Making cyclic RNAs easily available

Article abstract of DOI:10.1039/a904851k

A simple solid-phase procedure allows cyclic oligoribonucleotides to be obtained as long as the linear precursor attached to the support has a 2'-deoxyribonucleoside or a 2'-O-methylribonucleoside at the 3'-end.

Full text of DOI:10.1039/a904851k

Making cyclic RNAs easily available
Miriam Frieden, Anna Grandas and Enrique Pedroso*
Departament de Qu´ımica Orga`nica, Facultat de Qu´ımica, Universitat de Barcelona, Mart´ı i Franque`s 1-11, 08028
Barcelona, Spain. E-mail: pedroso@admin.qo.ub.es
Received (in Cambridge, UK) 17th June 1999, Accepted 13th July 1999
A simple solid-phase procedure allows cyclic oligoribonu-
cleotides to be obtained as long as the linear precursor
attached to the support has a 2A-deoxyribonucleoside or a 2A-
O-methylribonucleoside at the 3A-end.
attempts to prepare small cyclic RNAs (2- to 6-mer) employing
the Fpmp group were completely unsuccessful: very low yields
and impure crude products were obtained. We reasoned that the
key difference with respect to cyclic DNA synthesis was the
presence of the bulky 2A-O-Fpmp group at the 3A-end of 2 that
may be hindering the cyclization reaction. In order to test the
validity of this assumption, three nucleotide resins 1 having
differently hindered phosphate groups were prepared as pre-
viously described14 on an NH2-TentaGel™ resin, 1a (BA = T,
RA = H), 1b (BA = U, RA = OMe) and 1c (BA = U, RA - OFpmp),
and their homogeneity was assessed by gel-phase 31P NMR.†
On these nucleotide-resins a series of dinucleotides were
assembled, cyclized, cleaved and deprotected.‡ Nucleoside
sequences, yields and mass spectrometric data are indicated in
Table 1 (entries 1 to 5). These results clearly indicate that the
lowest yields were obtained when the nucleotide-resin 1c was
employed (entries 4 and 5). In fact, c(UU) could not be isolated
from the complex crude mixture, whilst the rest of the cyclic
dinucleotides were easily purified by C18-HPLC chromatog-
raphy and unequivocally characterized by mass spectrometry
and enzymatic digestion.14 Particularly striking is the difference
in yield between the two syntheses of c(UT) [ = c(TU)] (entries
3 and 4): much lower yield was obtained from the most hindered
resin 1c than from 1a, thus demonstrating that steric hindrance
in the vicinity of the 3A terminus phosphate of linear precursor 2
plays a key role. Nevertheless, comparing the results of entries
4 and 5 we can assert that the rest of the chain has a non-
negligible effect. Most probably, it hinders the dinucleotide in
attaining a reactive conformation.
Some of the smallest oligoribonucleotides with known bio-
logical activity are cyclic molecules. For instance, c(GG) is an
activator of cellulose synthase in Acetobacter xilinum,1 and
c(UU) and c(AU) are inhibitors of the DNA dependent RNA
polymerase of E. coli.2 At the other end of the scale, large cyclic
RNAs are formed in the splicing processes of ribonucleic acids
in certain organisms3 and viroids have circular single stranded
RNA as their genomic material.
The structure of cyclic RNAs has been determined either by
X-ray diffraction4 or by NMR5 only for very small molecules.
Cyclic RNAs may be useful models for the study of a variety of
RNA structural motifs,6 such as hairpin loops, which could
bring new insights into the structure–function relationship of
ribonucleic acids.
In spite of their wide potential applications, few efforts have
been dedicated so far to the development of chemical methods
for the preparation of cyclic oligoribonucleotides. The synthesis
of cyclic dinucleotides7 and tetranucleotides8,9 has been
reported using the phosphotriester7,8 or the H-phosphonate9
methods in solution, whereas a solid-phase method has been
employed for the preparation of cytidine homooligomers.10
Larger cyclic RNAs have been obtained either by template-
directed chemical11 or enzymatic12 ligation, and by rolling
circle transcription and self-processing of circular DNA
oligonucleotides encoding hairpin ribozymes.13
We have recently described a straightforward solid-phase
procedure for obtaining small to medium-sized cyclic oligo-
deoxyribonucleotides.14 The main advantage of the method is
that, after the cyclization and cleavage reactions, non-cyclized
chains, polymers and other by-products remain attached to the
solid matrix. Therefore, fairly pure crude products are obtained,
regardless of the size of the circles and the sometimes low
cyclization yields. Scheme 1 shows the key steps in the
extension of this methodology to the preparation of cyclic RNA.
Synthesis of the ribonucleotide-resin 1 and chain elongation by
the phosphite-triester approach using 2-cyanoethyl (CNE)
phosphoramidites yields the oligonucleotide-resin 2 which,
upon cyclization with 1-mesitylenesulfonyl-3-nitro-1,2,4-tria-
zole (MSNT), affords 3. After oximate cleavage of 3 and
deprotection the cyclic RNA is obtained.
The choice of a suitable 2A-OH protecting group is the most
crucial decision in RNA synthesis. It is well documented that
the last step of the synthesis must be the deprotection of the 2A-
hydroxy functions to avoid strand cleavage or 3A–5A to 2A–5A
migration of the phosphodiester linkages.15 The commonly
used TBDMS group was discounted because of its lack of
stability towards the oximate cleavage treatment.16 Early
elimination of the TBDMS group of the ribonucleoside directly
attached to the support may result in the above mentioned
undesirable side reactions.
For this reason, we turned our attention to the acid-labile
1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group (Fpmp),17
whose stability to oximate was confirmed using 2A-O-Fpmp-
uridine (data not shown). However, to our surprise, the first
Scheme 1
Chem. Commun., 1999, 1593–1594
1593

