Synthesis of 4-thiothymine based photolabels as new tools for nucleic acids structural studies in solution: Formation of long-range photo-cross- links within a hammerhead ribozyme domain

Article abstract of DOI:10.1016/S0040-4020(00)00024-7

The nucleosidic photolabels 2-5, able to form long-range photo-cross- links, have been used to further investigate the tertiary folding of a hammerhead ribozyme domain. These photolabels derive from 2'-deoxyuridine which is substituted at its C-5 position

Full text of DOI:10.1016/S0040-4020(00)00024-7

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 1197–1206
Synthesis of 4-Thiothymine Based Photolabels as New Tools for
Nucleic Acids Structural Studies in Solution: Formation of
Long-Range Photo-Cross-Links within a Hammerhead
Ribozyme Domain
a,b,†
a
b
b
Pascale Clivio,a, Jean-Louis Fourrey, Anne Woisard, Philippe Laugaa
*
´
ˆ
Carole Saintome,
and Alain Favreb
aInstitut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-Yvette, France
b
´
´
Laboratoire de Photobiologie Moleculaire, Institut Jacques Monod, CNRS, Universites Paris 6 et Paris 7, 2 Place Jussieu, 75251 Paris
Cedex 05, France
Received 15 October 1999; accepted 23 December 1999
Abstract—The nucleosidic photolabels 2–5, able to form long-range photo-cross-links, have been used to further investigate the tertiary
folding of a hammerhead ribozyme domain. These photolabels derive from 20-deoxyuridine which is substituted at its C-5 position with a
photoactivable 4-thiothymine unit linked by a chain of various length and rigidity. Derivatives 2–5 were inserted at strategic positions of a
deoxysubstrate analogue (dS) of a hammerhead ribozyme (Rz). Covalent cross-links were generated by 366 nm irradiation of the Rz–dS
complexes assembled under cleavage conditions. The Rz residues involved in these cross-links were mapped to give new sets of proximity
data extending those previously obtained by intrinsic photolabelling with 20-deoxy-4-thiouridine 1. Thus, compared to the zero-length cross-
linker 1, photolabels 2–5 exhibit a higher exploration capacity which depends critically upon the length and flexibility of the linker and their
site of incorporation. Interestingly, in contrast to 1, these photolabels were able to cross-link residues involved in the double G:A mismatch as
well as residues of the adjacent base pairs. These findings suggest that the mismatch domain exhibits an unexpected conformational
flexibility in solution. ᭧ 2000 Elsevier Science Ltd. All rights reserved.
Introduction
of thionucleosides, such as 20-deoxy-4-thiouridine (ds4U)7 1
and 20-deoxy-6-thioinosine (ds6I).8 These nucleosides were
incorporated at selected positions of an all-deoxy ribozyme
substrate analogue in place of thymidine to be irradiated in
the presence of a 35-mer ribozyme. These photoaffinity
studies allowed the identification of contacts between
residues of the substrate cleavage site and residues of the
50-conserved region of the ribozyme. Evidence for inter-
actions between the 30-end of the substrate and the stem
loop II has also been obtained by this method. A three
dimensional structure accounting for these proximity data
has been modelled.9 This model is not only in good overall
agreement with the crystalline structure but, additional
studies revealing that cross-links within complexes involv-
ing cleavable deoxysubstrate analogues retained activity,
underlined its biological significance.10 On the other hand,
whereas crystallographic studies showed magnesium ions to
induce a weak conformational motion of the catalytic core,11
it was subsequently suggested that more important confor-
mational changes in the global crystalline structure were
needed to reach the active conformation.12
Because of their remarkable endonuclease activity great
attention is currently paid to hammerhead ribozymes.1
When combined with their RNA substrate, these RNA
molecules form a triple junction with the three double-
stranded stems, I, II and III, connected to a core of
conserved residues which are essential for catalytic activity.
The crystal structures of two bipartite hammerhead
ribozyme domains have been solved.2 In both cases, the
system adopts a Y-shaped structure with stems I and II
side by side, in agreement with the results derived from
the conformation studies in solution.3–5 However, in the
crystal structure, the conformation at the cleavage site
does not allow an in-line attack of the scissile phospho-
diester bond by the adjacent deprotonated 20-hydroxyl as
shown by the experimental data.6 In order to approach the
problem of the solution structure of a hammerhead domain,
we have taken advantage of the photo-cross-linking ability
Keywords: photolabels; hammerhead ribozyme; nucleic acids.
* Corresponding author. Fax: ϩ33-1-69-07-72-47;
e-mail: pascale.clivio@icsn.cnrs-gif.fr
In order to gain further informative data on the conforma-
tional mobility of the hammerhead ribozyme domain, we
†
Fax: ϩ33-1-44-27-35-80; e-mail: saintome@jim.jussieu.fr
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00024-7

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C. Saintome et al. / Tetrahedron 56 (2000) 1197–1206
1198
have synthesized a new series of photoactivable oligo-
deoxynucleotide probes. These probes consist of 14-mer
substrate analogues dS of the 35-mer ribozyme Rz
incorporating the modified 20-deoxyuridine units 2–5
(Fig. 1)13 whose photo-cross-linking abilities have already
been tested in a double helix context.14 Herein, we describe
the preparation of 2–5 along with their incorporation into
oligonucleotide probes and establish their capacity to serve
as valuable structural tools to investigate the hammerhead
ribozyme domain folding.
Results and Discussion
Synthesis of photolabelled deoxysubstrate analogues
The preparation of the suitably protected phosphoramidites
18a, 18b, 19 and 20 (Scheme 1) is a prerequisite for a
convenient incorporation of nucleosides 2–5 into
Figure 1. Structure of modified nucleosides 1–5.
Scheme 1. (i) Phosphorus pentasulfide; (ii) 2N aqueous NaOH, H3Oϩ; (iii) Pivaloyloxymethyl chloride, K2CO3; (iv) DCC, N-hydroxysuccinimide; (v)
Propargylamine; (vi) Ethylenediamine; (vii) 1,4-Diaminobutane; (viii) 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine;
(ix) (Ph3P)4Pd, CuI, triethylamine.

