Base and sugar requirements for RNA cleavage of essential nucleoside residues in
internal loop B of the hairpin ribozyme: implications for secondary structure
Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure
Sabine
Schmidt
,
Leonid
Beigelman
1
,
Alexander
Karpeisky
1
,
Nassim
Usman
1
,
Ulrik S.
Sørensen
+
and
Michael J.
Gait*
Medical Research Council, Laboratory of Molecular Biology, Hills Road,
Cambridge
CB2 2QH,
UK
and
1
Department of Chemistry and Biochemistry, Ribozyme Pharmaceuticals Inc., 2950
Wilderness Place,
Boulder
, CO 80301,
USA
Received November 22, 1995
;
Revised and Accepted January 8, 1996
ABSTRACT
The hairpin ribozyme is a small self-cleaving RNA that can be engineered for RNA cleavage
in trans
and has potential as a therapeutic agent. We have used a chemical synthesis
approach to study the requirements of hairpin RNA cleavage for sugar and base
moieties in residues of internal loop B, an essential region in one of the two
ribozyme domains. Individual nucleosides were substituted by either a 2
'
-deoxynucleoside, an abasic residue, or a C3-spacer (propyl linker) and the abilities of the modified ribozymes
to cleave an RNA substrate were studied in comparison with the wild-type ribozyme. From these results, together with previous studies, we
propose a new model for the potential secondary structure of internal loop B of
the hairpin ribozyme.
INTRODUCTION
The hairpin ribozyme is a region of the negative strand of the satellite RNA
from tobacco ringspot virus that undergoes self-cleavage (
1
-
3
). The hairpin (reviewed in
4
) is one of several small RNAs, which include the hammerhead and hepatitis delta
virus RNAs, that can be engineered for cleavage
in trans
(
5
,
6
). In such cases, a substrate RNA strand is cleaved following incubation with a
catalytic RNA (ribozyme) strand in the presence of divalent metal ions such as
magnesium. The power of such ribozymes is being harnessed for gene therapy
applications through intracellular, vector-driven RNA production (for example, see
7
) as well as for exogenous delivery as potential therapeutics (
8
,
9
).
Much effort has centered on understanding the structures and mechanisms of
action of small RNA ribozymes in order that this will lead to the design of
more effective RNA-cleaving therapeutic agents (
6
,
10
). Particular success has come from crystallographic and other structural
studies of the hammerhead ribozyme where knowledge of the overall folding is
now leading to the first proposals for the structural basis of the cleavage
mechanism (
11
,
12
). By contrast, much less is known about the conformation and mechanism of
cleavage of the hairpin ribozyme and there are no data available yet as to its
three-dimensional structure.
Mutagenesis and
in vitro
selection studies have revealed that the hairpin ribozyme consists of four
regions of Watson-Crick duplex RNA and two internal loops (A and B) (Fig.
1
) (
13
-
18
). All of the essential nucleotides, the identity of which are required for
catalytic cleavage, are located in the two internal loop regions (
16
,
18
). It has recently become clear that the hairpin ribozyme can be divided into
two domains, the first containing the substrate strand (internal loop A and
helixes 1 and 2) and the second consisting of internal loop B and helixes 3 and
4. These domains appear to be hinged at the junction of helixes 2 and 3
(between residues 14 and 15) such that the ribozyme can bend and the two
internal loop regions can approach and presumably interact with each other
during catalytic cleavage. Such conclusions have been reached primarily from
studies where the two domains are constrained by linkers of variable length
between residue -5 on the substrate strand and residue 50 on the ribozyme strand (
19
,
20
) and also by linking the two domains in a completely different `reverse'
configuration (
21
). Very recently it has been shown that the two domains can be completely
separated into independent sections and ribozyme activity reconsitituted by
addition of one to the other (
22
). Although a 10
4
increase in
K
M
was found, there was remarkably little effect on
k
cat
. The domains may play somewhat different roles, however. Whereas internal loop
A may be conformationally flexibile (
23
), loop B appears to undergo an initial folding process which is independent of
binding of ribozyme to substrate (
24
).
MATERIALS AND METHODS
Preparation of oligoribonucleotides
Oligoribonucleotides were synthesized on a 1 [mu]mol scale using 2'-
O
-
t-
butyldimethylsilylnucleoside-3'-
O
-(2-cyanoethyl-
N,N
-diisopropyl) phosphoroamidite monomers having phenoxyacetyl amino group
protection for A and G and benzoyl protection for C (Glen Research via Cambio).
