Table Of ContentSmall Efficient Hammerhead Ribozymes
Philip Hendry, Trevor J. Lockett, and Maxine J. McCall
1. Introduction
The hammerhead ribozyme was discovered as a self-cleavmg RNA
molecule in certain plant vtroids and satellite RNAs (1). Shortly after its
conserved features were defined (2,3), the hammerhead was shown to be able
to act as a true enzyme, cleaving multiple substrates m a bimolecular reaction
(4). The self-cleaving hammerhead can be divided in a number of ways into
two, or even three, separate strands (45). The most useful form has almost
all of the conserved nucleotides on the ribozyme strand, leaving minimal
sequence requirements in the substrate strand. To be cleavable the substrate
must possess the sequence 5’ UH (H is C, U, or A), where cleavage occurs to
the 3’ stde of H (6) (Fig. 1). This particular configuration has been the para-
digm for hammerhead ribozyme design since 1988. Here we describe varia-
tions on this basic design, with the constant theme being to minimize the size
of the ribozyme.
The advantages of minimizing the size of the ribozyme are several-fold. An
obvious advantage, for ribozymes which will be used as exogenously-supplied
therapeutics, is that the cost of synthesis is reduced if the number of rtbonucle-
otides is mimmized. A second advantage, for the exogenously-supplied
ribozyme, is that delivery to cells may be aided if the ribozyme is small. An
additional, and unexpected, advantage is that some minimized ribozymes
cleave their substrates faster in vitro than the analogous, standard ribozymes
do, although it is not yet known if this advantage is carried over to produce an
enhanced effect in vivo. Two strategies for minimizing the size of the hammer-
head ribozyme are described in this chapter. The first involves shortening the
hybridizing arms of the ribozyme (which results m shortening helix I and/or
From Methods m Molecular Me&we, Vol It Therapeutic Appkahons of Rlbozymes
Edlted by K J Scanlon 0 Humana Press Inc , Totowa, NJ
1
Hendry, Lockeft, and McCall
Helix III Helix I
1
Substrate 5’ N N N N N N N U HI7 N N N N NM 3’
3’ r;r i il Ii Ii a,&, i.l I; il Ii i N 5’
A %
G
A
Ribozyme G12 AG uA6
C-G s
Helix II N-N
N.N
NON
Loop II N N
NN
Fig. 1 Schematic representation of the hammerhead rtbozyme m complex with its
complementary substrate. The ribozyme forms helix I with its 5’ arm and the substrate,
helix III with its 3’ arm and the substrate, and helix II and loop 2 with the nucleotldes
joming A, and G12 In the rtbozyme, all nucleotldes, except for the conserved C3 to A,
and GU to 4, I, may be erther ribonucleotrdes or deoxyrlbonucleotldes The site of
cleavage m the substrate is shown by the downward arrow, 3’ to HI7 (H = C, U or A)
helix III in the ribozyme-substrate complex), whtle mamtaming maximal cleav-
age rates. The second involves shortening, or completely elimmatmg, helix II
in the ribozyme. The consequences of each of these modifications on the cleav-
age activrties will be discussed.
2. Guidelines for Design
The work we describe here is based largely on observations made using
short substrates m vitro. To directly extrapolate these observations to the use of
ribozymes m vivo IS difficult because of the unknown factors that operate
wtthm a living organism. In particular, the ribozyme may be affected by the
activities of RNA binding proteins; these proteins can either enhance or retard
substrate binding and dissociation (7-9), may stabilize the ribozyme against
degradation (10,11), or may direct the rrbozyme to specific compartments
within the cell in a manner observed for some mRNAs (12). However, it 1s
important to understand the basic processes of ribozyme cleavage and, by mak-
ing some assumptions about the intracellular envtronment experienced by an
RNA molecule, tt should be possible to project some of this m vitro experience
mto therapeutic practice.
Hammerhead Ribozymes 3
2.7. Minimiring Arm Lengths
2.1.1. Introduction
The standard form of the hammerhead ribozyme, as used by most research-
ers, is shown in Fig. 1. It has the conserved nucleotides C3 to A, and Gi2 to
Ai5 i, 4 bp m helix II with a G C bp adjacent to A9 Glz, 4 nucleotides in loop II,
and a variable number of nucleotides in the 5’ and 3’ arms which, on bmdmg to
the substrate, form helix I and helix III, respectively. The goal here is to rede-
sign this hammerhead ribozyme so that it contains as few bases as possible in
the hybridizing arms without compromising the cleavage ability or specificity
of the ribozyme.