Table 1 Synthesized cyclic oligoribonucleotides
Mass spectrometry
calc. found
608.09
624.08
610.07
610.07
Crude
Purity (%)
(HPLC)
Entry
Oligonucleotide-resin
Cyclic oligonucleotide yield (%)
1
2
3
4
5
6
7
8
9
T-T-R
T-UOMe-R
UOFpmp-T-R
T-UOFpmp-R
c(TT)
50
43
48
14
3
19
33
7
> 90
> 90
> 90
80
—
75
69
57
70
80d
80d
608.29b
624.20b
610.39b
610.39b
c(TUOMe
c(UT)
c(TU)
c(UU)
c(UUUT)
c(CUCT)
c(UUUUUT)
c(UGCUUGCT)
c(UAUAGUOMe
)
UOFpmp-UOFpmp-R
612.09
—
UUU-T-Ra
1222.12
1220.15
1834.17
2523.49
1935.57
1934.26
1222.14c
1222.20c
1834.76c
2521.39c
1933.56c
1932.84c
CUC-T-Ra
UUUUU-T-Ra
UGCUUGC-T-Ra
UAUAG-UOMe-Ra
UAGCA-UOMe-Ra
20
5
4
10
11
)
)
c(UAGCAUOMe
a Ribonucleosides were 2A-O-Fpmp protected. b Electrospray MS. c MALDI-TOF MS. d Estimated by PAGE.
for the cyclization reaction are: 0.15 M MSNT in pyridine, three treatments
(4 h + 4 h + overnight) with 20 equiv. MSNT each. Conditions for the
deprotection and cleavage reactions are: (i) Et3N–pyridine (1+1), 3 3 1 h;
(ii) 0.2 M tetramethylguanidinium syn-pyridine-2-aldoximate in dioxane–
water (1+1), 4 h + 4 h + overnight with 50 equiv. oximate; (iii) conc.
aqueous NH3, 55 °C, 12 h; (iv) 0.5 M AcONa, pH 4, 12 h, and then
neutralization with 3 M Tris·HCl, pH 8.
The synthesis of some larger circles was undertaken to
further evaluate the possibilities of the method. Several cyclic
oligoribonucleotides were obtained at the 1–2 mmol scale from
resins 1a and 1b. Results are shown in Table 1 (entries 6 to 11).
The cyclic products were submitted to Sephadex G-10 gel
filtration and HPLC or PAGE purification, and were charac-
terized as above.
Yields of crude products are generally lower than those
typically obtained in the synthesis of cyclic DNA. The low yield
obtained for c(U5T) (entry 8) and the difference in yield
between the two cyclic tetramers (entries 6 and 7) reflect the
difficulty in getting high and reproducible yields in the
cyclization reaction with MSNT. A 2A-O-methylribonucleotide
at the 3A end of the linear precursor seems to have a negative
effect on the cyclization yield of the hexamers (entries 10, 11),
which was not observed for a dinucleotide (entry 2). The
homogeneity of the crude products detached from the resin is
also slightly lower than in cyclic DNA synthesis. However, a
major HPLC peak or PAGE band is always obtained, thus
allowing easy purification of the circle and showing that the key
advantage of the method is preserved.
In conclusion, cyclic RNA can be obtained provided that the
linear precursor attached to the support has a 2A-deoxy-
ribonucleoside or a 2A-O-methylribonucleoside at the 3A end.
Such single modification in the sequence of the cyclic RNAs
should have little relevance for many purposes. For instance,
enzymatically circularized hammerhead ribozymes containing
non-nucleoside linkers have been shown to display increased
biological activity and reduced divalent metal ion require-
ment.18
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Work is in progress to prepare larger, ‘all-ribonucleoside’
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hindrance at the 3A end phosphate.
This work was supported by the Ministerio de Educacio´n
(DGES, grant PB97-941) and the Generalitat de Catalunya
(Centre de Refere`ncia de Biotecnologia and SGR98-1).
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Notes and references
† Selected 31P NMR data (121.4 MHz, CDCl3) for 1a: dP 25.12. For 1b: dP
26.99. For 1c: dP 26.90.
‡ Oligonucleotide chains were assembled using 5A-O-DMT-nucleoside 3A-
cyanoethylphosphoramidites (2A-O-Fpmp-protected, when required) and
tetrazole for the coupling step, and ButOOH for the oxidation. Conditions
Communication 9/04851K
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Chem. Commun., 1999, 1593–1594