.0 ,2.5, 5, 10
8-50
Eurofins MWG Operon
0.05, 0.2, 1.0
10-40
www.eurofinsdna.com
IBA GmbH
0.2, 1.0, 15
up to 50-70
www.iba-go.com
(depending on scale and
sequence)
Integrated DNA technologies
0.1
10-50
www.idtdna.com
0.25, 1.0, 5, 10
5-50
Metabion
0.2, 1.0
2-80
www.metabion.com
Microsynth
0.04
10-30
www.microsynth.ch
0.2, 1.0
5-65
15
15-40
Midland Certified Reagent
0.2, 1.0, 10
up to 50
Company Inc.
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www.oligos.com
Primm Biotech Inc.
0.2, 1.0, 10
up to 50
www.primm.it
Sigma Genosys
0.05, 0.2, 1.0
7-45
www.sigmaaldrich.com
Trilink Biotechnolgies
0.2, 1.0, 15
no info on website
www.trilink.com
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.
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Summary
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Figure 1
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Figure 2
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