HIGHLIGHTS

Small Interfering RNAs and Their Chemical Synthesis

Ronald Micura*

Only few years ago, it was discovered that RNA plays a

fundamental role in post-transcriptional regulation of gene
expression. When double-stranded RNA (dsRNA) of around
300 to 1000 base pairs (bp) in length was injected into the
nematode Caenorhabditis elegans, the specific silencing of
genes highly homologous in sequence to the delivered dsRNA
was induced.[1] This phenomenon, termed RNA interference
(RNAi), paved the way for a straightforward determination
of gene function, once the sequence of the gene was
known.[2]

Mechanism of RNA Interference

Elucidation of the mechanism of RNAi is subject to
intensive research. Transfection with dsRNA results in the
degradation of the sequence-homologous mRNA, even in
only substoichiometric amounts. The initial step is the
processing of the dsRNA into fragments of 21 ± 23 nucleotide
(nt) strands. These short interfering RNA molecules (siRNA)
are the mediators of mRNA degradation, as was recently
established by T. Tuschl and co-workers, who demonstrated
that chemically synthesized RNA duplexes with the fragment
pattern mentioned above are capable of guiding mRNA
cleavage.[3]
These siRNA duplexes readily associate to form target-
cleaving RNA protein complexes (target ˆ sense or anti-
sense), which have not been well characterized to date. These
complexes are referred to as small interfering ribonucleopro-
tein particles (siRNPs; Figure 1). The model of Tuschl and co-
workers discriminates between two classes of siRNPs; namely, Figure 1. Model for RNA interference (RNAi) proposed by T. Tuschl and
one that cleaves only the antisense and another that cleaves co-workers. Small interfering RNAs (siRNA) are the key molecules for
only the sense strand. Depending on the orientation of the targeted RNA cleavage (Figure adapted from reference[3]).
duplex within an siRNP, only one of the two siRNA strands is
capable of target-RNA recognition (the siRNAs at the
proteins which are represented by black rounded rectangles
It is suggested that the relative orientation of the siRNA
in Figure 1). This model corresponds to earlier findings that
duplex within the siRNP is preserved from the preceding step
certain chemical modifications, such as 2'-aminouridine or 2'-
of dsRNA processing.[3] The proteins involved, among them a
deoxythymidine, are well tolerated in the sense strand, but not
nuclease of the RNase III family, assemble on the dsRNA in
in the mRNA cleavage-guiding antisense strand.[4]
an asymmetric fashion and remain at least partly associated
with the siRNA products. Thus, the molecular information of
[*] Dr. R. Micura directionality is passed on, which results in distinct roles of the
Leopold Franzens University siRNA strands, only one being capable of guiding targeted
Institute of Organic Chemistry
Innrain 52a, 6020 Innsbruck (Austria)
RNA cleavage. The siRNA duplex is thought to be tempo-
Fax: (‡ 43) 512-5072892 rarily disrupted during target recognition and reformed after
E-mail: [email protected] release of the cleaved target RNA. The position of RNA

