© 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 6 1183
Configurationally defined phosphorothioate-containing
ohgoribonucleotides in the study of the mechanism of
cleavage of hammerhead ribozymes
George Slim and Michael J.Gait*
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Received January 31, 1990; Accepted February 21, 1991
ABSTRACT
The chemical synthesis is described of
oligoribonucleotides containing a single phosphorothioate linkage of defined Rp and Sp configuration. The
oligoribonucleotides were used as substrates in the
study of the mechanism of cleavage of an RNA
hammerhead domain having the phosphorothioate
group at the cleavage site. Whereas the Rp Isomer was
cleaved only very slowly in the presence of magnesium
ion, the rate of cleavage of the Sp isomer was only
slightly reduced from that of the unmodified
phosphodiester. This finding gives further evidence for
the hypothesis that the magnesium ion is bound to the
pro-R oxygen in the transition state of the hammerhead
cleavage reaction. Also, Inversion of configuration at
phosphorus is confirmed for a two-stranded
hammerhead.
INTRODUCTION
A number of plant viroids and virusoids contain in their RNA
genomes a self-cleavage domain of about 50 residues known as
a hammerhead (1). Self-cleavage is an obligatory step in the
replication pathway of such plant pathogens but the reaction can
also be effected in vitro merely by addition of divalent metal ions
(usually magnesium) to an isolated hammerhead domain (2).
Because of their small sizes, it has become possible to prepare
synthetic RNA hammerheads and this has facilitated studies of
the mechanism of cleavage (3). A simplifying feature is that, in
contrast to many other RNA self-processing reactions (4, 5), there
is only a single chemical step and the reaction is generally
irreversible (6).
The secondary structure of a hammerhead (Figure 1) consists
of 11 non-helical nucleotides held together by three duplexes.
A total of 13 nucleotides in the structure are phylogenetically
conserved and cleavage takes place at a unique phosphodiester
bond. Whereas the reaction in vivo is unimolecular, cleavage can
be effected in vitro in trans by suitable arrangement of two (or
even three) separate oligoribonucleotides as long as the three
duplexes are preserved (2, 3, 6, 7, 8). Extensive mutagenesis
studies have shown that very few of these conserved nucleotides
* To whom correspondence should be addressed
can be altered without substantial loss of cleavage activity (9,
10, 11). For a two-stranded hammerhead, the cleavage reaction
has been found to be catalytic in terms of the non-cleaved strand
by virtue of successive cycles of annealing, cleavage and
dissociation. In effect the catalytic RNA strand can be thought
of as a sequence-specific endoribonuclease with potential
applications including, for example, use as an RNA-targeted
antiviral agent (12).
In the cleavage reaction, there is an absolute requirement for
divalent metal ions which may act to stabilize a proposed pentacoordinated phosphate in the transition state formed as a result
of attack of the 2'-hydroxyl group of the adjacent ribonucleoside
on the phosphodiester at the cleavage site (Figure 2)(13). The
outcome of the cleavage is a free 5'-hydroxyl group on one RNA
fragment and a cyclic-2',3'-phosphate on the other. Incorporation
of a single Rp-phosphorothioate at the site of cleavage has been
found to significantly reduce the rate of hammerhead self-cleavage
(14). By use of such phosphorothioate substrates, it has been
shown recently that a unimolecular hammerhead cleavage reaction
proceeds by an in-line attack leading to inversion of configuration
at the phosphorus atom (15). In line attack is found in the cleavage
mechanism of most polypeptide ribonucleases (16).
In the presence of magnesium ion, Rp-phosphorothioate is
found to be cleaved much more slowly than a natural
phosphodiester but in the presence of manganese, the cleavage
rate is restored (17). Based on this data, it has been suggested
that the metal ion complexes directly to the pro-R oxygen on
the phosphate at the cleavage site. This assumption has formed
the basis for a computer modelling study of the self-cleavage
domain (13). However, since the rate of cleavage of the Spphosphorothioate has not been separately measured, it is not
possible to rule out complexation of the metal ion to both oxygen
atoms.