There are a number of steps m defining the hybridizing arms of a hammer-
head ribozyme. First, the target site within the RNA of interest must be chosen.
Good targets have the UH cleavage site located within an accessible region of
the RNA, so that the ribozyme 1sr eadily able to hybridize to the site (see Note 1).
Second, the number of base pairs to be formed between the ribozyme and sub-
strate, or the extent of complementarity, must be chosen. The aspects to con-
sider here are catalytic activity and specificity. Ribozymes that form a large
number of base pairs with the substrate are unlikely to turnover. Smaller
ribozymes with relatively short hybridizing arms are able to turnover rapidly
and therefore have the potential for high catalytic activity. Therefore, the extent
of complementarity should be such that the ribozyme-substrate complex
formed is (relatively) stable under the conditions of the experiment, and this
typically requires 1 l-l 7 bp. For specificity, the number of bp formed between
the ribozyme and substrate should be large enough to make the target sequence
unique, but not so large that imperfectly matched substrates form stable com-
plexes (13). Statistically, about 13 nucleotides are required to uniquely define a
particular site m a mRNA pool in a mammalian cell (see Note 2). Finally, once
the target site and the number of nucleotides to be bound has been decided, the
disposition of the nucleotides about the cleavage site must be determined. By
far, the most common arrangement has been to target an equal number of nucle-
otides on either side of Hi7. A ribozyme of this design we call a symmetric
ribozyme. We have recently shown that this design does not produce the most
rapid cleavage rates m vitro.
2.1.2. The Optimum Length for Helix I
We observed that the length of helix I in the ribozyme-substrate complex
has a very profound effect on the cleavage rate constant for that complex (14).
In a number of systems we have varied the length of helix I, both by varymg
the number of nucleotides to the 3’ side of the cleavage site of the substrates
and by changing the length of the 5’ hybridizing arms of the nbozymes. All
Hendry, Lockett, and McCall
Length of Helix I (bp)
Fig 2 Dependenceo f rate constantso n length of helix II, with helix III constanta t
10 bp substratesw ith varying numbers of nucleotrdest o the 3’ side of the cleavage site
and 10 nucleotideso n the 5’ side are cleaved by their cognater tbozymes;I Kr RA- 101
10; A Kr RB-IO/lo; 0 TAT RB-lO/lO. Reactionsc ondlttons; 10 mA4MgCl,, 37°C
pH 7 13 From ref. 14
these experiments were performed under ribozyme excess conditions with the
rrbozyme-substrate complex fully formed prior to initiation of the reaction.
The cleavage rate constants for substratesw ith varying numbers of nucleottdes
3’ of Hi7 by three different symmetrrc (10 + 10) ribozymes are shown m Fig, 2.
Cleavage rate constants of 2 1- mer substratesb y ribozymes with varying lengths
of hybridizing arms are given m Table 1. Together these data demonstrate that
the optimum length for helix I m a hammerhead rrbozyme 1sa bout 5 or 6 nucle-
otides whether the length of the hehx 1sh mited by the length of the substrate or
ribozyme. Ribozymes wtth longer 5’ arms are potentially limited m their acttv-
tty by slow cleavage rates.
2.1.3. The Optimum Length for Helix III
To determine whether there was an optimum length for helix III, the cleav-
age rate constants were compared for ribozyme-substrate pairs with optimum,
or near optimum, helix I lengths and either 10 or 6 bp rn helix III. The variation
in rate constant observed is shown m Table 2. The effect is quite small, twofold
Hammerhead Ribozymes 5
Table 1
Cleavage Rate Constants for 21-mer Substrates
by Various Cognate Ribozymes
Substrate Rlbozyme III/P k&mm
TAT S21-lo/lob TAT RA- 1O /l Ob 10110 0 63”
TAT S21-IO/10 TAT RA- 1O /5 5110 0.09 -I- 0.01
TAT S21-lO/lO TAT RA-5110 1015 lo+ 1
Kr s21-lO/lO Kr RA-lO/lO lO/lO 0.10
Kr S21-lO/lO Kr RA-6110 1016 6.7
Condltlons; pH 7 13,37”C, 10 mMMgC12
ONumber of bp m hehces III and I, respectively
bThe nomenclature for the substrates and rlbozymes 1s as follows The sequences of the
substrate molecules are taken from naturally-occurrmg mRNAs and are identified by their on-
gin, The TAT series are from the TAT gene of HIV-l, and the Kr senes are from the Krdppel
gene of Drosophzla melanogaster Rlbozymes are denoted by an R followmg the identifying
prefix, and substratebsy the letter S anda numberw hich indicatest he numbero f nucleotldesm
the substrate There are three versionso f hammerheardlb ozyme usedm this chapter,a nd they
are denoteda sn bozymesA , B andC RlbozymesA (RA) are composeds olely of RNA (with
the exceptlon of the 3’ nucleotlde), rlbozymes B (RB) possessD NA m the arms that hybridize
to the substrate( with the exceptiono f nucleotldes1 5 1 and 15 2 which remama s RNA [Fig.