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C. Saintome et al. / Tetrahedron 56 (2000) 1197–1206
1199
Figure 2. Ribozyme residues involved in cross-links. (Conserved residues are indicated in bold letters, variable residues of Rz are in outlined letters and photo-
labelled nucleosides are circled.) A: Structure of the hammerhead ribozyme domain with nucleoside 4 at positions 1.6 and 16.7 on dS; B: Nucleoside 2 at
position 1.1; C: Nucleoside 5 at position 1.1; D: Nucleoside 2 at position 16.1; E: Nucleoside 5 at position 16.1. For ribozyme domain numbering see: Nucleic
Acids Res. 1992, 20, 3252.
oligonucleotides by chemical synthesis. Intermediates 15a,
15b and 16, the immediate precursors of 18a,b and 19, were
obtained by acylation of an amino substrate by 13, an
activated derivative of acid 8. Thus, the amino-nucleosides
12a15 and 12b, prepared by trans-amination of the ester
function of the known intermediate 1115 with ethylene
diamine or 1,4-diaminobutane (90% yield), were treated
with 13, obtained by means of standard reactions, to give
amides 15a (77% yield) and 15b (34% yield), respectively.
In the case of 16, nucleoside 10 was first obtained in 70%
yield by overnight treatment of 916 in ethylenediamine.
Compound 10 was subsequently acylated by 13 to provide
16 (54% yield).
gave amide 14 (50% yield) which upon reaction with 9
according to Hobbs17 and Tong,18 provided 17 (44%
yield). Nucleosides 15a, 15b, 16 and 17 were subsequently
treated with 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite to give the corresponding phosphoramidites 18a,
18b, 19 and 20. The latter were used to prepare 12 photo-
labelled analogues dS of the ribozyme substrate Rz contain-
ing at position 1.1, 1.6, 16.1 and 16.7, in place of thymidine,
one of the four modified 20-deoxyuridines 2–5 (Fig. 1).
Photolabelling studies of a hammerhead ribozyme
domain
Cross-linking characterizations. The herein studied
hammerhead ribozyme domain consisted of a 35-mer (or
36-mer) ribozyme Rz and its 14-mer (or 15-mer) substrate
A more convergent route was defined for the preparation of
nucleoside 17. Indeed, treatment of propargylamine with 13

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C. Saintome et al. / Tetrahedron 56 (2000) 1197–1206
1200
analogue dS7 containing one of the photolabels 2–5 inserted
at a specific position (Fig. 2). The folding of this system
has been previously studied using the parent 20-deoxy-4-
thiouridine 1 as zero-length photo-cross-linker.7 Moreover,
in the presence of 14-mer deoxysubstrates containing a
ribocytidine at the cleavage site, Rz was shown to exhibit
closely similar cleavage activities with respect to its all-
RNA 14-mer substrate.7,8 In the present study, photolabels
2–5 were site-specifically inserted at positions either 1.1 or
16.1 of dS adjacent to the putative cleavage site to allow
exploration of the central core, whereas the two shorter
ones, 4 and 5, were also introduced at the 50 (16.7) and 30
(1.6) ends of dS (Fig. 2).
A).21,22 These distances were measured, in the crystalline2c
and solution9 structures, between the C-5 position of the
substituted residue and the middle of the target double
bond of the Rz residue involved in each cross-link (Tables
1 and 2).20 Obviously, these distances are only indicative as
they do not take into account possible steric hindrance and
internal motion.
Examination of Fig. 2 and Table 1 shows that photolabels
2–5 fall into two categories. To the first one belongs photo-
labels 2 and 3, characterized with long and flexible linkers,
whose photo-cross-linking behaviour is reminiscent of ds4U
1 with a larger exploration capacity. Whereas photolabels 4
and 5, with short and more rigid linkers, constitute the
second category to give cross-links which differ from
those obtained with either 2, 3 (at position 16.1) or ds4U 1
(at position 1.1).
In all experiments, an excess of each dS (1.2 mM) was
combined with 0.2 mM 50-32P-labelled Rz in the cleavage
buffer in the presence of Mg2ϩ. The mixtures were
irradiated at 15ЊC with 366 nm light to completion of the
reaction to give intermolecular Rz–dS cross-links which
were separated from each other and from unreacted Rz by
denaturing PAGE-8 M urea. Controls indicated that there
were no such reactions in the dark, neither when the
complex was irradiated in the absence of Mg2ϩ nor in the
presence of unmodified dS. Long separating gels (40 cm)
were used since in some cases (with photolabels 2 and 3
at positions 1.1 and 16.1) up to 15 distinct cross-linked
species were detected (Fig. 3). These species were isolated,
repurified if necessary, and sequenced as previously
described.7,8,19 With a few exceptions, which may represent
either mixtures of cross-linked species or chemically
unstable cross-linked structures under alkaline conditions,
each of these species yielded clear-cut windows on the
sequencing gel allowing the determination of the ribozyme
residues involved in the cross-links. In a few cases, two
closely migrating cross-linked species were shown to
covalently bind the same ribozyme residue. These doublet
species may be due to distinct or isomeric photoadducts as
previously discussed.19 For each dS, the efficiency of cross-
linking formation and their localizations on Rz have been
collected in Tables 1 and 2, which also show the results
previously obtained with ds4U 1, for comparison.7
Position 16.1. Incorporation of the zero-length photo-cross-
linker ds4U 1 at position 16.1, prior to the cleavage site,
yielded three cross-links with A2.1, U4 and U7.7 The one
with A2.1, unaccountable on the basis of the published
structures,2,9 and attributed to alternative conformations,7
was not observed with any of photolabels 2–5. However,
the other two (with U4 and U7) were both detected with
2–5:
• Since the long and flexible chains of 2 and 3 allowed s4T
to sweep the upper border of the catalytic pocket (C3, U4
and U7, G8), the absence of cross-link in its lower part
(G5 and A6 forming the U-turn) is ascribed to the cross-
linking polarity of the photolabel already observed in
double stranded oligonucleotides.14 Indeed, it has been
shown that the photolabel which emerges from the helix
major groove yielded cross-links with residues on the 30-
(but not 50-) side of the strand bearing the photolabel.14
Therefore, whereas A13 and A14 are accessible, residues
G5 and A6 and those of helix III are not suitably disposed
to be cross-linked.
• On the other hand, the shorter chain of 4 and 5 did not
allow s4T to reach C3 as observed above. The location of
the pyrimidine C-5 atom into the major groove and the
straight acetylenic bond bring the s4T moiety of 5 into the
vicinity of the GA mismatches. This might explain that,
compared with 4, a higher cross-linking yield was
observed with G12 whose N-7;C-8 double bond is readily
accessible. Photolabel 4 behaviour, with a short but
rather flexible linker, is intermediate: its cross-linking
pattern is similar to that of 5 with a higher photo-cross-
linking yield with U7 as observed with 2 and 3. All these
cross-links, with the exception of those with A13, which
will be discussed in more details below, are consistent
with the published structures.