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HIGHLIGHTS
cleavage relative to the guide siRNA is near the center of the (Scheme 1 a,c, Table 1). The key feature is the 2'-O-triisopro-
region covered by the 21 or 22 nt siRNA. pylsilyloxymethyl (TOM) protecting group of the nucleotide
Synthetic oligoribonucleotides were also valuable, as it was building blocks which was first introduced in 1998, but was
observed that 21 or 22 nt siRNA duplexes with double recently published with the synthetic details.[10] The TOM
nucleotide 3'-overhangs were more efficient in degrading protecting group guarantees an average coupling yield of
target RNA than similar blunt-ended duplexes.[3] This obser- 99.4 % under DNA coupling conditions and the usage of
vation is consistent with the cleavage pattern of an RNase III- benzylthiotetrazole as activator. This superior coupling be-
like nuclease activity during dsRNA processing.[5] It is of havior, compared to the 2'-O-TBDMS building blocks (typical
further note that dsRNAs of less than 38 bp are inefficient coupling yield:  98 %), can be attributed to the minimal
mediators of RNAi, as the reaction rate of siRNA formation is steric demand of the TOM protecting group and allows the
significantly reduced compared with longer dsRNA.[3] synthesis of up to 80mers. Furthermore, the combination of
this coupling with the simple N-acetyl protection at the
siRNA in Mammalian Cells exocyclic amino groups of the nucleobases enables a reliable
Although RNAi has become routine in many laboratories and complete two-step deprotection, first with MeNH2 in
and has been studied for a wide range of organisms, its use in EtOH/H2O, followed by Bu4NF in THF, without concomitant
mammalian cells has long been considered problematic. degradation of the RNA products. The HPLC chromatograms
However, with the understanding of siRNAs as key compo- of these RNA species are comparable to those obtained for
nents in RNAi, the breakthrough for RNAi applications in the corresponding DNA sequence homologues. The TOM
mammalian cells has followed. The problem was that dsRNA method is offered commercially as an oligoribonucleotide
in mammalian cells activates a nonspecific viral defense production service, and the nucleotide building blocks are also
mechanism, the interferon response, which results in a global available commercially, which has contributed to the fast
shutdown of protein synthesis and leads to cell death.[6] This propagation of the method.[11] This availability resulted, for
pathway masks any sequence-specific effects that might arise example, in the development of a solid-phase approach for the
from an RNAi pathway. However, the nonspecific pathway preparation of small circular RNA species, and in the usage of
requires longer dsRNA, and does not occur with dsRNAs a variety of TOM-protected modified nucleoside building
shorter than 30 bp. So, Tuschl and co-workers tested the blocks.[12] A further strength of the TOM method is that it can
ability of siRNA to target various luciferase transgenes in be easily combined with the existing large pool of nucleoside
mammalian cell cultures (COS-7, NIH/3T3, HeLa, and labelling and marker building blocks.
293 cells) and indeed observed reproducible, sequence-spe-
cific siRNA inhibition, which was easily assayed by lumines- The 2'-O-ACE Method
cence spectroscopy.[7] In addition, siRNAs were also effective
when targeting endogenous genes. Non-sequence-specific The second novel method for the chemical synthesis of
effects started to emerge for the transfection with 50 bp and RNA was introduced by S. Scaringe and co-workers, and
longer RNAs, which suggested that both pathways (RNAi and represents an impressive strategy which was first communi-
interferon response) can operate simultaneously. However, cated in 1998, recently followed by the detailed procedures.[13]
gene expression in mammalian cells was not eliminated Based on the literature, mildly acidic aqueous conditions were
completely, as it was observed in the reference system of considered the most desirable for the final 2'-O deprotection
Drosophila cells.[7] That apart, the work above represents a of the synthesized RNA. The loss of orthogonality in the
landmark for siRNA as the upcoming gene-silencing method- combination with the 5'-O-DMT group was an obstacle to
ology which, at this time, seems to be more promising than the using a mildly acid-labile 2'-O protecting group. The new
alternatives, which include antisense and ribozyme-based RNA synthesis strategy is therefore based on the fluoride-
strategies. labile 5'-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether
(DOD), together with the 2'-O-bis(2-acetoxyethyloxy)methyl
Two Major Improvements in the Chemical Synthesis of RNA (ACE) orthoester (Scheme 1 b,c, Table 1). The 3'-OH group is
derivatized as the methyl-N,N-diisopropylphosphoramidite,
Chemically synthesized RNA oligonucleotides are key as the cyanoethyl group turned out to be unstable with
components of RNAi technology. Although the widespread fluoride reagents. The coupling yields are higher than 99 %, in
5'-O-dimethoxytrityl(DMT)-2'-O-tert-butyldimethylsi- less than 90 s and are therefore also superior to those
lyl(TBDMS) phoshoramidite chemistry enables their syn- observed for the TBDMS building blocks.
thesis, this method has not reached the level of chemical DNA After the oligonucleotide assembly, the phosphate methyl
synthesis in terms of product quality and the accessible protecting groups are removed with disodium 2-carbamoyl-2-
oligonucleotide length.[8, 9] In the need for more robust RNA cyanoethylene-1,1-dithiolate trihydrate (S2Na2) in DMF
routine-synthesis strategies, two novel methodologies have (Scheme 2). Then basic conditions (40 % aqueous MeNH2)
been developed and demonstrated to be successful. cause oligonucleotide cleavage from the solid support, along
with the removal of the acyl protecting groups on the
The 2'-O-TOM Method
exocyclic amino groups and, importantly, of the acetyl groups
The first improvement was achieved by S. Pitsch and co- on the 2'-orthoesters. The resulting 2'-O-bis(2-hydroxyethyl-
workers and maintains the principle of 5'-O-DMT-protection oxy)methyl orthoesters are ten times more acid labile than
and 3'-O-(2-cyanoethyl)diisopropylphosphoramidite coupling prior to the removal of the acetyl groups; very mild acidic