2'-deoxy-3'-
O
-(2-cyanoethyl-
N,N
-diisopropyl)phosphoramidites and the C3-spacer phosphoramidite, used as modifications, were also obtained
from Glen Research. 1-Deoxy-D-ribofuranose phosphoramidite was synthesised from D-Ribose as described recently (
35
,
36
). The syntheses of the modified and unmodified oligoribonucleotides were
undertaken using RNA synthesis procedures as previously described (
33
,
37
,
38
) but with some minor improvements as very recently described (
39
). In some syntheses, the standard tetrazole activator was replaced by the more
powerful 5-ethylthio-1H- tetrazole (
40
,
41
). Minor changes for deprotection and purification were also introduced. Thus,
oligoribonucleotides were deprotected by suspension of the controlled pore
glass in methanolic ammonia overnight, decanting and evaporation of the
resultant solution to dryness. Treatment with triethylamine trihydrofluoride at
room temperature overnight (
42
) or triethylamine trihydrofluoride/DMF (3:1) at 55oC for 1.5 h (
41
) was carried out to remove silyl groups, followed by desalting via Sephadex NAP-10 (Pharmacia) filtration. Oligonucleotides were purified by strong anion
exchange chromatography on a semi-preparative NucleoPac PA-100 column (Dionex) (
40
) using Buffer A: 10 mM and buffer B: 400 mM sodium perchlorate, 20 mM Tris-HCl (pH 6.8), 25% formamide, flow rate 3 ml min
-1
with gradients of 15-45%B over 20 min (
39
). Desalting was achieved via extensive dialysis against water. The purity of
oligoribonucleotides was assayed by 5'-
32
P-labelling (
43
) followed by electrophoresis on a 20% polyacrylamide gel (PAGE) in the presence
of 7 M urea.
Characterization of modified oligonucleotides
To verify the presence of the modified residues,
32
P-end-labelled oligoribonucleotides were subjected to partial alkaline
hydrolysis. Each sample (~20 pmol) was dissolved in 15 [mu]l 1 M sodium carbonate/sodium bicarbonate solution (pH 10) and
incubated at 80oC over 5 min. After cooling on ice, 1 [mu]l 2 M HCl solution was added and the mixture was kept at room
temperature for 2 h. Finally, 0.5 [mu]l 4 M sodium hydroxide solution was added to the reaction mixture and a 2 [mu]l aliquot of each sample was subjected to electrophoresis on a 20%
denaturing polyacrylamide gel for 2 h at 30 W followed by autoradiography. The
presence of a 2'-deoxynucleotide or a C3-spacer was indicated by a gap at the modified position within
the sequence ladder. By contrast in the sequence ladder for
oligoribonucleotides containing an abasic residue, a band of enhanced intensity
was seen at the site of modification, presumably due to better accessibility of
the abasic residue to alkali.
Determination of ribozyme steady state parameters
A stock solution equimolar in ribozyme strand A and ribozyme strand B (50 nM)
was prepared as well as a 2 [mu]M stock solution of
32
P-labelled substrate strand, each in 40 mM Tris-HCl (pH 7.5) and incubated separately at 90oC for 1 min followed by cooling to 37oC for 15 min. The ribozyme stock solution was adjusted to
10 mM MgCl
2
and incubated at 37oC for 15 min. The cleavage reactions were carried out at 37oC by adding appropriate volumes of substrate strand solution to a
reaction mixture containing the ribozyme strands followed by brief vortexing.
Final concentrations were 2-20 nM ribozyme strands and 20-300 nM substrate strand (for unmodified ribozyme or faster
cleaving modified ribozymes) or 5-30 nM ribozyme strands and 20-300 nM substrate strand (slower cleaving modified ribozymes) in a
final volume of 100 [mu]l, 10 mM MgCl
2,
40 mM Tris-HCl (pH 7.5). Aliquots (10 [mu]l) of the reaction mixture were removed at six suitable time
intervals and the reactions quenched by addition to 20 [mu]l of stop mix (7 M urea, 50 mM EDTA, 0.04% xylene cyanol, 0.04% bromophenol
blue). The samples were analysed by polyacrylamide gel electrophoresis on 20%
denaturing gels, the resultant gels dried and subjected to scanning using a
PhosphorImager (Molecular Dynamics). The data were processed using the
programme Image Quant (Molecular Dynamics) and quantitated by use of the
Geltrak programme (
44
; Smith, J. and Singh, M., manuscript submitted).