Structural implications of the cross-links. As expected,
the cross-linking efficiency and the positions of the residues
of Rz involved in the cross-links vary according to the type
of photolabel considered as well as its position within the dS
sequence. The overall cross-linking efficiency (1–17%) is
usually two to three times lower than that observed with
ds4U 1.7 This is likely due to the fact that the 4-thiothymine
substituted photolabel explores a larger space allowing it to
yield intramolecular cross-links with residues of the
substrate analogue14 as well as to photolytic decomposition.
In view of interpreting the cross-link data in relation to the
crystalline2c and solution9 structures, several points should
be considered: (a) one should keep in mind that, for labels
2–5, the 4-thiothymine reactive C–S bond is expected to
Position 1.1. Incorporation of ds4U 1 at position 1.1, next to
the cleavage site, yielded three cross-links with U4, G5
and A6 of the catalytic pocket. Two of them (with U4
and A6) could be explained on the basis of the published
structures assuming a high mobility of residue 1.1 and/or of
the catalytic loop.7 The last one (with G5) is puzzling since
(i) the inter-residue distance (12–13 A) is larger than in the
other cases; (ii) A6 is sandwiched between residue C17 and
G5 in the 3D structure. However, these cross-links and
˚
˚
explore a space ranging from 3.5 to 15.5 A (2), 3.5–18 A
˚
˚
(3), 4.5–12 A (4), 7–12 A (5), from the C-5 centre of the
20-deoxyuridine moiety;20 (b) the distances between Rz–dS
cross-linked residues can be evaluated from the presumed
reactive sites as revealed in model reactions (C-5;C-6
double bond for U and C, N-7;C-8 double bond for
˚

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C. Saintome et al. / Tetrahedron 56 (2000) 1197–1206
1201
Table 2. Photo-cross-link data obtained when photolabels 1,7 4 and 5 are
inserted either at the 50- or 30-end of dS. For each labelled position, Rz
cross-linked residues are reported in column 2; the overall and relative
yields of cross-linking obtained with photolabels 1, 4 and 5 are reported
˚
in columns 3–5. In the last two columns are reported the distances A
between both partner residues as determined from X-ray2 (XR) or photo-
labelling derived molecular modelling9 (hn) structures
˚
Distances A
Labelled position Cross-linked residue
Photolabel
1
4
5
XR
hn
16.7
1.6
Overall yield
C15.6
A15.7
Overall yield
AL2.4
A2.6
25
60
100 40
8
45
6
12
7
5
7
5
100
2
10 25
3.8
6
9
5
55 94
90
5
and rigid side chain pointing towards the mismatch domain,
yielded cross-links mainly with U7, G8 and G12. Molecular
modelling shows that, when its linker is extended, 5 fills the
gap between positions 1.1 and G8 whose orientation of the
photo-reacting bonds is unfavourable (90Њ vs. an ideal value
of 180Њ). Assuming that our proposed photochemical path-
way involving the N-7;C-8 double bond of the purine is
operating in the present case,22 our observation highlights
the dynamics of the system in solution with the mismatch
domain undergoing reorientation to allow cross-link forma-
tion. The dynamics of the system is further illustrated by the
photo-cross-links formed between 2 and 3 and residue A13
in the mismatch domain. These cross-links occurred what-
ever the substituted position (16.1 or 1.1) in substantial
yield. Though they are compatible with the distances
given in Table 1, it should be noted that (i) residue A13 is
located on the opposite side of the mismatch and its access is
Figure 3. Electrophoretic separation on denaturing 15% polyacrylamide/
8 M urea gel of the photo-cross-linked products (Rz–dS) obtained upon
irradiation of Rz with dS containing 3 at position 16.1. Ribozyme residues
involved in the cross-links are indicated.
others could be observed with 2 and 3 since their long and
flexible linkers allow the photolabel to sweep not only the
whole catalytic loop but also a large number of residues
within helices I and II. On the other hand, 5 with its short
Table 1. Photo-cross-link data obtained when photolabels 1,7 2–5 are inserted either prior (16.1) or next (1.1) to the cleavage site of dS. For each labelled
position, Rz cross-linked residues are reported in column 2; the overall and relative yields of cross-linking obtained with photolabels (1–5) are reported in
2
˚
columns 3–7. In the last two columns are reported the distances A between both partner residues as determined from X-ray (XR) or photolabelling derived
molecular modelling9 (hn) structures
˚
Labelled position
16.1
Cross-linked residue
Photolabel
Distances A
1
2
3
4
5
XR
hn
Overall yield
A2.1
C2.2
C3
U4
U7
G8
G12
A13
A14
38
37
12
11
17
3
2
19
21
17
14
5
20
22
18
14
5
2
6
44
38
7
10
42
24
11
52
18
42
20
17
13
14
28
40
7
9
9
10
11
9
3
3
4
6
10
9
1.1
Overall yield
C2.5
C2.2
C3
U4
G5
40
8
15
5
10
18
7
7
16
7
6
24
3
10
5.5
14
15
9
11
8
4
10
25
7
6
48
15
17
16
7
7
13
9
12
8
A6
5
5
U7
G8
G10.1
C10.3
G12
11
8
1.5
8
4
12
12
21
25
11
14
22
27
20
22
11
11
17
20
14
18
1
1.5
14
8
A13
10

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C. Saintome et al. / Tetrahedron 56 (2000) 1197–1206
1202
hindered by G8; (ii) according to the putative reaction
mechanism, the formation of the cross-link might involve
the N-7 atom of A13 which is engaged in an hydrogen bond
stabilizing the mismatched A13:G8 base pair. This strongly
suggests that this region is highly mobile.
Experimental
General
Methyl thymin-1-ylacetate 6a,23 50-O-(4,40-dimethoxytrityl)-
5-iodo-20-deoxyuridine 9,16 50-O-[4,40-dimethoxytrityl]-5-
[3-methoxy-3-oxopropyl]-20-deoxyuridine 1115 and 50-O-
[4,40-dimethoxytrityl]-5-[3-[(2-amino)ethyl) amino]-3-oxo-
propyl]-20-deoxyuridine 12a15 were synthesized following
literature procedures. 1H, 13C, 31P NMR spectra were
recorded on Bruker AC 200, AC 250, AM 300 or AC
Positions 1.6 and 16.7. The above results obtained with the
four different labels introduced at positions 1.1 and 16.1
indicated a rather large dispersion of the cross-links when
using those having 4-thiothymine at the end of a long chain.
