Professional Documents
Culture Documents
Slim 1991
Slim 1991
6 1183
ABSTRACT
for the preparation of hammerhead ribozymes containing single The 37-nucleotide catalytic strand (E) and the unmodified
phosphorothioates of Rp and Sp stereochemistry at the cleavage 15-nucleotide substrate strand (S) were prepared by T7 RNA
site. The methods involve sulphurization of a phosphite diester polymerase-mediated transcription of synthetic DNA templates
formed as an intermediate during solid-phase oligoribonucleotide by the method of Milligan et al. (18). Substrate strand was
synthesis. This results in an approximately 1:1 mixture of Rp labelled by use of a-^P-CTP in the transcription reaction. Each
and Sp phosphorothioate isomers. After assembly, the completed strand was purified by polyacrylamide gel electrophoresis under
oligonucleotide containing a mixture of Rp and Sp denaturing conditions. Unmodified 13-nucleotide substrate strand
phosphorothioate isomers can be resolved by reversed-phase (S) was synthesized by a phosphoramidite procedure (22) using
chromatography. The individual isomers have been used in a commercially available ribonucleoside phosphoramidite
hammerhead cleavage reaction and the results compared to the monomers on an automated DNA Synthesizer, purified by hplc
cleavage of the unmodified ribozyme. (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
RESULTS
in the presence of 20mM magnesium at 50°, transcribed 15-mer
Synthesis and cleavage of an unmodified hammerhead (S) strand (0.5 pM) was cleaved with a t 1/2 of 1 —2 minutes
The hammerhead ribozyme sequence chosen for study has a short whereas chemically synthesized 13-mer (S) strand (0.5 /tM) was
substrate strand (S) and a longer 37-residue catalytic strand (E) cleaved with t 1/2 of < 1 minute. Both transcribed and
(Figure 3). The catalytic strand contains the loop sequence chemically synthesized (S) strands were cleaved to >99%
completion within 1 hour (data not shown) which indicates that
AGUCCC
Figure 1. Hammerhead consensus structure. Boxed residues signify those
UCAGGGppp
conserved in most hammerheads. Dots indicate Watson-Crick base pairing.
,GPPP
C- G
b)
E G- C
G- C
s
\ Phosphodiester or
yS Rp Pboiphorothioate
A^i
C G GC G
u C G C GG
cd G ' 'AGUCCC
\ U C A G G Gppp
u U/S A
Figure 2. Proposed stereochemical course of phosphate cleavage showing Figure 3. a) Hammerhead comprising transcribed 37-mer catalytic strand (R)
pentacoordinated intermediate. When X = Y = O : unmodified phosphodicster, when and chemically synthesized 13-mcr substrate strand (S). The 5'-end of S is
X = O, Y = S : Sp-phosphorothioate and exo isomer of cCMPS; when X = S, enzymatically phosphorylated. b) Hammerhead comprising transcribed 37-mer
Y = O : Rp-phosphorothioate and endo isomer of cCMPS. 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 radiography on polyacrylamide gels) was cleaved to completion
of ^P-labelled Rp-thio 15-S with unlabelled 37-mer E catalytic using unlabelled 37-mer catalytic strand (0.15 /iM) in the presence
strand in the presence of magnesium showed a very slow cleavage of manganese ion (20mM) at pH 7.4 for 4 hours at 50°. The
rate (Figure 4b). After 240 minutes, the cleavage was only 12% cleavage products were separated by denaturing PAGE and the
complete and significant non-specific degradation was observed. slower band eluted and salts removed by extraction with n-butanol
In the presence of manganese, the cleavage rate approximately (23). The RNA fragment was digested with nuclease PI and then
doubled. alkaline phosphatase treated. This procedure should give rise only
Although it has been shown in a single-stranded transcribed to nucleosides and to 35S cytidine 2',3'-cyclophosphorothioate
hammerhead that inversion of configuration at phosphorus takes (cCMPS) which results from the nucleoside 5' to the cleaved
place during self-cleavage, it was important to confirm that this phosphorothioate bond. The products of digestion were added
is still the case for a two-stranded hammerhead. Accordingly two to a mixture of the exo and endo isomers of unlabelled cCMPS
batches of Rp-thio 15-S were prepared, one in which 35 S- and separated by reversed-phase hplc. Material eluting at the
adenosine a-thiotriphosphate was used in the transcription position of each isomer was collected and counted by liquid
reaction to incorporate radioactivity into the thiophosphate at the scintillation and ah" the radioactivity (as 35S) was found to have
cleavage site and another in which a small amount of a-32P-C- eluted at the position of the endo isomer (data not shown). This
TP was used. A mixture of 35S and 32P-labelled Rp-thio 15-S result can only have been obtained by complete inversion of
(0.5 fiM) (just enough 32P to be able to follow by auto- configuration at phosphorus during cleavage of the Rp-
phosphorothioate. Since this is the same result as obtained from
cleavage of the single-stranded hammerhead (15), it follows that
* 20-
200 300
eluted in 0.5 mM ammonium acetate, 10 mM magnesium acetate reaction was quenched with triethylammonium acetate (0.1 M,
in sterile water. The oligonucleotides were desalted using OPC 5 ml) to give a homogeneous solution (any insolubility at this
cartridges (Applied Biosystems) following manufacturers stage indicates incomplete desilylation) and dialysed immediately
instructions. Transcription reactions were carried out at 37° for against distilled water. The resulting product was lyophilized and
3 hours in 250/xL-lml reactions containing 40 mM Tris HO (pH purified by strong anion exchange (SAX) chromatography on a
8.2 at 37° at 1M concentration), 7 mM MgCl2, 5 mM DTT, semipreparative Partisil P-10 SAX column (Hichrom) using a
1 mM spermidine, 0.01 % Triton X100, 50 /xg/ml acetylated BSA gradient of buffer A: 1 mM KH2PO4 (pH 6.3)/60% formamide
(Anglian Biotech), 6% polyethylene glycol 6000 (Koch Light), and buffer B: 300 mM KH2PO4 (pH 6.3) /60% formamide
2 mM each of ATP, GTP, UTP and CTP and oligonucleotides (0%B 5', 0-50%B 10', 50-100%B 15'). Elution time of (Rp
at 0.2 pmol //xl using T7 RNA polymerase (15-20 units / /xl). + Sp)-thio 13-mer was 23.6' (cf unmodified 13-mer was 22.4').
32
P-labelled transcripts were made by incorporation of a-32P- After dialysis and lyophilization, the 13-mer product was further
CTP (40 /xCi /ml). Thio-substituted transcripts were made by purified by reversed phase chromatography on a semipreparative
replacement of ATP by Sp-ATPas (Amersham) at 0.5 mM. ^S- /x-Bondapak C-18 column (Waters/Millipore) using gradients of
thio-substituted transcripts were made by addition of 35S-Sp- buffer A: 0.1 M ammonium acetate and buffer B: 20% buffer
ATPas (Amersham) to 40 /xCi /ml. Reactions were stopped by A/ 80% acetonitrile (Figure 5). Material in the product peak was
addition of EDTA to 30 mM, phenol extracted and ethanol isolated by evaporation. Full details of these procedures are
precipitated. Pellets were taken up in 0.1 mM EDTA and published elsewhere (34).
transcripts were purified by PAGE (15% denaturing gels, 1.5 Small aliquots (10—200 pmol) of synthetic oligoribonucleotides