Tài liệu Báo cáo khoa học: Efficient RNA ligation by reverse-joined hairpin ribozymes and engineering of twin ribozymes consisting of conventional and reverse-joined hairpin ribozyme units ppt

In contrast to the hammerhead ribozyme, the conformation of the hair-pin ribozyme–substrate complex does not change signi-ficantly upon cleavage: the two cleavage fragments Keywords ratio

and engineering of twin ribozymes consisting of

conventional and reverse-joined hairpin ribozyme units

Sergei A Ivanov, Ste´phanie Vaule´on and Sabine Mu¨ller

Ruhr-Universita¨t Bochum, Bochum, Germany

In recent years RNA has become the focus of

develop-ment into new diagnostic and therapeutic schemes

Antisense-RNA, ribozyme, aptamer and siRNA

tech-nologies have been developed and have found

applica-tion in molecular medicine [1–7] Signalling aptamers

and aptazymes have been constructed that can sense a

number of molecules in real time and thus are valuable

diagnostic tools [8–10] Furthermore, recently

discov-ered riboswitches that regulate gene expression in vivo

in response to specific metabolites [11–13] or

tempera-ture [14] may lead to new RNA-based therapeutic

strategies

Elucidation of the molecular principles of RNA

functioning in a specific context has led to the

engi-neering of RNA molecules with new functions Two

complementary strategies can be used in RNA

engi-neering: rational design and directed evolution

Whereas directed molecular evolution relies on the

cre-ation of a repertoire of modified RNAs from which

beneficial variants are filtered, in a rational design

experiment, defined changes in the nucleotide sequence

and⁄ or secondary structure of a specific RNA are

planned on the basis of a preconceived idea This requires detailed structural and mechanistic tion on the parent RNA In cases where this informa-tion is available, rainforma-tional design has contributed to the development of new functional RNA, for example, sig-nalling aptamers and aptazymes [8–10]

Work in our laboratory has focused on the rational design of functional RNA, in particular on the development of hairpin-derived twin ribozymes for site-specific alteration of RNA sequences, and fluores-cent and affinity labelling of large RNA molecules [15–18] The hairpin ribozyme catalyses the reversible site-specific cleavage of suitable RNA substrates, gen-erating fragments with a 2¢,3¢-cyclic phosphate and, respectively, a free 5¢-OH terminus [19,20] In the reverse reaction, the oxygen atom of the free 5¢-OH group of one RNA fragment attacks the phosphorous

of the cyclic 2¢,3¢-phosphate group of another, result-ing in ligation of the two fragments In contrast to the hammerhead ribozyme, the conformation of the hair-pin ribozyme–substrate complex does not change signi-ficantly upon cleavage: the two cleavage fragments

Keywords

rational design; RNA catalysis; RNA ligation;

sequence alteration; twin ribozyme

Correspondence

S Mu¨ller, Ruhr-Universita¨t Bochum,

Fakulta¨t Chemie, Universita¨tsstrasse 150,

D-44780 Bochum, Germany

Fax: +49 234 321 4783

Tel: +49 234 322 7034

E-mail: sabine.w.mueller@rub.de

(Received 13 June 2005, accepted 15 July

2005)

doi:10.1111/j.1742-4658.2005.04865.x

In recent years major progress has been made in elucidating the mechanism and structure of catalytic RNA molecules, and we are now beginning to understand ribozymes well enough to turn them into useful tools Work in our laboratory has focused on the development of twin ribozymes for site-specific RNA sequence alteration To this end, we followed a strategy that relies on the combination of two ribozyme units into one molecule (hence dubbed twin ribozyme) Here, we present reverse-joined hairpin ribozymes that are structurally optimized and which, in addition to cleavage, catalyse efficient RNA ligation The most efficient variant ligated its appropriate RNA substrate with a single turnover rate constant of 1.1 min)1 and a final yield of 70% We combined a reverse-joined hairpin ribozyme with a conventional hairpin ribozyme to create a twin ribozyme that mediates the insertion of four additional nucleotides into a predetermined position of a substrate RNA, and thus mimics, at the RNA level, the repair of a short deletion mutation; 17% of the initial substrate was converted to the inser-tion product