Accordingly, we reasoned that a higher selectivity should be
observed with probes having labels 4 and 5 at positions 1.6
and 16.7. When position 16.7 was labelled with 5, the sole
cross-linked residue was A15.7 (as observed with 1) while
both A15.7 and C15.6 were cross-linked with 4 having a
flexible side chain (Table 2). Cross-links were observed
between the ends of stems I and II (with photolabels 1, 4
or 5 placed at position 1.6), whereas none were detected
between the ends of stem III and either stem I or II, thus
further confirming the existence of the Y form in Mg2ϩ
solutions.10
1
300-P spectrometers. H chemical shifts (d) are reported
relative either to tetramethylsilane in CDCl3 or residual
solvent traces in CD3OD (3.3 ppm) or in DMSO-d6
(2.6 ppm). 13C chemical shifts (d) are reported relative to
CDCl3 (77.7 ppm) or to DMSO-d6 (39.7 ppm). 31P chemical
shifts (d) are reported relative to external 85% phosphoric
acid. Interchangeable attributions are asterisked. Mass
spectroscopy was carried out using AEI MS 50 (EI spectra)
or AEI MS 9 (CI spectra) or Kratos MS 80 (HRMS, L-SIMS
or FAB (thioglycerol matrix)) instruments. Tetrahydrofuran
(THF), dichloromethane and dimethylformamide (DMF)
were dried by heating, under reflux, with sodium/benzo-
phenone, calcium hydride and barium oxide after azeotropic
distillation with toluene, respectively. Acids, amines and
30-OH nucleosides were dried at room temperature (RT)
in a dessicator over phosphorus pentoxide under vacuum
overnight before amide condensation and phosphitylation.
Oligonucleotide syntheses were performed on an Applied
Biosystems 392 synthesizer. Nucleoside b-cyanoethyl-
phosphoramidites, columns and reagents were from
PerSeptive Biosystems. All reactions were carried out at
room temperature (RT) unless otherwise stated. Column
chromatography was performed on Merck 7729 silica gel.
Thin layer chromatography was run on precoated silica gel
sheets with fluorescent indicator (254 nm) manufactured by
Schleicher and Schuell (ref 394 732). Lichroprep RP 18
(Merk 13 900) was employed for reverse phase chromato-
graphy.
Conclusion
When photolabels 2–5 were inserted at a position, either
prior or next to the cleavage site, the cross-linking results
could be easily explained by analysis of the structures
derived from the crystal or solution model studies. While
most of the cross-linked sites were found in the catalytic
loop, one notices that when using 2 and 3, with long and
flexible linkers allowing the 4-thiothymine to sweep a large
domain, residues within helices I and II were reached. In
contrast, photolabels 4 and 5, with short and more rigid
linkers, were shown to exhibit a higher selectivity. More-
over, unlike ds4U 1, both photolabels were able to cross-link
residues involved in the double G:A mismatch. Short and
rigid photolabels cross-linked residues G8 and/or G12,
while long and flexible photolabels cross-linked A13. The
latter result can be accommodated with the published struc-
tures if one assumes important local motions involving (i)
opening the G8:A13 mismatch base pair which will give
access of the 4-thiothymine to the N-7;C-8 double bond of
A13; (ii) bringing this bond at a distance compatible with
the linker length and taking into account the steric hindrance
arising from the presence of G8 on the pathway followed by
the photoreactive moiety. We thus suggested that, under
cleavage conditions, the mismatch domain exhibits an
unexpected high mobility.
Methyl 4-thiothymin-1-ylacetate 6b. Methyl thymin-1-
ylacetate 6a23 (9.92 g, 50 mmol) was dissolved in dioxane
(250 mL) and the solution brought to reflux. Phosphorus
pentasulfide (14.6 g, 1.3 equiv.) was then added and the
mixture maintained to reflux for 1.5 h. After cooling to
RT, the reaction mixture was filtered on Celite and the
filtrate concentrated. Crystallization from methanol lead to
9.41 g of bright yellow crystals (88% yield). Recrystalliza-
tion from ethanol gave an analytical sample. Mp: 176ЊC. 1H
NMR (200 MHz; DMSO-d6), d (ppm): 12.92 (s,1H, N3–H),
7.80 (s, 1H, H6), 4.65 (s, 2H, N1–CH2), 3.80 (s, 3H, OCH3),
2.05 (s, 3H, CH3). 13C NMR (50 MHz; DMSO-d6), d (ppm):
191.5 (C4), 168.4 (CO2CH3), 148.6 (C2), 139.2 (C6), 117.5
(C5), 52.6 (CO2CH3), 49.2 (N1–CH2), 16.7 (CH3). CI-MS:
m/z 215 (MϩH)ϩ HRMS (CI) calculated for C8H11N2O3S
215.0490, found 215.0502. Calculated for C8H10N2O3S: C,
44.86; H, 4.71; N, 13.08; S, 14.94, found C, 45.14; H, 4.56;
N, 13.07; S, 14.88.
Photolabel 1 has already been inserted in a cleavable
deoxysubstrate analogue at the positions under study. It
has been observed that none of the species containing a
cross-link involving residues of the ribozyme core retained
cleavage activity,10 in contrast to those generated at the
ends of helices I and III or between helices I and the
loop closing helix II. The length and flexibility depen-
dent photo-cross-linking ability of photolabels 2–5 can
now be exploited to examine whether any of the cross-
linked species retain cleavage activity as a consequence of
enhanced conformational freedom. Work along these lines
is in progress.
4-Thiothymin-1-ylacetic acid 7. A suspension of methyl(4-
thiothymin-1-yl)acetate 6b (8.75 g, 41 mmol) in 2 N
aqueous NaOH (50 mL) was submitted to sonication for
10 min. Then, the solution was chilled to 0ЊC, acidified to

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C. Saintome et al. / Tetrahedron 56 (2000) 1197–1206
1203
pH 1 with 9.7N HCl and the resulting suspension filtered.
The precipitate was washed with cold water and dried at RT
in a dessicator over phosphorus pentoxide under vacuum to
to a gum which was purified by reverse phase chromato-
graphy eluting with H2O (50 mL), then 20% CH3CN in H2O
(240 mL) affording 12b (94 mg, 90% yield) as a foam. H
1
1
give 8.53 g of yellow crystals (96% yield). Mp: 228ЊC. H
NMR (300 MHz, DMSO-d6), d (ppm): 7.77 (t, 1H, NH
amide, Jˆ5.4 Hz), 7.53–7.27 (m, 10H, H6; ar DMT), 6.98
(d, 4H, ar DMT, Jˆ8.4 Hz), 6.26 (t, 1H, H10, Jˆ6.4 Hz),
NMR (300 MHz, DMSO-d6), d (ppm): 12.86 (s, 1H, N3–H),
7.77 (s, 1H, H6), 4.53 (s, 2H, N1–CH2), 2.05 (s, 3H, CH3).