Introduction of an Rp-phosphorothioate can be effected by
transcription of a synthetic DNA template using T7 RNA
polymerase (18) and an appropriate Sp-nucleoside athiotriphosphate (15). However it is a general property of
polymerases that Rp-a-thiotriphosphates are not substrates (19)
and thus the Sp-phosphorothioate diester cannot be accessed
enzymatically. We now describe a synthetic chemistry approach
1184 Nucleic Acids Research, Vol. 19, No. 6
for the preparation of hammerhead ribozymes containing single
phosphorothioates of Rp and Sp stereochemistry at the cleavage
site. The methods involve sulphurization of a phosphite diester
formed as an intermediate during solid-phase oligoribonucleotide
synthesis. This results in an approximately 1:1 mixture of Rp
and Sp phosphorothioate isomers. After assembly, the completed
oligonucleotide containing a mixture of Rp and Sp
phosphorothioate isomers can be resolved by reversed-phase
chromatography. The individual isomers have been used in a
hammerhead cleavage reaction and the results compared to the
cleavage of the unmodified ribozyme.
RESULTS
Synthesis and cleavage of an unmodified hammerhead
The hammerhead ribozyme sequence chosen for study has a short
substrate strand (S) and a longer 37-residue catalytic strand (E)
(Figure 3). The catalytic strand contains the loop sequence
CUUCGG (20) and also a G:C-rich stem (helix II). These were
chosen so as to encourage formation of a stable hairpin and to
prevent kinetic dependence on interconversion between
incorrectly and correctly folded structures (21). A 6-base pair
G:C-rich helix HI was chosen to avoid possible dimerisation into
a double-hammerhead conformation (10). A chemically
synthesized substrate strand (Figure 3a) was compared with an
enzymatically prepared substrate strand (Figure 3b). The
sequences of each substrate are identical except that in the case
of the transcribed substrate strand, two additional G residues were
added at the 5'-end to facilitate reasonable yields in T7 RNA
polymerase-mediated transcription (18). Also a single A residue
3' to the cleavage site allows for convenient introduction of a
unique Rp-phosphorothioate diester.
The 37-nucleotide catalytic strand (E) and the unmodified
15-nucleotide substrate strand (S) were prepared by T7 RNA
polymerase-mediated transcription of synthetic DNA templates
by the method of Milligan et al. (18). Substrate strand was
labelled by use of a-^P-CTP in the transcription reaction. Each
strand was purified by polyacrylamide gel electrophoresis under
denaturing conditions. Unmodified 13-nucleotide substrate strand
(S) was synthesized by a phosphoramidite procedure (22) using
commercially available ribonucleoside phosphoramidite
monomers on an automated DNA Synthesizer, purified by hplc
(see experimental section) and end-labelled by treatment with T4
polynucleotide kinase and -y-^P-ATP.
When incubated with 37-mer (E) strand (0.25 /xM) at pH 7.4
in the presence of 20mM magnesium at 50°, transcribed 15-mer
(S) strand (0.5 pM) was cleaved with a t 1/2 of 1 —2 minutes
whereas chemically synthesized 13-mer (S) strand (0.5 /tM) was
cleaved with t 1/2 of < 1 minute. Both transcribed and
chemically synthesized (S) strands were cleaved to >99%
completion within 1 hour (data not shown) which indicates that
in both cases all the substrate is available for cleavage.
Kinetic parameters for the cleavage of excess chemically
synthesized 13-mer substrate (S) at pH 7.4 and 30° determined
from an Eadie—Hofstee plot were Km = 0.023 /tM and k^ =
2.8 /min. These results indicate that this hammerhead cleavage
is an extremely efficient one (cf. hammerheads reported in
reference 21). The cleavage rate of the (S) strand (0.6 nM) with
(E) strand (0.02 /iM) at pH 7.4 and 30° in the presence of 20
mM manganese was slightly faster (tl/2 = 12 minutes) than that
measured in the presence of 20 mM magnesium (tl/2 = 32
minutes) (Figure 4a).