l]), andr lbozymesC (RC) are the samea sr lbozymesB except that their helix II andl oop II are
also composed of DNA. The number of nucleotldes m the hybridlzmg arms (for nbozymes) or
on eachs ideo f Cl7 (for substratesa)r e addedt o the name,w ith the first numberr eferrmg the 5’
side and the secondto the 3’ side For example,T AT I&4-5/10 IS an all-RNA rlbozyme with 5
nucleotldesm its 5’ arm and 10 nucleotldesm its 3’ arm, TAT S21-lO/lO 1sa 21-nucleotlde
substratew ith 10 nucleotldese achs ideo f C,,, andT AT RA-5/10 and TAT S21-lO/lO form a
complexw ith 10 bp m helix III and 5 bp m helix I (III/I = 10/5)
CAtp H 8 00
Table 2
Effect of Length of Helix Ill on Cleavage Rate Constants
S17-10/6 S13-616
Ribozyme III/I k,b,/min III/I k&mm
TAT RB-lO/lO 1 O/6 1.8 f 0.4 616 0.92 f 0 1
Kr RA-lO/lO 1016 4.8 f 0 6 616 3.4 f 1.0
Kr RB-lO/lO 1016 3 2 f 0.3 616 17+04
Condltlons 10 mMMgQ, 37”C, pH 7 13
at most, with the more efficient cleavage occurring in ribozyme-substrate pairs
possessing the longer helices III. Although we have not pursued this further,
there is no reason to suspect that cleavage efficiency will be impaired rf the
ribozymes are able to form even longer helices III with their substrates. How-
6 Hendry, Lockett, and McCall
ever, there is the danger that excessrvely long hybridizing arms are able to form
folded, stable structures that prevent substrate binding.
2.1.4. Multiple Turnover
The observations above relate to reaction condttrons m which the rrbozyme
is m excess of the substrate, and the rrbozyme and substrate were preannealed
before mrtration of the reaction by addition of Mg2+. Under these condmons,
substrate binding and product drssociatton have no effect on the observed cleav-
age rate constants, However, under multiple turnover conditions, a desirable
situation for therapeutic uses, the rates of substrate binding and product disso-
ciation must also be considered. The rate of substrate association 1sd ifftcult to
predict for large substratesa nd will be largely dependent on the structure of the
RNA m that region (15). On the other hand, for a given sequence, the rate of
dissociation of the cleavage product 1s expected to consistently decrease with
increasing length (15,16). Given that the optimum length for helix I is around 5
or 6 bp, and that duplexes of this length usually drssocrate quite rapidly, It 1st he
length of helix III that is most crucial m this respect. The rate constant for
dissociation of either cleavage product in vitro may be readily esttmated by a
number of techniques (see Note 3). As a rough guide, m condmons like that
encountered m biological systems, (pH 7.0, 37°C 100 mM NaCl), the rate
constants for drssociation of the helix III (after cleavage) are hkely to approach
that observed for the cleavage step when the length of helix III is m the range
5 -9 bp, depending on the sequence.
2.1.5. Summary
The most efficient hammerhead rtbozymes have 5 or 6 nucleotrdes in their 5’
hybridizing arms, so that they may form a helix I of 5 or 6 bp in complex with
their substrates. On the other hand, the ribozymes should have a minimum of 5
or 6 nucleotides m then 3’ hybridrzmg arms, so that they may form a helix 3 of
at least 5 or 6 bp m the complex. No diminution of cleavage rate constant under
rrbozyme excess condmons 1s expected for ribozymes with longer 3’ hybrrdiz-
mg arms, but the turnover rate under substrate excess conditions will be
adversely affected by excessrvely long helices III.