Kinetic parameters were determined from non-linear regression fitting of the data to the Michaelis-Menten equation. Thus:
{v over {{roman {[ E ]}}}} = {{{k sub {{roman {c a t}}}} {roman {cdot [ S ]}}}
over {{K sub roman M} {roman {+ [ S}} ]}}
where
v
= initial rate, [S] = substrate concentration and [E] = total enzyme
concentration. The values of
k
cat
and
K
M
are shown in Table
1
.
Magnesium ion titrations
The effect of increasing magnesium ion concentration on the kinetic parameters
of the cleavage reaction was determined using a substrate concentration of 200
nM and an enzyme concentration of 10 nM (for fast-cleaving ribozymes) or 25 nM (for slow-cleaving ribozymes) at 37oC and with magnesium chloride concentrations ranging from 2 to
150 mM. The Tris concentration was 40 mM. Rate constants for cleavage were
calculated by measuring the initial cleavage rates at varying magnesium ion
concentrations (Table
2
). The apparent magnesium dissociation constant
K sub roman {{M g} sup {2 +}}
was calculated graphically from the magnesium ion concentration at which the
observed cleavage rate was half-maximal.
RESULTS
Assembly of modified hairpin ribozymes and measurement of kinetic parameters
A three-stranded ribozyme was chosen for the nucleotide substitution studies (Fig.
1
). This is identical to that used in our previous purine nucleotide substitution
studies (
33
) and as also utilised in investigations of the effect of 2'-deoxynucleotide substitution in ribozyme strand A (
34
). In this way the new results could be compared directly with previous studies
in consideration of loop B structure. We decided to concentrate in particular
on the nucleotides in loop B located on ribozyme strand B. There was no
previous information as to the requirements of individual 2'-hydroxyl groups in this strand. In addition, although we had
previously obtained functional group data on the three A residues (A
38
, A
40
and A
43
) (
33
), very little information was available as to the base requirements of the five
pyrimidine residues present in this strand.
Ribozyme strand B substitution results
U
37
is not amongst those nucleotides previously identified as being essential for
hairpin cleavage. Our results (Fig.
3
and Table
1
) show a 10-fold reduction in cleavage efficiency for the abasic analogue, reflected
entirely in
k
cat
reduction. Thirteen to sixteen-fold reductions in cleavage efficiency were found for both the 2'-deoxynucleoside and the propyl linker, each primarily due to
increases in
K
M
. These modest though significant effects show that both the base and sugar have
minor structural roles to play in attaining an active conformation, yet are
unlikely to play a significant part in catalysis.
Substitution of A
38
by 2'-deoxy or propyl resulted in 50- and 100-fold reductions in cleavage respectively, the majority
of these effects being seen in
k
cat
. Abasic residue substitution led to a 1000-fold decrease in
k
cat
/
K
M
, the only position where abasic subsititution was more harmful than propyl
substitution and which also showed the largest increase in cleavage rate (6-fold above the unmodified hairpin) when magnesium ion concentration was
increased (Table
2
). Both base and sugar therefore must play important roles. Since the correct
identity of the base at A
38
is essential also (
15
,
16
) and both purine and N
7
-deaza substitution are also harmful (
33
), this base is likely to be involved in hydrogen-bonding contacts.
U
39
is the only position we have found in loop B where none of the three
substitution mutants had a significant effect on catalytic cleavage. It is
clear from the tolerance we have found to both abasic and propyl linker
substitution that U
39
merely acts as a spacer and plays no significant role either in folding or
catalysis. The results at A
40
show that, whereas there is no effect of deoxy-substitution, substitution by an abasic residue or propyl linker resulted
in 100-fold or more reductions in cleavage efficiency. Thus the presence of a
base here is required but, when taken together with other data (see below), do
not indicate specific base-pair formation required for catalytic activity.
The identities of U
41
and U
42
are amongst those said to be essential for cleavage based on the fact that no
mutations at these sites were found in selection experiments (
15
). Our results show that deoxy-substitution of U
41
results in a 20-fold reduction in cleavage efficiency. Similar substitution at U
42
results in a small but significant increase, due to improvements in both
k
cat
and
K
M
. This is the only position that we have found that is actually improved by
analogue substitution and may reflect a preference of the sugar for the 2'-endo configuration. By contrast, both abasic and propyl
substitution resulted in >100-fold drops in catalytic efficiency for both U positions. Thus it is clear
that these two residues play important structural roles and do not act merely
as spacers.