remain oriented close to each other in the ribozyme–

product complex before dissociating to leave the free

ribozyme behind [21–23] Therefore, in the hairpin

ribozyme reaction the entropic cost of ligation is rather

low and can be compensated for by the favourable

reaction enthalpy of formation of the

5¢,3¢-phospho-diester bond via ring opening of the 2¢,3¢-cyclic

phos-phate [24] This specific feature basically makes the

hairpin ribozyme a better ligase than it is an

endonuc-lease However, dissociation of cleavage fragments

from the ribozyme has to be considered and thus the

ability to preferentially cleave or ligate a specific RNA

substrate strongly depends on the stability of the

pro-perly folded substrate–ribozyme complex Strikingly,

the hairpin ribozyme is an efficient ligase if the

ribo-zyme substrate complex is folded into a stable

secon-dary and tertiary structure By contrast, if seconsecon-dary

and tertiary structure elements are less stable (yet

sta-ble enough to form a catalytically competent complex)

cleavage is favoured [25,26] We exploited this specific

feature of the hairpin ribozyme in a scheme of

site-directed and patchwise exchange of RNA sequences

[16,17] Combination of two hairpin ribozymes into

one molecule leads to twin ribozymes with two

pro-cessing sites at a suitable substrate RNA Because of

the specific hairpin ribozyme cleavage–ligation

charac-teristics described above a fragment of residing

sequence is removed in the first part of the reaction

followed by binding of another separately added RNA

fragment in the gap left behind and its ligation to the

final product [16]

In addition to the conventional hairpin ribozyme,

we studied hairpin ribozymes with the two domains

(loop A and loop B) joined in reverse order [15,27,28] (Fig 1) To further complement our work on the rational design of ribozymes for RNA sequence alter-ation we were interested in using reverse-joined hairpin ribozymes as building blocks for the construction of twin ribozymes In order to evaluate the structural properties of reverse-joined hairpin ribozymes for func-tional design, we first studied the cleavage and ligation activity of reverse-joined hairpin ribozyme variants Based on the results, the most suitable ribozyme was chosen for construction of a twin ribozyme The ability

of this twin ribozyme to mediate site-specific alteration

of RNA sequence is demonstrated

Results

RNA ligation by reverse-joined hairpin ribozymes Reverse-joined hairpin ribozymes, first introduced by Ohtsuka and colleagues [29,30], are derived from the conventional hairpin ribozyme by dissecting the two domains at the hinge between helix 2 and helix 3 and rejoining helix 4 to helix 1 via a linker of six unpaired residues [27] (Fig 1) Linkers consisting of cytidine or adenosine have been studied and it has been found that a linker of six unpaired residues is suitable for connecting the two domains [29,30] Further extending the linker length to 9, 12 or 18 residues increased the cleavage rate by only a factor of two [29] Therefore,

as well as to confine the conformational freedom of the ribozyme structure, we initially used an A6-linker for the design of reverse-joined hairpin ribozymes and stabilized helix 3 by a UUCG tetraloop cap [15,27]

Fig 1 (A) Schematic presentation of how reverse-joined hairpin ribozymes are derived from the conventional hairpin ribozyme Loop A and loop B domains of the conventional hairpin ribozyme are separated between helix 2 and helix 3 The loop B domain is turned through 180
and helix 4 is rejoined with helix 1 via a single-stranded linker to generate a reverse-joined hairpin ribozyme (B) Secondary structure of reverse-joined hairpin ribozymes with substrates for ligation experiments The circle indicates a 5¢-terminal fluorescein moiety used for detec-tion cp, 2¢,3¢ cyclic phosphate.

Under appropriate conditions, reverse-joined hairpin

ribozymes with an A6-linker designed in our

laborat-ory efficiently catalysed RNA cleavage [27] However,

ligation activity was rather poor; only about 1% of

cleavage products⁄ ligation substrates were ligated

(S A Ivanov & S Mu¨ller, unpublished results)

Pre-vious linker length variation experiments were carried

out to study the cleavage activity of reverse-joined

hairpin ribozymes without looking at ligation [29,30]

Therefore, to search for reverse-joined hairpin

ribo-zymes with improved ligation activity we reinvestigated

the influence of linker length using the constructs

shown in Fig 1B In order to favour ligation over

cleavage, helix 2 was extended to 16 bp (Fig 1B) This

design should allow for a higher ligation yield due to

more stable binding of the 5¢-ligation fragment [25,26]