Synthesis and cleavage of Rp-phosphorothioate-substituted
oligoribonudeotides
Enzymatic synthesis of Rp-phosphorothioate-substituted S (Rpthio 15-S) was carried out in a similar way to the unmodified
strand described above (18) except that adenosine a-
Ocavige Site
Helix
Helix I
Phosphodiester,
Rp or Sp Pbosphorothioate
AGUCCC
UCAGGGppp
Figure 1. Hammerhead consensus structure. Boxed residues signify those
conserved in most hammerheads. Dots indicate Watson-Crick base pairing.
,GPPP
b)
C- G
E
cd G '
C G GC G
u C G C GG
u
Figure 2. Proposed stereochemical course of phosphate cleavage showing
pentacoordinated intermediate. When X = Y = O : unmodified phosphodicster, when
X = O, Y = S : Sp-phosphorothioate and exo isomer of cCMPS; when X = S,
Y = O : Rp-phosphorothioate and endo isomer of cCMPS.
s
G- C
G- C
U/S
A^i
\
yS
Phosphodiester or
Rp Pboiphorothioate
'AGUCCC
\ U C A G G Gppp
A
Figure 3. a) Hammerhead comprising transcribed 37-mer catalytic strand (R)
and chemically synthesized 13-mcr substrate strand (S). The 5'-end of S is
enzymatically phosphorylated. b) Hammerhead comprising transcribed 37-mer
catalytic strand (R) and transcribed 15-mer substrate strand (S).
Nucleic Acids Research, Vol. 19, No. 6 1185
thiotriphosphate was used in the transcription reaction. Incubation
of ^P-labelled Rp-thio 15-S with unlabelled 37-mer E catalytic
strand in the presence of magnesium showed a very slow cleavage
rate (Figure 4b). After 240 minutes, the cleavage was only 12%
complete and significant non-specific degradation was observed.
In the presence of manganese, the cleavage rate approximately
doubled.
Although it has been shown in a single-stranded transcribed
hammerhead that inversion of configuration at phosphorus takes
place during self-cleavage, it was important to confirm that this
is still the case for a two-stranded hammerhead. Accordingly two
batches of Rp-thio 15-S were prepared, one in which 35 Sadenosine a-thiotriphosphate was used in the transcription
reaction to incorporate radioactivity into the thiophosphate at the
cleavage site and another in which a small amount of a-32P-CTP was used. A mixture of 35S and 32P-labelled Rp-thio 15-S
(0.5 fiM) (just enough 32P to be able to follow by auto-
a
o
20
100
200
300
TlnWmln
Unmodfflec1 substrata with Mg.
Unmodfflsd substrata wtttl Mn.
*
radiography on polyacrylamide gels) was cleaved to completion
using unlabelled 37-mer catalytic strand (0.15 /iM) in the presence
of manganese ion (20mM) at pH 7.4 for 4 hours at 50°. The
cleavage products were separated by denaturing PAGE and the
slower band eluted and salts removed by extraction with n-butanol
(23). The RNA fragment was digested with nuclease PI and then
alkaline phosphatase treated. This procedure should give rise only
to nucleosides and to 35S cytidine 2',3'-cyclophosphorothioate
(cCMPS) which results from the nucleoside 5' to the cleaved
phosphorothioate bond. The products of digestion were added
to a mixture of the exo and endo isomers of unlabelled cCMPS
and separated by reversed-phase hplc. Material eluting at the
position of each isomer was collected and counted by liquid
scintillation and ah" the radioactivity (as 35S) was found to have
eluted at the position of the endo isomer (data not shown). This
result can only have been obtained by complete inversion of
configuration at phosphorus during cleavage of the Rpphosphorothioate. Since this is the same result as obtained from
cleavage of the single-stranded hammerhead (15), it follows that
there is no intrinsic difference in reaction pathway between
intermolecular and intramolecular hammerhead cleavage.