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 HIGHLIGHTS

Scheme 1. a) Nucleoside building blocks used in the 2'-O-TOM method (other nucleobases: N4-acetylcytosine, N2-acetylguanine, uracil); b) nucleoside
building block used in the 2'-O-ACE method (other nucleobases: N4-acetylcytosine, N2-isobutyrylguanine, uracil); c) General scheme for the automated
synthesis of oligoribonucleotides. PG ˆ protecting group.

conditions (pH 3.8, 30 min, 60 8C) are therefore required approach as this precursor RNA is water soluble, can be
for the final deprotection step. The HPLC chromatograms analyzed by HPLC, and purified if necessary. Of further
of the crude RNAs impress by showing hardly any by- significance is that the 2'-O-bis(2-hydroxyethyloxy)methyl
products. protection of the precursor oligoribonucleotide appears to
Unequivocally, the access to the 2'-O-bis(2-hydroxyethyl- interrupt secondary structures. This was demonstrated by the
oxy)methyl oligoribonucleotide is a major strength of the new synthesis of a 23mer homopolymer of guanosine.[13d]

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 HIGHLIGHTS
Table 1. Conditions for a complete coupling cycle in the 2'-O-TOM method and in the 2'-O-ACE method, and for the subsequent deprotection of the
synthesized oligoribonucleotide.
2'-O-TOM method (1.5 mmole scale) 2'-O-ACE method (0.2 mmole scale)
Removal of the 5'-O-PG 4 % dichloroacetic acid in 1,2-dichloroethane, 90 s 1.1 m HF/2.9 m triethylamine in dimethylformamide, 35 s
Activation/Coupling 0.25 m benzylthiotetrazole (65 equiv)/0.1m cyanoethyl- 0.5 m ethylthiotetrazole (75 equiv)/0.1m methylphosphor-
phosphoramidite (6 equiv) in CH3CN, 90 s amidite (15 equiv) in CH3CN, 90 s
Capping Ac2O/2,6-lutidine/THF (1/1/8; v/v) and N-methylimidazole/ Ac2O/CH3CN (1/9; v/v) and N-methylimidazole/CH3CN
THF (16/84; v/v), 60 s (1/9; v/v), 30 s
Oxidation I2/H2O/pyridine/THF (3/2/20/75; w/w), 45 s 1m tert-butylhydroperoxide in toluene, 45 s
Oligonucleotide deprotection 1) 10 m MeNH2 in EtOH/H2O; 1 ± 24 h, 25 ± 33 8C 1) 1m disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate
2) 1m Bu4NF ¥ 3 H2O in THF; 1 ± 50 h, 25 8C trihydrate (S2Na2) in DMF; 15 min
3) 1m Tris ¥ HCl, H2O, pH 7.4 2) 40 % MeNH2 in H2O; 1 h, 55 8C
3) 100 mm tetramethylethylendiamine/acetic acid, pH 3.8;
30 min, 60 8C