13C NMR (62 MHz, DMSO-d6), d (ppm): 191.5 (C4), 169.3
(COOH), 148.8 (C2), 139.6 (C6), 117.4 (C5), 49.4 (N1–
CH2), 16.8 (CH3). FABϩ-MS: m/z 201 (MϩH)ϩ. Calculated
for C7H8N2O3S·H2O: C, 38.53; H, 4.62; N, 12.84; S, 14.66;
found C, 38.66; H, 4.48; N, 12.53; S, 14.65.
0
0
4.31 (m, 1H, H3 ), 3.95 (m, 1H, H4 ), 3.83 (s, 6H, OCH3
DMT), 3.28 (m, 2H, H50; H500), 3.06 (m, 2H, 2 H11), 2.60
(m, 2H, 2 H14), 2.43–2.17 (m, 6H, H20; H200; 2 H7 and 2
H8), 1.42 (m, 4H, 2 H12 and 2 H13). 13C NMR (75 MHz,
DMSO-d6), d (ppm): 170.9 (C9), 163.4 (C4), 158.3 (quat
DMT), 150.5 (C2), 145.0 (quat DMT), 136.5 (C6), 135,7
(quat DMT), 129.9 (ar DMT), 128.0 (ar DMT), 127.8 (ar
DMT), 126.9 (ar DMT), 113.4 (ar DMT), 113.1 (C5), 85.9
(quat DMT), 85.4 (C40), 84.1 (C10), 70.5 (C30), 64.1 (C50),
55.2 (OCH3 DMT), 41.5 (C14), 39.4 (C20), 38.5 (C11), 34.4
(C8), 30.7 (C12ء), 26.8 (C13ء), 23.0 (C7). FABϩ-MS: m/z
303 (DMT), 673 (MϩH)ϩ, 695 (MϩNa)ϩ.
S-Pivaloyloxymethyl-4-thiothymin-1-ylacetic acid 8. To
a solution of 7 (2 g, 10 mmol) in dimethoxyethane
(200 mL) was added K2CO3 (5.52 g, 4 equiv.), water
(80 mL) and pivaloyloxylmethyl chloride (POMCl) (4.3 mL,
3 equiv.). The reaction was stirred at RT for 24 h then
diluted with ethyl acetate (300 mL) and washed with 1N
HCl. The aqueous phase was extracted twice with ethyl
acetate. The combined organic layers were dried over
sodium sulfate and concentrated. Crystallization of the
residue from ethanol gave 13 as colourless crystals
(2.25 g, 74% yield). Mp: 210ЊC. 1H NMR (200 MHz,
DMSO-d6), d (ppm): 7.96 (s, 1H, H6), 5.84 (s, 2H, CH2
POM), 4.61 (s, 2H, N1–CH2), 2.04 (s, 3H, CH3), 1.22 (s,
9H, CH3 POM). 13C NMR (50 MHz, DMSO-d6), d (ppm):
177.5 (C4), 174.2 (CyO POM), 169.3 (COOH), 153.3 (C2),
146.2 (C6), 110.2 (C5), 60.9 (CH2 POM), 50.9 (N1–CH2),
38.4 (C(CH3)3 POM), 26.8 (C(CH3)3 POM), 13.2 (CH3).
CI-MS: m/z 315 (MϩH)ϩ. HRMS (CI): calculated for
C13H19N2O5S: 315.1014, found 315.1013. Calculated for
C13H18N2O5S: C, 49.68; H, 5.77; N, 8.91; S, 10.18.
Found: C, 49.66; H, 5.74; N, 8.84; S, 10.21.
N-Succinyl S-pivaloyloxymethyl-4-thiothymin-1-ylacetate
13. S-pivaloyloxymethyl-4-thiothymin-1-ylacetic acid 8
was dissolved in THF (10 mL/mmol) and N-hydroxy-
succinimide (1 equiv.) was added under a dry atmosphere.
The reaction was chilled to 0ЊC and dicyclohexylcarbodi-
imide (1 equiv.) was added. After stirring overnight at 5ЊC,
the resulting suspension was filtered. The filtrate was either
used directly or the solvent evaporated to give a colourless
powder which was used without purification for the next
step.
General procedure for amide bond formation
To a THF solution of activated acid 13 (3 equiv.) under a
dry atmosphere was added amine 10, 12a, 12b or 3-amino-
propyne and the reaction stirred at RT. Evolution of the
reaction was monitored by TLC. The reaction mixture was
diluted with CH2Cl2, the organic phase washed with a
saturated sodium hydrogen carbonate solution, brine, dried
over sodium sulfate and concentrated prior to purification by
flash chromatography.
50-O-[4,40-Dimethoxytrityl]-5-[(2-aminoethyl)amino]-20-
deoxyuridine 10. A solution of 50-O-(4,40-dimethoxytrityl)-
5-iodo-20-deoxyuridine 9,16 (1.35 g, 2.06 mmol) in freshly
distilled ethylenediamine (3 mL) was stirred under argon for
40 h. The mixture was concentrated under reduced pressure
to a gum that was purified by reverse phase chromatography
(Lichroprep RP 18) using a gradient of CH3CN in H2O
(0–100%) which afforded 10 (848 mg, 70% yield) as a
3-[(S-Pivaloyloxymethyl-4-thiothymin-1-ylacetyl)amino]-
propyne 14. 3-Aminopropyne (210 mL, 3.77 mmol) treated
with 13 as described in the general procedure gave after
purification by flash chromatography (CH3OH in CH2Cl2
0–2%) 14 (770 mg, 58% yield) as a foam. 1H NMR
(250 MHz, DMSO-d6), d (ppm): 8.81 (t, 1H, NH amide,
Jˆ5.3 Hz), 7.92 (s, 1H, H6), 5.84 (s, 2H, CH2 POM), 4.54
(s, 2H, N1–CH2), 4.00 (dd, 2H, CH2 prop, Jˆ5.2, 2.4 Hz),
3.26 (t, 1H, H prop, Jˆ2.4 Hz), 2.04 (s, 3H, CH3), 1.22 (s,
9H, CH3 POM). 13C NMR (75 MHz, DMSO-d6), d (ppm):
177.5 (C4), 173.8 (CyO POM), 166.5 (CyO amide), 152.8
(C2), 146.8 (C6), 109.9 (C5), 80.8 (quat prop), 73.6 (CH
prop), 60.9 (CH2 POM), 51.6 (N1–CH2), 38.4 (C(CH3)3
POM), 28.3 (CH2 prop), 26.8 (C(CH3)3 POM), 13.2
(CH3). CI-MS: m/z 352 (MϩH)ϩ. HR-MS (CI): found
352.1344, calculated for C16H22N3O4S 352.1331.