Chemically synthesized oligoribonucleotides containing Rp
and Sp phosphorothioates
13-mer substrate strand S with a phosphorothioate at the cleavage
point was chemically synthesised using commercially available
RNA phosphoramidite monomers on an automated Synthesizer
but substituting the iodine/water oxidation of the phosphorus at
the cleavage site with sulphurization using either elemental
sulphur (24) or the new and more reactive reagent
tetraethylthiuram disulphide (25). The result of either treatment
was the same but tetraethylthiuram disulphide is soluble in
acetonitrile and the use of noxious carbon disulphide is avoided.
Following complete deprotection, strong anion exchange (SAX)
hplc of the crude synthetic product showed one major product
indicating that both 13-mer phosphorothioate isomers eluted
together. The elution position was later than would be expected
for the corresponding unmodified 13-mer. However, the isomers
were well resolved by reversed phase hplc (Figure 5). The
separation of Rp and Sp isomers of singly thio-substituted
20-
200
300
Transcript Rp-thio substrata wtth Mg.
Transcript Rp-thio subctrat* with Mn.
Figure 4. a) Tune course of cleavage reaction of chemically symhesized unmodified
13-mer S by 37-mer R in the presence of magnesium or managanese. b) Time
course of cleavage reaction of transcribed 15-mer Rp-thio S by 37-mer R in the
presence of magnesium or manganese.
20
Time (minutes)
30
Figure 5. Reversed-phase h.p.l.c. chromatogram of chemically synthesized thio
13-mer substrate S showing separation of Rp and Sp isomers. Gradient conditions:
0%B 5', 0-20%B 30' (see experimental section).
1186 Nucleic Acids Research, Vol. 19, No. 6
DISCUSSION
S
u
20
Synthetic Rp-tHo subdnta with Mg.
Synthetic Rp-thto substrate with Mn
Synthetic Sp-thlo mbunte with Mg
Synthetic Sp-lhio njbstnte with Mn
Figure 6. Time course of cleavage reaction of chemically synthesized 13-mer
Rp-thio S and Sp-thio S by 37-mer R in the presence of magnesium or manganese.
oligodeoxyribonucleotides has been well documented (24) and
in general it has been found that the best separations are obtained
when the thio substitution is near the 5'-end and when a terminal
dimethoxytrityl group is present (26). To our knowledge this is
the first report in the oligoribonucleotide series. Since we have
obtained an excellent separation with a centrally thio-substituted,
fully deprotected oligoribonucleotide, it is likely that reversed
phase separation should be applicable to most singly thiosubstituted oligoribonucleotides.
The assignment of configuration of the two isomers was
accomplished by enzymatic digestion. The later eluting isomer
was more resistant to cleavage by snake venom phosphodiesterase
(27) whereas the earlier eluting isomer was more resistant to
cleavage by nuclease PI (28). These results indicate that the
earlier eluting compound is the Rp-phosphorothioate and the later
eluting isomer has the Sp configuration.
Cleavage of the Rp-phosphorothioate-substituted 13-mer S (Rpthio 13-S) by E in the presence of magnesium gave a very slow
rate of cleavage (6% cleavage after 240 minutes) which was very
similar to that obtained for the transcribed Rp-thio 15-S described
above (Figure 6). The cleavage rate in the presence of manganese
improved by a similar margin also (26% cleavage after 240
minutes). By contrast, the cleavage of the Sp-thio 13-S in the
presence of magnesium proceeded much faster than Rp-thio 13-S
(tl/2 = 60 minutes, 98% complete in 240 minutes), although
not as fast as the unmodified phosphodiester substrate. Once again
the rate improved in the presence of manganese (tl/2 = 16
minutes, 93% complete after 120 minutes) (Figure 6). It is
interesting to note that the rate of cleavage of Sp-thio 13-S in
the presence of manganese was almost identical to the rate of
cleavage of unmodified phosphodiester in the presence of
magnesium.