2.2. Minimizing or Eliminating Helix II
2 2.1. Introduction
Apart from the hybridizing arms, the other region of the hammerhead
rrbozyme which may be reduced m size,o r even eliminated, IS helix II and loop
II. When hehx II and loop II are completely eliminated, so that the rrbozyme
consists of two strands with free ends at the conserved nucleotides Ag and Glz,
very slow cleavage of the substrate is observed (17,18). When Ag and Gr2 are
Hammerhead Ribozymes 7
linked, either by nucleotides or non-nucleotide chains, slow cleavage is
observed with linkers containing just 13 atoms, whereas more reasonable rates
are observed with linkers containing 25 atoms (I 7). These results demonstrate
that helix II is not essential for cleavage activity. However, the nature of the
linker which replaces helix II greatly affects the cleavage activity of the small
ribozyme (17,19).
Experiments to determine rate constants for the cleavage of short substrates
by ribozymes that have a truncated helix II have shown that the number of base
pairs m helix II can be reduced to two without loss of cleavage activity, relative
to the standard ribozyme with a four base-pan helix II (20). A further reduction
to one base pan m helix II results in a lo-fold loss of activity, and ehmmation
of helix II (where A9 and Gla are connected by a 4-nucleotide loop) results m
around a loo-fold loss of activity, relative to the standard ribozyme (20-22).
The short substrates with which these experiments have been carried out were
about 13 ribonucleottdes m length. When the substrate length, increases, for
example to 2 1 nt, with a concomitant increase m the lengths of hehces I and III,
the cleavage rates of ribozymes with one or no base pairs in helix II increases
relative to that of the standard ribozyme. Furthermore longer RNA transcripts
are cleaved in vitro by these small ribozymes faster than they are cleaved by
standard ribozymes (22). It is not known if the same relative rates of cleavage
by the small and standard ribozymes also occur in viva.
In order to distinguish these small ribozymes from the standard ribozymes,
we define a ribozyme with one base pair m helix II as a mmiribozyme, and a
ribozyme with no helix II as a minizyme.
2.2.2. Miniribozymes
A miniribozyme with nucleotides of sequence 5’rGUUUUC joining A9 and
Gt2 is shown m Fig. 3A. Maximal cleavage activity is conferred on the
mmiribozyme when the single base-pair that replaces helix II is a G.C in the
orientatron shown in the figure. The optimal sequence for the loop connecting
the G and C has not been determined, but a flexible loop of 4 ribouridmes gives
good activity, whereas a loop of three ribouridines has about 70% the effi-
crency of the 4-ribouridme loop in cleaving short substrates (unpubhshed data).
A loop of sequence 5’ UUUG had a cleavage rate constant about 10% that of
the parent ribozyme (21).
The mmnibozyme may consist solely of ribonucleotides, or it may be syn-
thesized with a mixture of deoxyribonucleotides and ribonucleotides. A simple
configuration for the DNA/RNA hybrid has DNA in all positions except for
C3-A9 and Gi2-Ai5 2. The inclusion of DNA in the molecule reduces the costs
of synthesis, and may also give a degree of protection against degradation in
human serum (23) or in cells (24).
8 Hendry, Lockett, and McCall
3’ N N N N N PJ2A,u N N N N N N 5’
A cq~
G
A
& AGuA
C-G 9
u u
Miniribozyme uu
3’ N N N N N f&Am N N N N N N 5’
A kG
A
Gu AG”A
T Gg
Minizyme TTT
Fig. 3. Schematic representatton of a mmmbozyme and a mmizyme Formally, the
mmmbozyme has single G.C base pair replacing helix II, and the minizyme has no
helix II The sequences of nucleotides shown joining As and G12 in the miniribozyme
and mmizyme confer good cleavage activities on these molecules. The numbers of
nucleotides m the 5’ and 3’ arms of the mmnibozyme and mmizyme may be larger than
indicated here, without dtmnushing cleavage activity m vitro. All nucleotides, except
for the conserved CJ to A9 and G12 to A15 1, may be either ribonucleotides or deoxyri-
bonucleottdes.
DNA-contammg mimribozymes with d(GTTTTC) m place of helix II and
loop II, and with DNA hybridizing arms, cleave 13-mer substrates at approx
40% of the rate of analogous ribozymes with DNA in the hybridizing arms and
m helix II (Table 3). In these examples, both the mmn-ibozymes and ribozymes
form heltces I and III each of 6 base pairs m complex with the substrate. The
same mimribozymes cleave 21-mer substrates about twofold faster than the
analogous ribozymes (Table 3), and here they form a helix I and a helix III
each of 10 base pairs m complex with the longer substrate. Of more relevance
to biological applications, an all-RNA mmmbozyme cleaves an 809-nt RNA mol-
ecule in vitro much faster than does an analogous all-RNA ribozyme (Fig. 4),
Hammerhead Ribozymes
Table 3
Cleavage Rate Constants for 13.Mer and Pi-Mer Substrates
by DNA-Containing Miniribozymes (Mgttttc) and Ribozymes (FE)
k&mm
Substrate Mgttttc RC-lo/10 Ratio Mgttttc/RC
TAT S 13 -6J6 0 175+0003 0.43 5 0 08 0.41
TAT S21-lo/10 0.9 Ik 0.2 0.45 * 0 09 20
Kr S13-6/6 0.59 f 0.09 1.6 2 0.3 0 37
Kr S21-lO/lO 3.0 zk 0.2 1.34 zk 0.08 22
Condltlons 10 mh4 MgCl,, 37”C, 50 mh4 Trls-HCl, pH 7 13
Sequences of nbozymes, with upper-case letters representing nbonucleotldes, and lower-
case letters representing deoxyribonucleotides, are as follows.