Furthermore, we used two different short substrates,

SG9 and SU9, for ligation Whereas SU9 forms a 6 bp

duplex with the ribozyme, binding of SG9 generates

a duplex of only 5 bp due to the terminal G-A

mis-match Thus, the nonpaired adenosine residue at the

hinge point can be integrated into the linker leading to

further enhancement of the degrees of conformational

freedom We prepared reverse-joined hairpin

ribo-zymes with single-stranded linkers of 6, 7, 8 and 12

adenosine residues and compared their activity for

ligation of S17F5 cp with either SU9 or SG9 (Fig 1B,

Table 1) This experimental design allowed us to look

at eight ribozyme–substrate complexes varying in the

length of the single-stranded linker and⁄ or the length

of the duplex between the ribozyme and the 3¢-ligation

substrate Ligation reactions were carried out under

conditions involving equimolar concentrations of

ribo-zyme and ligation fragments, as well as under single

turnover conditions (Table 1) in the presence of 10 mm

MgCl2 and 2 mm spermine; the polyamine was

previ-ously found to be essential for efficient reverse-joined

hairpin ribozyme catalysis [27] Initially, we used a

reaction temperature of 32
C because this has been

shown to be optimal [29,30] However, we observed that ribozyme activity varied only slightly in measure-ments at 32 and 37
C The construct HP–RJWTA7 showed the fastest reaction kinetics and gave the high-est yields for ligation of the short fragment SG9 to S17F5 cp under equimolar concentrations, as well as under ribozyme saturation (Table 1) Thus, a linker length of eight adenosine residues in combination with

a duplex of 5 bp between the 3¢-ligation substrate and the ribozyme seems to be most favourable for

an efficient reaction among the studied species HP–RJWTA7 is also an efficient endonuclease; it cleaves the substrate S40F3F5 with kobs¼ 0.4 min)1at equimolar concentrations of ribozyme and substrate (according to conditions used for sequence exchange reaction, see below)

Design of a twin ribozyme and kinetic analysis

As a result of the studies described above, the opti-mized structure of the reverse-joined ribozyme unit for twin ribozyme design consists of two domains joined

by a single-stranded linker of eight adenosine residues and binds the 3¢-terminal part of its substrate via a duplex of 5 bp (Fig 2A) The second part of the twin ribozyme consists of a three-way junction hairpin ribo-zyme as used in the twin riboribo-zyme approach described recently [16]

In order to learn about the activity of both ribozyme units in the twin ribozyme, we determined the kinetic parameters for cleavage as well as ligation at the two sites in individual experiments (Fig 3) Substrate RNAs S40F5dA15 and S40F5dA31 were synthesized

to be cleaved at either of the specific sites Cleavage at the second site was abolished by replacing the attack-ing 2¢-OH group with a hydrogen atom Specifically, either A15 or A31 was substituted by deoxyadenosine Cleavage reactions were carried out under single turn-over conditions delivering the kinetic constants shown

Table 1 Ligation parameters of reverse-joined hairpin ribozymes with single stranded linkers of varying lengths.

Ligation yield (%)

B

Fig 2 (A) Reaction scheme for HP–TWRJ-mediated fragment exchange Substrate RNA S40F3F5 is annealed to HP–TWRJ (left) and cleaved

at two defined sites The fragment extending between the two cleavage sites (16-mer, shown in red) is replaced on the ribozyme by the oligonucleotide S20 cp (20-mer, shown in green) which subsequently becomes ligated to the flanking substrate fragments to form the HP–TWRJ–product complex (right) Green circles indicate 5¢- and 3¢-terminal fluorescein moieties used for detection Reaction was carried out in the absence and presence, respectively, of the oligonucleotide S6-anti, which is complementary to the six 5¢-terminal nucleotides of HP–TWRJ (for details refer to main text) (B) Model duplexes used for determination of melting points The sequence of the educt and prod-uct duplex corresponds to the sequence of HP–TWRJ with initial substrate S40F3F5 and with prodprod-uct P44F3F5, respectively.