We have shown that there is no intrinsic difference in reaction
mechanism between a single-stranded hammerhead ribozyme and
one in which the catalysis is provided in trans by a second strand
in that both occur with complete inversion of configuration. This
finding allows the use of short chemically synthesized
oligoribonucleotide substrates to be used in studies of the
mechanism of hammerhead cleavage.
It has been known for some time that metal ion (normally
magnesium) is crucial to the activity of hammerhead ribozymes
but the exact role of metal ion in catalytic cleavage has remained
elusive. The demonstration that cleavage of Rp-phosphorothioate
substrate is enhanced in the presence of manganese (17) suggested
that the metal ion may be closely complexed to the pro-R oxygen
of the phosphodiester bond at the cleavage site. This conclusion
is based on the assumption that the complexation of sulphur (a
softer Lewis base than oxygen)(29) is poorer with magnesium
than with manganese (a softer Lewis acid than magnesium) (19).
Complexation of magnesium with the pro-R oxygen presumably
increases the rate of cleavage of substrate by stabilizing the
pentacoordinated phosphorus in the transition state by electron
withdrawal (30). Our results are in general agreement with this
hypothesis since the rate of cleavage of the Sp-thio 13-S in the
presence of magnesium is only slightly reduced compared to the
unmodified phosphodiester, whereas the cleavage rate of the Rpthio 13-S is substantially reduced. The slight reduction in cleavage
rate for the Sp-thio 13-S can be explained by the reduction in
electronegativity afforded by sulphur substitution and hence a
reduction of electrophilicity of the phosphorus atom. This would
lead to a slight destabilization of the transition state of the cleavage
reaction (31).
Very recent NMR studies of a hammerhead composed of an
RNA catalytic strand and a non-cleavable DNA substrate strand
have shown that at least in one case there appears to be very
little perturbation of structure when magnesium is added (32).
These results suggest that a defined 'pocket' for metal ion is
formed by the hammerhead and the role of metal is therefore
primarily catalytic rather than structural. We have confirmed that
at least part of the catalytic enhancement is due to complexation
with the pro-R oxygen of the cleaved phosphodiester. From
theoretical calculations of the energetics of RNA cleavage, Taira
et al. (33) have argued that magnesium participates by acting as
a genera] acid catalyst to stabilize the leaving 5'-oxygen atom.
Experimental evidence for the hypothesis is not yet available but
this could be tested by thio substitution at the 5'-oxygen. It would
also be of interest to measure the relative effects of thio
substitution at bridging and non-bridging oxygen atoms and to
correlate the effects with cleavage efficiency.
EXPERIMENTAL SECTION
Transcription of ribozyme (E) and Rp-phosphorothioate
substrate (Rp-thio 15-S)
RNA transcripts were prepared by the method of Milligan et al.
(18) but with minor modifications. Synthetic DNA template
(54-mer and 32-mer) and primer (18-mer) strands were
chemically synthesized by the phosphoramidite method on an
Applied Biosystems DNA Synthesizer following manufacturers
instructions and purified by electrophoresis on preparative
(1.5mm thick) 12% or 20% denaturing polyacrylamide gels (PAGE) respectively. Bands were located by UV shadowing and
Nucleic Acids Research, Vol. 19, No. 6 1187
eluted in 0.5 mM ammonium acetate, 10 mM magnesium acetate
in sterile water. The oligonucleotides were desalted using OPC
cartridges (Applied Biosystems) following manufacturers
instructions. Transcription reactions were carried out at 37° for
3 hours in 250/xL-lml reactions containing 40 mM Tris HO (pH
8.2 at 37° at 1M concentration), 7 mM MgCl2, 5 mM DTT,
1 mM spermidine, 0.01 % Triton X100, 50 /xg/ml acetylated BSA
(Anglian Biotech), 6% polyethylene glycol 6000 (Koch Light),
2 mM each of ATP, GTP, UTP and CTP and oligonucleotides
at 0.2 pmol //xl using T7 RNA polymerase (15-20 units / /xl).