1
foam. H NMR (250 MHz, CD3OD), d (ppm): 7.50–7.16
(m, 9H, ar DMT), 6.85 (d, 4H, ar DMT, Jˆ8.3 Hz), 6.62 (s,
1H, H6), 6.40 (dd, 1H, H10, Jˆ6.2, 7.8 Hz), 4.44 (m, 1H,
H30), 3.98 (m, 1H, H40), 3.76 (s, 6H, OCH3 DMT), 3.46 (dd,
1H, H50, Jˆ3.0, 10.3 Hz), 3.24 (dd, 1H, H500, Jˆ3.7,
10.3 Hz), 2.42 (s, 4H, 2H 8 and 2H 9), 2.30 (m, 2H, H20;
H200). 13C NMR (50 MHz, CDCl3), d (ppm): 161.8 (C4),
159.2 (quat DMT), 149.8 (C2), 145.2 (quat DMT), 136.3
(quat DMT), 136.2 (quat DMT), 130.8 (ar DMT), 128.9 (ar
DMT), 128.5 (ar DMT), 127.6 (ar DMT), 126.5 (C5), 113.8
(ar DMT), 111.1 (C6), 87.0 (quat DMT), 86.4 (C40), 85.1
(C10), 72.6 (C30), 64.4 (C50), 55.9 (OCH3 DMT), 46.6 (C8ء),
40.7 (C20 and C9ء). L-SIMSϩ-MS: m/z 303 (DMT), 611
(MϩNa)ϩ.
50-O-[4,40-Dimethoxytrityl]-5-[3-[(4-aminobutyl)amino]-
3-oxopropyl]-20-deoxyuridine 12b. A solution of 50-O-
[4,40-dimethoxytrityl]-5-[3-methoxy-3-oxopropyl]-20-deoxy-
uridine 1115 (100 mg, 0.162 mmol) in freshly distilled
1,4-diaminobutane (800 mL) was stirred under argon for
18 h. The mixture was concentrated under reduced pressure
50-O-[4,40-Dimethoxytrityl]-5-[3-[[2-[(S-pivaloyloxy-
methyl-4-thiothymin-1-ylacetyl)amino]ethyl]amino]-3-
oxopropyl]-20-deoxyuridine 15a. 50-O-[4,40-Dimethoxy-
trityl]-5-[3-[(2-amino)ethyl amino]-3-oxopropyl]-20-deoxy-
uridine 12a15 (129 mg, 0.2 mmol) was treated with 13 as

´
C. Saintome et al. / Tetrahedron 56 (2000) 1197–1206
1204
100
described in the general procedure. Purification of the reac-
tion product by flash chromatography (EtOH/CH2Cl2, 8:92,
1% TEA) gave 15a (144 mg, 77% yield) as a foam. 1H NMR
(300 MHz, CDCl3), d (ppm): 7.87 (br s, 1H, NH amide),
CH2 POM), 4.55 (s, 2H, N –CH2), 4.44 (m, 1H, H30), 4.06
(m, 1H, H40), 3.76 (s, 6H, OCH3 DMT), 3.50–3.00 (m, 6H,
H50; H500; 2 H8; 2 H9), 2.37 (m, 2H, H20; H200), 1.99 (s, 3H,
CH3), 1.15 (s, 9H, CH3 POM). 13C NMR (62 MHz, CDCl3),
d (ppm): 179.0 (C400), 176.7 (CyO POM), 168.1 (CyO
amide), 161.4 (C4), 159.2 (quat DMT), 155.3 (C200), 149.5
(C2), 145.5 (C600), 145.1 (quat DMT), 136.2 (quat DMT),
136.1 (quat DMT), 130.8 (ar DMT), 128.8 (ar DMT), 128.6
(ar DMT), 127.7 (ar DMT), 126.0 (C5), 113.8 (ar DMT),
113.0 (C500), 111.6 (C6), 87.0 (quat DMT), 86.5 (C40), 85.2
(C10), 72.7 (C30), 64.5 (C50), 61.3 (CH2 POM), 55.9 (OCH3
ء
7.55ء (s, 1H, H6), 7.45–7.15 (m, 10H, H600 ; ar DMT), 7.03
(br s, 1H, NH amide), 6.83 (d, 4H, ar DMT, Jˆ8.7 Hz), 6.34
(t, 1H, H10, Jˆ6.6 Hz), 5.77 (s, 2H, CH2 POM), 4.54 (m,
00
3H, H30; N1 –CH2), 4.06 (m, 1H, H40), 3.77 (s, 6H, OCH3
DMT), 3.45–3.26 (m, 6H, H50; H500; 2 H11; 2 H12), 2.50–
2.14 (m, 6H, H20; H200; 2 H7 and 2 H8), 1.98 (s, 3H, CH3),
1.16 (s, 9H, CH3 POM). 13C NMR (62 MHz, CDCl3), d
(ppm): 179.0 (C400), 176.7 (CyO POM), 173.6 (C9), 167.7
(CyO amide), 165.0 (C4), 159.3 (quat DMT), 155.3 (C200),
151.3 (C2), 145.3 and 145.1 (C600 and quat DMT), 138.0
(C6), 136.2 (quat DMT), 130.8 (ar DMT), 128.8 (ar DMT),
100
ء
DMT), 53.9 (N –CH2), 40.6 (C20 ), 39.5 (C8; C9; C(CH3)3
POMء), 14.4 (CH3). FABϩ-MS: m/z 303 (DMT); 653
(MϩNaϪ255ϩ1)ϩ; 675 (MϪHϩ2NaϪ255ϩ1)ϩ, 789
(MϩNaϪ119ϩ1)ϩ, 907 (MϩNa)ϩ.
ء
128.6 (ar DMT), 127.7 (ar DMT), 114.4 (C500 ), 113.9 (ar
DMT), 113.0 (C5ء), 87.4 (quat DMT), 86.8 (C40), 85.6
(C10), 72.8 (C30), 64.5 (C50), 61.4 (CH2 POM), 55.9
50-O-[4,40-Dimethoxytrityl]-5-[3-[(S-pivaloyloxymethyl-
4-thiothymin-1-ylacetyl)amino]prop-1-yn-1-yl]-20-deoxy-
uridine 17. To a solution of 50-O-(4,40-dimethoxytrityl)-5-
iodo-20-deoxyuridine 916 (374 mg, 0.57 mmol) in DMF
(4 mL) was added CuI (26 mg, 0.24 equiv.), Et3N
(150 mL, 2 equiv.) and compound 14 (400 mg, 2 equiv.).