Both methodologies, the 2'-O-TOM method and

O O the 2'-O-ACE routes are major improvements in the
BN-Acyl BN-Acyl
synthesis of RNA oligonucleotides and offer unpre-
O O
O O
cedented product quality. With respect to the incor-
O
poration of modifications, the TOM chemistry ap-
O O O O O
O +_ O pears to have a slight advantage at the moment, as it
H3CO P O Na O P O
O
S2Na2 O benefits from the large existing ™modifier∫ pool
O O O O
BN-Acyl BN-Acyl developed for use with TBDMS. It will be interesting
O O O O to see if, in the long term, the 2'-O-ACE method is
O O able to take the place of the 5'-O-DMT method, which
O O O O O O O O is now strengthened by the TOM protecting group.
O O
O O
O O
[1] A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver,
O C. C. Mello, Nature 1998, 391, 806 ± 811.
O
CH3NH2 [2] For a Highlight on RNA interference see: a) U. Schepers, T.
Kolter, Angew. Chem. 2001, 113, 2503 ± 2505; Angew. Chem.
Int. Ed. 2001, 40, 2437 ± 2439; for reviews see, for example:
O
O NH 2
b) T. Tuschl, ChemBioChem 2001, 2, 239 ± 245; c) R. Barn-
B
BNH 2 stead, Curr. Opin. Chem. Biol. 2001, 5, 63 ± 66.
O
O [3] S. M. Elbashir, W. Lendeckel, T. Tuschl, Genes Dev. 2001, 15,
188 ± 200.
O OH + _ O O O
OH [4] S. Parrish, J. Fleenor, S. Xu, C. Mello, A. Fire, Mol. Cell 2000,
+_ Na O P O 6, 1077 ± 1087.
Na O P O pH 3.8
O [5] E. Bernstein, A. A. Caudy, S. M. Hammond, G. J. Hannon,
O OH
O Nature 2001, 409, 363 ± 366.
BNH 2
BNH 2 [6] G. R. Stark, I. M. Kerr, B. R. Williams, R. H. Silverman,
O
O R. D. Schreiber, Annu. Rev. Biochem. 1998, 67, 227 ± 264.
[7] S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K.
OH O O
OH OH OH Weber, T. Tuschl, Nature, 2001, 411, 494 ± 498.
water-soluble [8] G. M. Blackburn, M. Gait, Nucleic Acids in Chemistry and
O
precursor RNA OH Biology, 2nd ed., Oxford University Press, Oxford, UK,
1996.
Scheme 2. Deprotection of an oligoribonucleotide synthesized by the 2'-O-ACE
[9] K. K. Ogilvie, K. L. Sadana, E. A. Thompson, M. A. Quil-
method proceeds via a water-soluble precursor RNA which cannot form strong
liam, J. B. Westmore, Tetrahedron Lett. 1974, 15, 2861 ± 2863.
secondary structures and is therefore easy to analyze by HPLC.
[10] a) X. Wu, S. Pitsch, Nucleic Acids Res. 1998, 26, 4315 ± 4323;
b) ™Ribonucleoside-derivative and method for preparing the
same∫: S. Pitsch, P. A. Weiss, L. Jenny, US Patent 5,986,084,
The 2'-O-ACE method can be used on commercial 1999; c) S. Pitsch, P. A. Weiss, L. Jenny, A. Stutz, X. Wu, Helv. Chim.
Acta 2001, 84, 3773 ± 3795.
automated DNA synthesizers after some technical adjust-
[11] a) M. H¸nges, C. Rolz, R. Gschwind, R. Peteranderl, F. Berglechner,
ments. This chemistry has been commercialized as an G. Richter, A. Bacher, H. Kessler, G. Gemmecker, EMBO J 1998, 17,
oligoribonucleotide production service, however, the corre- 4092 ± 4100; b) M. C. Nagan, P. Beuning, K. Musier-Forsyth, C. J.
sponding nucleotide building blocks are not yet commercially Cramer, Nucleic Acids Res. 2000, 28, 2527 ± 2534; c) M. Br‰nnvall,
available. First reports on the incorporation of nucleoside N. E. Mikkelsen, L. A. Kirsebom, Nucleic Acids Res. 2001, 29, 1426 ±
1432.
modifications also exist and, just recently, the combined [12] Small circular RNA species: a) R. Micura, Chem. Eur. J. 1999, 5,
chemical and enzymatic synthesis of tRNAs for high-through- 2077 ± 2082; b) R. Micura, W. Pils, K. Grubmayr, Angew. Chem. 2000,
put crystallization was published.[14] 112, 956 ± 959; Angew. Chem. Int. Ed. 2000, 39, 922 ± 926; modified