32
P-labelled transcripts were made by incorporation of a-32PCTP (40 /xCi /ml). Thio-substituted transcripts were made by
replacement of ATP by Sp-ATPas (Amersham) at 0.5 mM. ^Sthio-substituted transcripts were made by addition of 35S-SpATPas (Amersham) to 40 /xCi /ml. Reactions were stopped by
addition of EDTA to 30 mM, phenol extracted and ethanol
precipitated. Pellets were taken up in 0.1 mM EDTA and
transcripts were purified by PAGE (15% denaturing gels, 1.5
mm thick). Transcripts were located by UV shadowing and eluted
in 0.5 M ammonium acetate, 1 mM EDTA (pH 7.4), 0.5% SDS
made up in sterile water, and desalted by extraction with n-butanol
as described (23). Transcripts were stored in 1 mM EDTA (pH
7.4) and concentrations estimated by UV spectroscopy.
Chemical synthesis of unmodified 13-mer substrate (S) and
thio-substitued substrates (Rp-thio 13-S and Sp-thio 13-S)
Solid-phase chemical synthesis of RNA was carried out on an
Applied Biosystems 380 B Synthesizer using a standard 1.0 /xmol
DNA assembly cycle except that the coupling wait time was
increased to 10 minutes. Empty columns (Applied Biosystems)
were packed with 2'-0-TBDMS-5'-0-dimethoxytrityl ribonucleoside-derivatised CPG obtained from Peninsular
Laboratories. 2'-O-TBDMS-5'-O-dimethoxytrityl ribonucleoside
3'-O-phosphoramidites were purchased from Milligen-Biosearch
and dissolved to 0.1 M in anhydrous acetonitrile (Applied
Biosystems). Assemblies were carried out using the 'trityl off
mode and oligonucleotides cleaved from the support using 35 %
ammonia (BDH Aristar)/ absolute ethanol (3:1) using the standard
programmed end procedure except that the total wait time was
extended to 120 minutes. Full details of the methods of synthesis
are given elsewhere (34).
Phosphorothioate-containing oligoribonucleotides were
prepared in an identical manner except for replacement of the
oxidation step by a manual sulphurization step at the cycle at
which the thio-modification was to be introduced. After the
capping step and immediately before the oxidation step, the
column was removed from the machine and treated by syringe
addition of either 1) elemental sulphur (0.4g, Aldrich Gold Label,
in carbon disulphide: 2,6-lutidine, 1:1,6 ml) for 5 - 6 hours and
washed with carbon disulphide:2,6-lutidine (1:1, 10 ml) and
acetonitrile (20 ml), or 2) tetraethylthiuram disulphide (Aldrich,
0.5 g in acetonitrile, 5 ml) for 1 hour and washed with acetonitrile
(10 ml). The column was reattached to the Synthesizer and
assembly continued at the next cycle entry. Both procedures gave
comparable yields of product.
Deprotection and isolation of synthetic oligoribonucleotides
The ammonia/ethanol solution was heated in a sealed tube at 55°
for 16 hours to remove base protecting groups and evaporated
to dryness (Speedvac, Savant). Silyl groups were removed by
treatment with tetrabutylammonium fluoride (Aldrich, 1 M in
THF containing less than 5% water, 1 ml) for 24 hours. The
reaction was quenched with triethylammonium acetate (0.1 M,
5 ml) to give a homogeneous solution (any insolubility at this
stage indicates incomplete desilylation) and dialysed immediately
against distilled water. The resulting product was lyophilized and
purified by strong anion exchange (SAX) chromatography on a
semipreparative Partisil P-10 SAX column (Hichrom) using a
gradient of buffer A: 1 mM KH2PO4 (pH 6.3)/60% formamide
and buffer B: 300 mM KH2PO4 (pH 6.3) /60% formamide
(0%B 5', 0-50%B 10', 50-100%B 15'). Elution time of (Rp
+ Sp)-thio 13-mer was 23.6' (cf unmodified 13-mer was 22.4').