Fig 3 Secondary structures of ribozyme substrate complexes used for measuring cleavage (A) or ligation (B) rates at either site Cleavage and ligation at the second site was abolished by replacing the essential adenosine in the substrate strand (A15 or A31) by deoxyadenosine Grey circles indicate 5¢- and 3¢-terminal fluorescein moieties used for detection.

in Table 2 A similar setup was used to determine

liga-tion parameters: fragments with dA instead of A

(S35F5dA15cp and S29dA31) were used for ligation

with the corresponding substrate SG9 or S15F5 cp,

respectively The results show that the twin

ribozyme-mediated cleavage, as well as ligation, proceeds with

similar activity at both individual sites; cleavage rates

are virtually identical, ligation rate constants vary by a

factor of only about 2.5

Alteration of RNA sequence by the twin ribozyme

HP–TWRJ

The twin ribozyme HP–TWRJ was designed to

pro-mote the insertion of four specific nucleotides into a

predetermined site of an arbitrarily defined substrate

RNA as illustrated in Fig 2A In the ribozyme–

substrate complex, a stretch of four nucleotides in the

central part of the ribozyme strand (GAUU) bulges

Cleavage at both predefined sites releases a 16-mer that

can easily dissociate from the ribozyme because of the

destabilizing bulge The added RNA fragment S20 cp

contains the four additional nucleotides

complement-ary to the GAUU loop in the ribozyme strand Hence,

binding of this oligonucleotide to the gap left by

disso-ciation of the 16-mer, converts the previously

inter-rupted duplex into a continuous one extended by 4 bp

For thermodynamic characterization of the system, we

determined the melting points of model duplexes

cor-responding to the central part of the twin ribozyme–

substrate and ribozyme–product complexes (educt and

product duplex, Fig 2B) There was a significant

dif-ference in the melting temperature of the two duplexes

(Table 3), supporting our strategy to drive the reaction

by a change in duplex stability On the basis of the

melting temperatures obtained using varying duplex

concentrations, thermodynamic parameters were

deter-mined The binding enthalpy for both duplexes varied

by 71.4 kcalÆmol)1 and fragment exchange was

associ-ated with a favourable DG of )15.2 kcalÆmol)1 (see

Experimental procedures and Table 3) This should

drive the reaction in the desired direction and favour product formation

The validity of the experimental design was checked

by analysing the time course of the reaction using a fluorescence assay as described previously [15] The substrate RNA S40F3F5 was incubated with an equi-molar amount of ribozyme at 37
C We used a reac-tion temperature of 37
C in order to compare the results with our previous twin ribozyme studies [16] The reaction was allowed to proceed for 30 min, after which S20 cp was added in equimolar quantities to ribozyme and initial substrate, and the reaction was left to proceed at 37
C for another 120 min We ini-tially chose this experimental design in order to be able

to observe the individual reaction steps However, as found later, the reaction occurs in a similar manner when initial substrate, ribozyme and S20 cp are present

in the reaction mixture from the beginning (S Vaule´on

& S Mu¨ller, unpublished observations) Data for the original setup are given in Fig 4 After 30 min, char-acteristic cleavage products were detected (Fig 4B, lane 2) Addition of the fragment S20 cp led to the for-mation of new products detected as three additional bands (lanes 3 and 4) These signals correspond to the 29- and 35-mer resulting from ligation of the 20-mer

to either the 9- or 15-mer cleavage product, and to the desired product RNA (P44F3F5) resulting from liga-tion of the 20-mer to both fragments as shown in Fig 4A Continuing the reaction for 15 min after the addition of S20 cp (total reaction time: 45 min) resul-ted in conversion of 5% of starting material into the 44-mer product P44F3F5, whereas 13% of the sub-strate S40F3F5 remained unprocessed (Fig 4B, lane 3, Fig 4C) After another 105 min of incubation, further enrichment of products involving ligation to the 20-mer fragment S20 cp was observed The ratio of product to initial substrate increased; 9% of substrate RNA was converted to the 44-mer and 5% was left unchanged (Fig 4B, lane 4, Fig 4C) These results demonstrate favourable cleavage of the substrate fol-lowed by dissociation of the 16-mer versus favourable ligation of the 20-mer

The yield of product RNA P44F3F5 could be fur-ther increased by stabilization of the ribozyme active

Table 3 Thermodynamic parameters of model duplexes.

Tm(
C) a

DH (kcalÆmol)1)

DS (calÆK)1Æmol)1)

DG37
C (kcalÆmol)1)

Product duplex 72.7 )149.8 )404.2 )24.4

a At 500 n M oligonucleotide concentration.