2268 ¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002 1433-7851/02/4113-2268 $ 20.00+.50/0 Angew. Chem. Int. Ed. 2002, 41, No. 13
 HIGHLIGHTS
nucleosides: c) R. Micura, W. Pils, C. Hˆbartner, K. Grubmayr, M.-O. c) S. A. Scaringe, Methods Enzymol. 2000, 317, 3 ± 18; d) S. A.
Ebert, B. Jaun, Nucleic Acids Res. 2001, 29, 3997 ± 4005; d) V. Boudou, Scaringe, Methods 2001, 23, 206 ± 217.
J. Langridge, A. Van Aerschot, C. Hendrix, A. Millar, P. Weiss, P. [14] Nucleoside modifications: a) M. Meroueh, P. J. Grohar, J. Qiu, J.
Herdewijn, Helv. Chim. Acta 2000, 83, 152 ± 161; e) C. Hˆbartner, M.- SantaLucia, Jr., S. A. Scaringe, C. S. Chow, Nucleic Acids Res. 2000,
O. Ebert, B. Jaun, R. Micura, Angew. Chem. 2002, 114, 619 ± 623; 28, 2075 ± 2083; b) H. M.-P. Chui, M. Meroueh, S. A. Scaringe, C. S.
Angew. Chem. Int. Ed. 2002, 41, 605 ± 609. Chow, Bioorg. Med. Chem. 2002, 325 ± 332; tRNAs: c) L. D. Sherlin,
[13] a) S. A. Scaringe, F. E. Wincott, M. H. Caruthers, J. Am. Chem. Soc. T. L. Bullock, T. A. Nissan, J. J. Perona, F. J. Lariviere, O. C. Uhlen-
1998, 120, 11 820 ± 11 821; b) S. A. Scaringe, US Patent 6,111,086, 1998; beck, S. A. Scaringe, RNA 2001, 7, 1671 ± 1678.

Hydrides and Iodides of Gold

Margaret-Jane Crawford* and Thomas M. Klapˆtke*

The synthesis of gold hydrides in the solid state has long tions.[10] The structures of AuI3 and AuH3 , however, were
been desirable. Despite early investigations by Wiberg found to be a complex problem, not least because of the
et al.,[1] to prepare AuH3 utilizing a variety of reducing agents decreasing stability of the gold trihalides with increasing mass
such as LiAlH4 , AlH3 , and LiBH4 , no direct evidence for the of the halide.[11±14] For AuH3 , the lowest-energy isomer was
elusive AuH3 could be obtained and only decomposition found not to be either the T-shaped or linear structure, but
products, that is, Au and H2 could be detected. Despite, or rather, a Y-shaped structure (singlet electronic state) which is
possibly because, of the lack of experimental evidence for better viewed as an adduct between AuH and H2 .
gold hydrides in the solid state, a considerable number of Bayse recently reported detailed quantum-chemical DFT
theoretical studies have probed the structure of AuH and by studies of the AuH3/Au2H6 system, and suggested that in
using density functional methods.[2±6] Moreover, in the past AuH3 the AuH and H2 units would be only loosely bound
few years, the chemistry of the gold halides and hydrides has together [Eq. (1)].[3]
received a great deal of attention, and utilizing a combination
DEdiss ˆkJ mol 1

of computational and experimental techniques, the structures HAu(h2-H2) ! AuH ‡ H2 (1)

of many of the gold halides have been shown to agree with
Moreover, with respect to the dimerization of AuH3 to form
earlier structural predictions made by Schwerdtfeger et al.[5±7]
Au2H6 , the ™classical∫ square-planar, D2h structure of Au2H6
The similar electronegativities (cAR I ˆ 2.2; cAR H ˆ 2.2;
was reported to be the only isomer located. The dimerization
cAR Au ˆ 2.4)[6 b] of I and H, makes a comparison between
of both the Y- and T-shaped isomers of AuH3 , which formed
gold hydride and iodide compounds relevant. Whereas AuH is
D2h Au2H6 , was found in both cases to be an exothermic
a stable diatomic molecule that has been characterized in the
process by 84 and 305 kJ mol 1, respectively (Figure 2).
gas phase, and although the analogous diatomic AuX species
The bonding in the lowest-energy Y-shaped isomer of AuH3
(X ˆ F, Cl, Br) have been know in the gas phase for some time,
it was only very recently that the gas-phase structure of AuI
was determined by microwave spectroscopy.[8] However,
whereas AuH was until very recently unknown in the solid
state, AuI is a well-known and
even commercially available (!)
polymer, which is constructed of
a zigzag chain with linear I-Au-I
units (Figure 1).[9] The unusual
chainlike structure found for AuI
can be explained by relativistic
effects (as opposed to correlation
Figure 1. Solid state struc- effects) which show an increased
ture of gold(i) iodide. covalency in the Au I interac-

[*] Dr. M.-J. Crawford, Prof. Dr. T. M. Klapˆtke

Department of Chemistry
Ludwig-Maximilians University, Munich
Butenadtstrasse 5 ± 13 (Haus D), 81377 Munich (Germany)
Fax: (‡ 49) 89-2180-7492 Figure 2. Relative energies of various gold hydride species (TS ˆ transi-
E-mail: [email protected], [email protected] tion state).

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