After dialysis and lyophilization, the 13-mer product was further
purified by reversed phase chromatography on a semipreparative
/x-Bondapak C-18 column (Waters/Millipore) using gradients of
buffer A: 0.1 M ammonium acetate and buffer B: 20% buffer
A/ 80% acetonitrile (Figure 5). Material in the product peak was
isolated by evaporation. Full details of these procedures are
published elsewhere (34).
Small aliquots (10—200 pmol) of synthetic oligoribonucleotides
were 5'-phosphorylated using T4 polynucleotide kinase (New
England Biolabs) and >-32P ATP (Amersham, 10/xCi / /il) as
previously described (35) and stored in 1 mM EDTA at 1.5
pm/id. 5'-phosphorylated, unlabelled substrate (1 nmol) was
prepared by treatment with T4 polynucleotide kinase and a 3-fold
excess of ATP. The phosphorylated substrate was separated from
unincorporated ATP by electrophoresis on a denaturing 20%
polyacrylamide gel and the products located by UV shadowing.
After excision and extraction, the substrate was concentrated by
butanol extraction (23) and purified on a Sephadex NAP-10
column (Pharmacia). The amount of unlabelled substrate was
determined by measuring the absorbance at 260 nm.
Assignment of configuration of thio-substituted
oligoribonucleotides
a) Digestion by Snake Venom Phosphodiesterase. An aliquot of
each thio-substituted oligonucleotide (earlier and later eluting by
reversed-phase hplc) (0.5 nmol) was treated for 8 hours at 37°
with snake venom phosphodiesterase (0.1 /xg, Boehringer) and
calf alkaline phosphatase (6.0 /xg, Boehringer) in 0.1M Tris.HCl
(pH 8.5), 0.3 mM DTT, 0.3 mM MgCl2 in a reaction volume
of 150 /xl. The products were analysed directly by reversed-phase
hplc on an analytical Spheri-5 RP-18 column (220x4.6 mm,
Applied Biosystems) using a gradient of buffer A: 0.1 M
triethylammonium acetate (pH 7.0) and buffer B: 60% buffer
A, 40% acetonitrile (5%B 15', 5-50% 30'). Retention times:
cytidine4.48', undine 5.75', guanosine 11.41', adenosine 24.48'.
The digestion products of the later eluting isomer showed a peak
at 34.3 minutes corresponding to Rp-CpsA.
b) Digestion by nuclease PL An aliquot of each thio-substituted
oligonucleotide (0.5 nmol) was digested with nuclease PI (2.0
/xg, Boehringer) in distilled water (120 /xl) for 1 hour at 37°.
The solution was buffered with 16 /xl 0.1M Tris HC1 (pH 8.5)
and digested with calf alkaline phosphatase (6.0 /xg, Boehringer)
for 1 hour at 37°. The product was analysed by reversed phase
hplc as above. The products of digestion of the earlier eluting
isomer showed a peak at 36.6 minutes corresponding to
Sp-CpsA.
Ribozyme cleavage reactions
a) Comparison of cleavage of unmodified transcribed 15-mer
substrate with chemically synthesized 13-mer substrate.
Unlabelled 37-mer ribozyme E (12.5 pmol) and radiolabelled
1188 Nucleic Acids Research, Vol. 19, No. 6
either transcribed 15-mer S or chemically synthesized 13-mer
S in 40 /tl water were incubated at 50° and cleavage initiated
by addition of 10 /tl of prewarmed 5 Xcut buffer (250 mM Tris
HC1 (pH 7.4), 100 mM MgCl2). Samples (3 /il) were taken at
intervals and the reaction quenched by the addition of 1 y\ 0.2M
EDTA (pH 7.4) and stored at - 2 0 ° . Products were separated
by PAGE (20% denaturing gel) and bands visualized by
autoradiography.
b) Determination of Km and k^ of unmodified hammerhead.