The reaction was degassed then purged under argon and
(Ph3P)4Pd(0) (80 mg, 0.12 equiv.) was added and the
reaction again purged with argon and stirred for 6.5 h. The
reaction was concentrated, diluted with ethyl acetate
(50 mL) and the organic phase washed with 3×25 mL of a
saturated hydrogen carbonate solution and dried over
sodium sulfate. Then it was concentrated and the residue
chromatographed on silica gel column eluting with a
gradient of 0–10% of methanol in CH2Cl2 to afford 17
(223 mg, 44% yield) as a foam. 1H NMR (300 MHz,
DMSO-d6), d (ppm): 11.82 (s, 1H, N3-H), 8.85 (t, 1H,
NH amide, Jˆ4.8 Hz), 8.08 (s, 1H, H600), 7.94 (s, 1H,
H6), 7.64–7.31 (m, 9H, ar DMT), 7.02 (d, 4H, ar DMT,
Jˆ8.3 Hz), 6.24 (t, 1H, H10, Jˆ6.5 Hz), 5.88 (s, 2H, CH2
100
(OCH3 DMT), 53.2 (N -CH2), 41.4 (C20), 40.4–39.5 and
39.4 (C(CH3)3 POM; C11 and C12), 36.3 (C8), 27.6
(C(CH3)3 POM), 23.7 (C7), 14.4 (CH3). FABϩ-MS: m/z
303 (DMT), 653 (MϩNaϪ312ϩ1)ϩ, 675 (MϪ1ϩ2NaϪ
312ϩ1)ϩ, 963 (MϩNa)ϩ.
50-O-[4,40-Dimethoxytrityl]-5-[3-[[4-[(S-pivaloyloxy-
methyl-4-thiothymin-1-ylacetyl)amino]butyl]amino]-3-
oxopropyl]-20-deoxyuridine 15b. Nucleoside 12b (500 mg,
74 mmol) was treated with 13 as described in the general
procedure. Purification of the reaction product by flash
chromatography (MeOH/CH2Cl2, 6:94, 1% TEA), gave
1
15b (240 mg, 34% yield) as a foam. H NMR (300 MHz,
CDCl3), d (ppm): 7.72 (br s, 1H, NH amide), 7.51 (s, 1H,
ء
H600 ), 7.46–7.14 (m, 10H, H6ء; ar DMT), 6.83 (d, 4H, ar
DMT, Jˆ8.5 Hz), 6.56 (br s,1H, NH amide), 6.37 (t, 1H,
100
H10, Jˆ6.6 Hz), 5.78 (s, 2H, CH2 POM), 4.52 (m, 3H, N –
CH2 and H30), 4.08 (m, 1H, H40), 3.76 (s, 6H, OCH3 DMT),
3.43–3.06 (m, 6H, 2 H11; 2 H14; H50 and H500), 2.50–2.10
(m, 6H, H20; H200; 2 H7 and 2 H8), 1.99 (s, 3H, CH3), 1.60–
100
POM), 5.47 (d, 1H, OH 20, Jˆ4.3 Hz), 4.57 (s, 2H, N –
CH2), 4.39 (m, 1H, H30), 4.06 (m, 3H, H40; 2 H9), 3.84 (s,
6H, OCH3 DMT), 3.38 (dd, 1H, H50, Jˆ10.2, 5.3 Hz), 3.20
(br d, 1H, H500, Jˆ10.2 Hz), 2.35 (m, 2H, H20;H200), 2.05 (s,
3H, CH3), 1.24 (s, 9H, CH3 POM). 13C NMR (75 MHz,
DMSO-d6), d (ppm): 177.5 (C400), 173.7 (CyO POM),
166.3 (CyO amide), 161.7 (C4), 158.2 (quat DMT), 153.2
(C200), 149.5 (C2), 146.8 (C600), 145.0 (quat DMT), 143.7
(C6), 135.8 (quat DMT), 135.4 (quat DMT), 129.8 (ar
DMT), 128.0 (ar DMT), 127.7 (ar DMT), 126.8 (ar
DMT), 113.4 (ar DMT), 109.8 (C500), 98.3 (C5), 89.0
(C7), 86.0 (quat DMT; C40), 85.2 (C10), 74.7 (C8), 70.6
(C30), 63.9 (C50), 60.8 (CH2 POM), 55.2 (OCH3 DMT),
1.34 (m, 4H, 2 H12 and 2 H13), 1,16 (s, 9H, CH3 POM). 13
C
NMR (75 MHz, CDCl3), d (ppm): 179.0 (C400), 176.4 (CyO
POM), 172.8 (C9), 167.3 (CyO amide), 164.7 (C4), 159.1
(quat DMT), 155.2 (C200), 151.3 (C2), 145.4 (C600), 145.1
(quat DMT), 137.8 (C6), 136.1 (quat DMT), 130.6 (ar
DMT), 128.7 (ar DMT), 128.5 (ar DMT), 127.5 (ar
ء
DMT), 114.0 (C5ء), 113.8 (ar DMT), 112.8 (C500 ), 87.1
(quat DMT), 86.6 (C40), 85.3 (C10), 72.4 (C30), 64.4
100
(C50), 61.3 (CH2 POM), 55.8 (OCH3 DMT), 53.1 (N –
CH2), 41.0 (C20), 39.9; 39.4; 39.3 (C11; C12; C(CH3)3
POM), 35.6 (C8), 27.4 (C(CH3)3 POM), 27.1 and 26.8
(C12 and C13), 23.9 (C7), 14.2 (CH3). FABϩ-MS: m/z
303 (DMT), 681 (MϩNaϪ312ϩ1)ϩ, 703 (MϪ1ϩ2NaϪ
312ϩ1)ϩ, 991 (MϩNa)ϩ.
100
51.4 (N –CH2), 40.0 (C20), 38.3 (C(CH3)3POM), 29.0
(C9), 26.7 (C(CH3)3POM), 13.2 (CH3). FABϩ-MS: m/z
902, (MϩNa)ϩ.
50-O-[4,40-Dimethoxytrityl]-5-[[2-[(S-pivaloyloxy-
methyl-4-thiothymin-1-ylacetyl)amino]ethyl]amino]-20-
deoxyuridine 16. Nucleoside 10 (74 mg, 0.13 mmol) was
treated with 13 as described in the general procedure. Puri-
fication of the reaction product by flash chromatography
(MeOH/CH2Cl2, 0–5%, 1% TEA), gave nucleoside 16
(60 mg, 54% yield) as a foam. 1H NMR (250 MHz,
CDCl3), d (ppm): 7.64 (br s, 1H, NH amide), 7.50–7.10
(m, 10H, H600; ar DMT), 6.81 (d, 4H, ar DMT, Jˆ7.2 Hz),
6.52 (s, 1H, H6), 6.46 (t, 1H, H10, Jˆ6.6 Hz), 5.78 (s, 2H,
Preparation of phosphoramidites 18a, 18b, 19, 20. To a
solution of each nucleoside (15a, 15b, 16 and 17) in CH2Cl2
(8.9 mL/mmol) under argon, was added N,N-diisopropyl-
ethylamine (4 equiv.) and 2-cyanoethyl N,N-diisopropyl-
chloro-phosphoramidite (3 equiv.). Upon completion of
the reaction, the mixtures were concentrated and the
residues dissolved in CH2Cl2. The solutions were washed
with sodium hydrogen carbonate, dried over sodium
sulfate and the desired products (18a, 18b, 19 and 20)
characterized. 31P NMR (121.49 MHz, CDCl3): d ppm

´
C. Saintome et al. / Tetrahedron 56 (2000) 1197–1206
1205
18a: 149.5–149.3 ppm, 18b: 149.7–149.5 ppm, 19: 149.5–
149.1 ppm, 20: 149.6–149.3 ppm.