Both abasic and propyl substitution at A
43
causes 300-500-fold loss of activity and it is clear that this residue must play
an important role. However, the 2'-hydroxyl group is not involved since deoxy-substitution was without effect. Surprisingly, neither abasic
nor propyl mutants showed any significant effect of magnesium ion concentration
on cleavage rate, in contrast to what has been previously found for N
7
-deazaA
43
where
K sub roman {{M g} sup {2 {back 20 {+ a p p}}}}
was seen to increase 5-fold (
33
). A similar result was found also for all other analogues tested in strand B
(Table
2
). An insignificant effect of abasic substitution at C
44
was found and deoxy-substitution was also tolerated. By contrast, propyl linker substitution
caused a 50-fold decrease in catalytic efficiency. These results demonstrate that the
base at this position is not required for hairpin catalytic cleavage.
DISCUSSION
Analogue substitution in the hairpin ribozyme
To determine the requirements for both sugar and base in loop 2 of the hairpin
ribozyme we chose to use three analogues, namely a 2'-deoxynucleoside, an abasic residue, and a C3-spacer (propyl linker). Single 2'-deoxynucleoside substitution has been used
previously to test the requirements for 2'-hydroxyl groups in the case of the hammerhead ribozyme (
46
,
47
) and also more recently in parts of the hairpin ribozyme (
34
).
The synthesis and use of an abasic residue was recently described as a
replacement for stem-loop II of the hammerhead ribozyme (
35
). It was further used as a replacement for two of the conserved U residues in
the catalytic core of the hammerhead ribozyme and in the case of U
4
replacement there was hardly any effect observed in hammerhead ribozyme
cleavage (
36
). This was interesting because the nucleotide at the cleavage site of the
hammerhead (C
17
) was found in the crystal structure to make a stacking interaction with the
exocyclic O-2 of U
4
(3 Å aromatic-n[pi]-interaction) (
12
). This demonstrates that abasic chemical substitution information can be
helpful in assessing whether base interactions seen in the ground state
(crystal structure) are also likely to be present in the transition state
(cleavage).
For those residues where a heterocylic base has little or no involvement in
attaining a catalytically active structure, substitution by a C3-spacer (propyl linker) tests the need for the 2'-hydroxyl group and/or sugar conformation in maintaining a
particular sugar-phosphate backbone geometry. Tolerance to propyl substitution shows that
a residue acts merely as a structural separator between the adjacent
nucleotides, as has been found in HIV-1 for two pyrimidine residues in the bulge of the trans-activation response region (TAR) RNA for recognition by the tat
protein (
48
) and of a U residue in the `bubble' structure of the rev-responsive element (RRE) RNA for recognition by the rev protein (
38
). The number and types of atoms separating sequential phosphates is identical
for both a ribose and a C3-spacer.
More precise functional group mapping can be effected with base analogues. For
the hairpin ribozyme, this has been effectively carried out for essential
purine residues (
32
,
33
). By contrast, although one useful pyrimidine analogue (2-pyrimidinone-1-[beta]- D-riboside) which is an exocyclic deletion
mutant of both uridine and cytosine has recently been prepared as an amidite
suitable for oligoribonucleotides synthesis (
49
), a range of pyrimidine analogues that satisfactorily tests hydrogen-bonding potential is not yet available as amidite derivatives.
Requirements for sugars and bases in loop B
In strand A of the hairpin ribozyme, the identities of residues G
21
, A
22
, A
23
, A
24
and C
25
were previously thought to be essential (
16
,
18
). Our new data for A
20
and G
21
are consistent with this. On the three A residues, each of the exocyclic amino
groups and two of the N
7
-positions (A
22
and A
23
) are required for efficient cleavage (
33
). For G
21
, 100-fold reductions in catalytic efficiency were observed for inosine (loss of
exocyclic amino group) or N
7
-deaza-substitution (
33
).