Table 2 Kinetic parameters of HP–TWRJ catalyzed cleavage and

ligation reactions.

Substrates

kreact, (min)1) K (n M )

kreact⁄ K, (min)1l M )1)

Cleavage S40F5dA15 0.57 ± 0.01 16.1 ± 2.4 35.4

S40F5dA31 0.32 ± 0.02 12.0 ± 4.6 26.6

Ligation SG9 +

S35F5dA15cp

0.55 ± 0.03 22.9 ± 8.5 24.0 S29dA31 +

S15F5 cp

1.38 ± 0.09 34.8 ± 8.1 39.6

structure Theoretical analysis of HP–TWRJ folding

(using software rna structure 4.0) showed that

HP–TWRJ, in addition to the desired minimal energy

structure (DG¼ –70.7 kcalÆmol)1), can fold into an

alternative nonfunctional structure with a virtually

identical Gibbs’ free energy (DG¼ –70.5 kcalÆmol)1)

A short antisense oligonucleotide 3¢-CCCTCT-5¢,

complementary to the six 5¢-terminal nucleotides of

HP–TWRJ (Fig 2A), assists proper folding; folding

analysis of this system revealed an energy difference

between both competing structures of 10 kcalÆmol)1 in

favour of the desired functional structure Carrying

out the sequence-exchange reaction described in the

presence of this antisense oligonucleotide increased the

yield of the final product to 17% (Fig 4C)

To validate our data we repeated the reaction, using

for sequence exchange a 20-mer fragment internally

labelled with a Cy5 moiety [S20 cp(Cy5)] (Fig 5) This

experimental setup allowed us to detect ligation

pro-ducts not only by fragment lengths analysis with an

ALF DNA sequencer (detection of fluorescein

emis-sion upon excitation at 488 nm), but also by virtue of

their unique fluorescence at 700 nm using a LI-COR

DNA sequencer (detection of Cy5 emission upon

exci-tation at 680 nm) As shown in Fig 5A, there is a

clear conversion of the fast-running Cy5-labelled

20-mer into three slower running species corresponding

to ligation products of the Cy5-labelled 20-mer with

fluorescein-labelled 9-mer [29-mer (F3, Cy5)] and

15-mer [35-mer (F5, Cy5)], respectively, and to final

44-mer RNA product labelled with Cy5 and two

fluo-rescein moieties [44-mer (F3, F5, Cy5)]

In both experiments (compare Figs 4B and 5A)

there is a strong 35-mer signal resulting from ligation

of the 15-mer produced by cleavage in the first step of

the reaction to the added 20-mer fragment S20 cp

This illustrates that ligation at the site of the

conven-tional hairpin ribozyme proceeds somewhat faster

(2.5-fold, Table 2) than ligation at the reverse-joined

ribozyme site

Discussion

The twin ribozyme HP–TWRJ was designed to mediate the specific exchange of two RNA fragments (Fig 2)

A

B

C

Fig 4 (A) Fragmentation and ligation scheme (compare with

Fig 2) cp, 2¢,3¢-cyclic phosphate (B) Monitoring the reaction Lane

1, start of cleavage reaction Lane 2, mixture after 30 min at 37
C

(immediately before adding S20 cp) Lane 3, mixture 15 min after

addition of S20 cp (incubation at 37
C continued, total reaction

time: 45 min) Lane 4, mixture after additional 105 min at 37
C

(areas of peaks corresponding to S40F3F5 and P44F3F5 indicate

9% conversion to full length product and 5% remaining substrate).

For additional details see main text Peak heights are standardized

by the data processing software, such that total peak integrals

of different lanes are not constant (C) Time course of fragment

exchange reaction in the absence (solid lines) and presence

(dashed lines) of the antisense oligonucleotide S6-anti.