TAT Mgttttc: 5’ gtcctaggctCUGAUGAgtttttcGAAACttcctgga.
TAT RC 5’ gtcctaggctCUGAUGAgtccttttggacGAAACttcctgga
Kr Mgttttc 5’ ctccagtgtgCUGAUGAgttttcGAAACtcgcaaat
Kr RC 5’ ctccagtgtgCUGAUGAgtccttttggacGAAACtcgcaaat
and shows good activity over a much wider temperature range than does the
ribozyme (Fig. 5). Thus, at least m the examples studied to date, minirlbozymes
seem to be superior to full-size hammerheads m cleaving long transcripts in
vitro.
The relative rates at which mimribozymes cleave 13-mer and 21 -mer swb-
strates indicates that unlike full-stzed ribozymes, mimrlbozymes are not hm-
dered in their cleavage activity when forming extensive base pan-mg with the
substrate, particularly with respect to helix I. This gives an advantage to
mmiribozymes over ribozymes in that, for a target sequence of defined length,
the distribution of nucleotides on either side of the cleavage site is not restricted
in any way, whereas, for optimal cleavage by the ribozyme, the number of
nucleotides in the 5’ arm is restricted to 5 or 6 (see Subheading 2.1.2.). There-
fore the miniribozyme may be made with hybridizing arms of equal length,
which may assist turnover.
2.2.3. Minizymes
A minizyme is a hammerhead ribozyme in which helix II and loop II have
been replaced by a short linker that contains no Watson-Crick base pairs (25’.
Minizymes have been made with linkers consisting of nucleotides (20,25,26),
or of short polymers of ethyleneglycol and phosphopropanedlol (17,19). The
rates of cleavage by mimzymes increase as the number of atoms m the chain
lmking Ag and G12 increases, with optimal rates being achieved with 25-31
atoms in the chain (27). For all-nucleotide linkers, this corresponds to 4 or 5
nucleotides conferring optimal activity.
IO Hendry, Lockett, and McCall
60
time (hr)
Fig 4. Rates of cleavage of an 809-nucleotlde interleukm-2 transcript by
mterleukm-2 mimrlbozyme and ribozyme, at 37”C, 50 mA4 Tris-HCl, pH 8.0, 10 mA4
MgC12, with no heat pretreatment.T he minmbozyme hast he sequence5 ’ r(GUUUUC)
in place of helix II and loop II. Both mmiribozyme and ribozyme are made of RNA,
and have 8 nucleotides in the 5’ hybridizing arms and 6 nucleotides in the 3’ arms
Cleavage of the transcript occurs 82 nucleotldes from the 5’ end.
The first mmizymes synthesized had linkers of sequence d(TTTT) or
r(UUUU) (25). These minizymes were about loo-fold less-active than analo-
gous standard ribozymes m cleaving short substrates of about 13 nucleotides
(20-22). Like the mimrlbozymes however, symmetric (10 + 10) mmizymes
actually improve in cleavage activity when cleaving 21-mer substrates, such
that the mimzymes are typically only IO-fold less active than analogous
ribozymes (22). Thus it appears that mmizymes, like miniribozymes, and unlike
ribozymes, are not inhtbtted in their cleavage rates by the formation of long
(>6 bp) hehces I. Against a 428-nucleotide RNA target derived from the HIV-
1 TAT coding sequence, minizymes with these lmkers cleaved faster than did
the analogous ribozymes (22). Apparently, the small size and/or flexibility
enjoyed by the ribozyme variants that lack or possess substantially truncated
hehces II gives them some advantage over full sized ribozymes at the cleavage
of long RNA transcripts.
Recently, a mimzyme with the lmker of sequence d(GTTTT) has been
described (271, and it is shown in Fig. 3. The inclusion of the G at the 5’ posi-