precipitated. Localization of the RNA cross-links was
achieved by limited alkaline hydrolysis as previously
described.19
Photolabelled deoxysubstrate analogues and ribozyme
synthesis
Acknowledgements
Synthesis of all dS were carried out using conventional
phosphoramidite chemistry.24 In the case of substrate
analogues with the label in position 1.6, a 15-mer bearing
an extra A in position 1.7 was synthesized. Sequences were
prepared on a 1 mmol scale using the trityl off end procedure
and employing the standard DNA synthesis cycle provided
by Applied Biosystems. Modified 20-deoxyuridine phos-
phoramidites (18a, 18b, 19 and 20) were used as 0.1 M
acetonitrile solutions. Coupling time for their incorporation
was extended to 10 min. After synthesis, the solid supports
were treated at room temperature with NH4OH 33% for two
days, then the solutions were concentrated to dryness.
Complete purification of the oligomers was performed in
two steps: first truncated oligonucleotides were separated
from the full-length oligomers by gel electrophoresis on a
denaturing 20% polyacrylamide gel; then thiolated-
oligomers were separated from non-thiolated ones on an
affinity gel slowing down the desired oligomers.25
We are indebted to Dr M. Thomas for the chemical
synthesis of all-deoxy ribozyme substrate analogues dS
and ribozyme Rz. This work was supported by grants of
Ligue Nationale contre le Cancer and ANRS.
References
1. (a) Vaish, N. K.; Kore, A. R.; Eckstein, F. Nucleic Acids Res.
1998, 26, 5237–5242. (b) Eckstein, F., Lilley, D. M. J., Eds. 1997,
Catalytic RNA; Springer: Berlin. (c) Burlina, F.; Favre, A.;
Fourrey, J.-L. Bioorg. Med. Chem. 1997, 5, 1999–2010.
2. (a) Wedekind, J. E.; McKay, D. B. Annu. Rev. Biomol. Struct.
1998, 27, 475–502. (b) Scott, W. G.; Finch, J. T.; Klug, A. Cell
1995, 81, 991–1002. (c) Pley, H. W.; Flaherly, K. M.; McKay,
D. B. Nature 1994, 372, 68–74.
3. (a) Bassi, G. S.; Mollegaard, N. E.; Murchie, A. I. H.; Lilley,
D. M. J. Biochem. 1999, 38, 3345–3354. (b) Bassi, G. S.;
Mollegaard, N. E.; Murchie, A. I. H.; Kitzing, E.; Lilley, D. M.
J. Nature Struct. Biol. 1995, 2, 45–55.
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1998, 431, 154–160. (b) van Tol, H.; Buzayan, J. M.; Felstein, F.;
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7. Woisard, A.; Fourrey, J.-L.; Favre, A. J. Mol. Biol. 1994, 239,
366–370.
Rz ribozyme was synthesized either chemically (see above,
deprotection was accomplished with NH4OH 33%/EtOH
2:1) or by in vitro transcription. In this latter case transcrip-
tion was effectued by T7 RNA polymerase in the presence
of a-32P-dCTP and synthetic single strand DNA templates
that are double-stranded at the promoter site. The products
were isolated by running on 15% PAGE-8 M urea. The
bands of interest were cut, eluted, precipitated and quanti-
fied by Cerenkov counting. The ribozyme transcripts were
dephosphorylated using Calf Intestine Alkaline Phos-
phatase: 30 min at 37ЊC, 15 min at 45ЊC (in order to expose
the 50 extremity) and finally 30 min at 37ЊC with a new
equivalent of CIP. Then the mixture was treated with 10
units of Proteinase K in the presence of 2% SDS. After
phenol extraction, ribozyme was precipitated and then
32P-labelled at the 50-end as previously described.19 The
only difference between these two methods of ribozyme
synthesis being that ribozyme obtained by enzymatic
synthesis contains an extra G residue at its 50-end. Photo-
labelling studies realized with these two kind of ribozyme
yielded the same results.
8. Woisard, A.; Favre, A.; Clivio, P.; Fourrey, J.-L. J. Am. Chem.
Soc. 1992, 114, 10072–10073.
ˆ
9. Laugaa, P.; Woisard, A.; Fourrey, J.-L.; Favre, A. C. R. Acad.
Sci., Life Sci. 1995, 318, 307–313.
ˆ
10. Bravo, C.; Woisard, A.; Fourrey, J.-L.; Laugaa, P.; Favre, A.
Biochimie 1999, 81, 201–212.
11. Scott, W. G.; Murray, J. B.; Arnold, J.; Stoddard, B. L.; Klug,
A. Science 1996, 274, 2065–2069.
12. (a) Murray, J. B.; Terwey, D. P.; Maloney, L.; Karpeisky, A.;
Usman, N.; Beigelman, L.; Scott, W. G. Cell 1998, 92, 665–673.
(b) Peracchi, A.; Beigelman, L.; Scott, E. C.; Uhlenbeck, O. C.;
Herschlag, D. J. Biol. Chem. 1997, 272, 26822–26826.
Ribozyme photolabelling studies
´
Oligodeoxynucleotides dS (1.2 mM) were placed in 50 mM
Tris–HCl pH 8, 0.5 mM EDTA and 20 mM MgCl2 in the
presence of 50-32P-labelled ribozyme (0.2 mM).19 Each
sample (5 mL) was introduced in a siliconized glass
capillary and placed 5 cm from the exit slit of a Bausch
and Lomb monochromator equipped with a HBO 200 W
superpressure mercury lamp and a Schott WG 345 filter.
The samples thermostated at 15ЊC were irradiated at
366 nm during 45 min. (Total fluenceˆ200 kJ/m2.) Large
gels (40 cm) were used to separate multiple cross-linked
species on 15% PAGE-8 M urea. Cross-linked and non-
cross-linked species were detected by autoradiography,
excised, quantified by Cerenkov counting and by
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