Chemically synthesized phosphorylated 13-mer S (20—400 pmol
unlabelled and 0.05 - 0 . 1 pmol 32P-labelled) and 37-mer E,
each at the appropriate concentration in 20 /tl water, were heated
separately to 95° for 1 minute, cooled to 30° and 5/d of 5 xcut
buffer added. After 15 min, the cleavage reaction was initiated
by mixing E and S and samples taken and treated as described
in a). Autoradiographs of the polyacrylamide gels were scanned
using a Molecular Dynamics laser scanning densitometer. The
initial concentration of substrate was varied from 10 to 400 nM
whilst maintaining a substrate:ribozyme ratio of at least 10. Km
and k^ were calculated from Eadie-Hofstee plots (21).
c) Demonstration of inversion of configuration in cleavage of
hammerhead. 35S-thio S (252 pmol) and 32P-thio S (58 pmol)
was treated with E (93 pmol) in 625 /d of 50 mM Tris HC1 (pH
7.4), 20 mM MnCl2 for 4 hours at 50°. The reaction was
quenched with 0.5 M EDTA (30 /il), extracted with butanol (23)
and products separated by electrophoresis on a 20% denaturing
polyacrylamide gel (1.5 mm thick). The slower band as identified
by autoradiography was eluted and butanol extracted as described
above. The pellet was dissolved in 30 /xl of pyridine/acetic
acid/water (2:1:330) and treated with 5 /tg of nuclease PI at 37°
for 1 hour. The sample was evaporated (SpeedVac), taken up
in 30 /tl of 0.1M ammonium bicarbonate, 34 mM ammonia (pH
9) and treated with 0.5 /tl calf alkaline phosphatase (Boehringer,
28 units/ml) at 37° for 30 minutes. After heat inactivation of
the phosphatase, the sample was added to a mixture of equal
amounts of cytidine 2',3'-cyclophosphorothioate (cCMPS, exo
and endo isomers) (36) and products separated on a Spherisorb
ODS 5/t column using a gradient of buffer A: 0.1M ammonium
acetate and buffer B: 0.1M ammonium acetate/ acetonitrile (2:8)
as follows: 0%B, 2', 0 - 12%B, 20'. Retention times: 4.5' (exo)
and 10.3' (endo). Material in each peak was collected, evaporated
(SpeedVac), dissolved in water (20/tl), spotted on a GF/C filter
(Whatman) and counted by liquid scintillation. All the
radioactivity (as ^S) was found in the peak corresponding to the
endo isomer of cCMPS.
d) Tune-course of cleavage reactions with thio substrates. 1 /tl
of 32 P-phosphorylated S (1.5 pmol) was added to
unphosphorylated S (30 pmol) and the volume made up to 20 /tl
with water. This solution and a solution of E (1 pmol in 20 /tl
water) were heated separately to 95° for 1 minute and incubated
at 30° for 5 minutes. To each solution was added 5 /il of a
solution containing 250 mM Tris HC1 (pH 7.4) and either
MgCl 2 or MnCl2 (100 mM) as appropriate. After 15 minutes at
30°, the cleavage reaction was started by mixing the solutions
of E and S . 3 /d samples were taken at intervals and quenched
with 1 /d EDTA (0.5 M, pH 7.4). Products were separated by
electrophoresis on a 20% polyacrylamide gel (0.3 mm thick),
visualized by autoradiography and quantified by laser scanning
densitometry.
ACKNOWLEDGEMENTS
We are very grateful to Dr Fritz Eckstein and Olaf Heindreich
(Max Planck Institut fur Experimentelle Medizin, Gottingen) for
provision of samples of exo and endo isomers of cCMPs, for
critical reading of the manuscript, and for much helpful advice
and encouragement. We would also like to thank Clare Pritchard
and Terry Smith with help and advice on oligoribonucleotide
synthesis and Sir Aaron Klug for the original impetus for this
work.
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