Our data concur also with previous results showing that neither hydroxyl group
is required at residues A
20
and G
21
(
34
). The same authors have also found that deoxy-substitution of either A
22
or A
23
had no significant effect but very large (300-1000-fold) reductions in catalytic efficiency were noted for both 2'-deoxy and 2'-
O
-methyl substitution at positions A
24
or C
25
(
34
). Activities of the A
24
analogues could be rescued by increasing magnesium ion concentration suggesting
an important contact of the hydroxyl group to magnesium in the transition state
of the reaction.
For strand B, U
37
was thought previously not be essential, based on the wild-type activity of a U
37
deletion mutant (
16
). However, this isolate also contained simultaneous G
36
C and U
38
G mutations. Three other cases of near wild-type cleavage were reported where U
37
had been changed to A or C, but G
36
was also mutated in these constructs (
16
). Other mutants of U
37
were also double with either A
24
(
16
,
18
) or A
43
(
18
) and both these double mutants were inactive. Thus no single point mutation of
U
37
has been reported. U
37
was also not included in
in vitro
selection experiments (
15
,
17
). Our new results are consistent with a minor, yet significant role of both
base and sugar.
The important role of the base of A
38
in catalysis (
15
,
16
) is confirmed by our results and in addition there is a possible contact
involving the 2'-hydroxyl group. By contrast, neither base nor sugar of U
39
plays a significant role. This agrees with and extends the previous
observations that U
39
C is a general up mutant (
16
) and that both C and A were selected as active alternatives in
in vitro
selection experiments (
15
).
The role of A
40
has been difficult to assess up to now. Single point mutations at this site
that maintained activity were not selected
in vitro
, but a double mutant U
39
C, A
40
C was active at a reduced level (
15
). Substitution of A
40
by U or G has been reported to result in a reduced (3%) activity in each case (
18
) but no significant reductions in cleavage were noted for either purine
substitution or N
7
-deazaA substitution (
33
). Our results confirm that specific base-pair formation is unlikely but that the base nevertheless plays an
important structural role. The only tolerated substitutions at positions 41 and
42 are U
41
C and U
42
C having 25 and 3% cleavage activities respectively (
18
). U
41
is also the site shown to be capable of intra-strand UV cross-linking to G
21
(
26
). Our results now show strong evidence for the participation of these residues
in base-pairing.
Another residue which has been somewhat difficult to assess is A
43
. This residue is not invariant in
in vitro
selection, A
43
G being found regularly, preferably associated with the U
39
C up mutation (
15
). However A
43
U was inactive (
18
). Purine substitution had no significant effect which is indicative of no
strong intra-loop base-pairing. By contrast, N
7
-deazaA substitution was extremely harmful (
33
). Since the K sub roman {{M g} sup {2 {back 20 {+ a p p}}}} of the N
7
-deazaA mutant was increased 5-fold, it was thought previously that the N
7
-position might be a magnesium binding site in the ground state. Our new
data are more consistent with the N
7
-position being involved in a hydrogen-bonding interaction of some sort. Note that the N
7
-position is preserved by G substitution and by purine substitution but not
by substitution with other bases. C
44
had been proposed to be an invariant nucleotide based on the fact that no
mutations were found at this site during
in vitro
selection (
16
). However, no C
44
mutagenesis data has been reported either. Our results show clearly for the
first time that the base at this position plays no significant role in hairpin
cleavage.
Implications for the secondary structure of loop B
Two models have been suggested recently for the secondary structure of sections
of loop B. Butcher and Burke have proposed three non-canonical base-pairs (G
21
:A
43
, A
22
:U
42
and A
23
:A
40
) based on homology with other UV sensitive RNA domains and with interpretation
of accessibility of the region to chemical reagents. Our previous results
showing the tolerance of A
40
and A
43
to removal of the exocyclic amino group (
33
) were not consistent with this model. We proposed instead that Watson-Crick pairing might take place in the lower part of loop B between
residues A
24
:U
37
and non-canonical pairing between A
23
:A
38
and either A
22
with U
39
or U
41
. However, our new data is not consistent with a A
22
:U
39
pair. Nor is the relatively small (10-fold) effect of abasic site substitution at U
37
consistent with a strong Watson-Crick pair involving U
37
. Thus this model too is unsatisfactory.
ACKNOWLEDGEMENTS
We thank Richard Grenfell, Jan Fogg and Terry Smith for assembly and
purification of oligoribonucleotides and Mohinder Singh for technical
assistance. We acknowledge grateful support to DAAD (Germany) for a fellowship
grant to Sabine Schmidt.
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