During the process, 16 nucleotides of residing substrate

sequence are exchanged for 20 nucleotides, which are

added to the reaction mixture as a separate synthetic

RNA fragment Recently, we communicated the

devel-opment of a twin ribozyme consisting of two hairpin

ribozymes connected in tandem that can catalyse the

same fragment-exchange reaction [16] The twin

ribo-zyme described here was more challenging, because it

involves a reverse-joined hairpin ribozyme unit and

required more extensive design and evaluation

Reverse-joined hairpin ribozymes were introduced

nearly 10 years ago [29,30] Since then they have

attrac-ted little attention: there has been no follow-up

demon-strating the catalytic potential of these interesting

ribozyme structures beyond the work of the initial

developers and our laboratory We studied the ligation

activity of reverse-joined hairpin ribozyme variants

Interestingly, reverse-joined hairpin ribozymes act as

highly efficient ligases The most active variant

HP–RJWTA7 ligated two substrates with 43% yield

when all reactants (ligation substrates and ribozyme)

were incubated at equimolar concentrations; under

single turnover conditions the yield increased to 70%

(Table 1) A conventional hairpin ribozyme variant corresponding to the 3¢-terminal region of the twin ribozyme HP–TWRJ delivered only 29% of ligated product (compared with 43% for HP–RJWTA7, data not shown) No high-resolution structure is available for reverse-joined hairpin ribozymes However, the observed functionality implies that the active confor-mation of reverse-joined hairpin ribozymes involves

a similar relative orientation of the two ribozyme domains to that seen in the crystal structure of the con-ventional hairpin ribozyme [21,31] Variation in linker length and⁄ or the length of the duplexes flanking the single-stranded linker will, therefore, influence the posi-tions of the two loops in the folded structure Our results indicate that a single-stranded linker of eight adenosine residues, as in HP–RJWTA7, and a 5 bp duplex between the 3¢-ligation substrate and the ribo-zyme allows proper folding of the complex into the act-ive conformation with the required contacts between loops A and B Thus, to the best of our knowledge, this

is the first example of a reverse-joined hairpin ribozyme that, under appropriate conditions, can ligate two suit-able substrates with up to 70% yield

Fig 5 (A) Monitoring the HP–TWRJ mediated fragment exchange involving a Cy5-labelled oligonucleotide S20 cp(Cy5) Lane 1, S20 cp(Cy5) control Lanes 2 and 3, incubation of S20 cp(Cy5) with HP–TWRJ in the absence of initial substrate under conditions of fragment exchange reaction after 5 and 80 min Lane 4, incubation of S20 cp(Cy5) with initial substrate S40F3F5 in the absence of HP–TWRJ under conditions

of fragment-exchange reaction Lane 5, mixture after 30 min cleavage reaction at 37
C (immediately before adding S20 cp(Cy5)) Lanes 6, 7 and 8, mixture 15, 120 and 180 min, respectively, after addition of S20 cp(Cy5) (incubation at 37
C continued) Lane 9, Cy5 labelled 29-mer

as length control Double bands result from an isomeric mixture (cis- ⁄ trans-isomers) of the Cy5 moiety used for labelling (B) Schematic presentation of HP–TWRJ mediated fragment exchange (compare with Fig 2) Green circles indicate 5¢- and 3¢-terminal fluorescein moieties; the red circle indicates a Cy5 moiety used for detection.

The reverse-joined hairpin ribozyme HP–RJWTA7

was combined with a three-way junction hairpin

ribo-zyme to generate the twin riboribo-zyme HP–TWRJ The

newly designed structure mediates the site-specific

exchange of two patches of RNA sequence with up to

17% yield, which is a remarkable improvement

com-pared with earlier versions of this twin ribozyme

[15,32] The yield can possibly be increased further by

the additional destabilization of ribozyme–substrate

complexes in the region containing the sequence to be

exchanged (Fig 2B, red) Although a bulge of four

nucleotides has been introduced to weaken binding of

this sequence, there is still a duplex of eight contiguous

base pairs hampering dissociation of the fragment to be

exchanged after cleavage Further reduction of the

length of this duplex would facilitate dissociation

However, this implies that the two ribozyme units of

the twin ribozyme are located closer together and this

may interfere with proper folding of the twin ribozyme

due to a sterical clash between both catalytic units In

our previous studies, we observed that the sequence

and length of the helix between the two loops

contain-ing the cleavage⁄ ligation site, as well the size of the

bulge, influence the exchange efficiency [16,32,33]

Therefore, the specific design of a custom-designed twin

ribozyme will strongly depend on the substrate to be

processed, such that variation of the distance between

the two sites seems reasonable only at this stage

In summary, the demonstration of functionality of

HP–TWRJ provides proof of the principle that, in

addition to the conventional hairpin ribozyme,

reverse-joined hairpin ribozymes can also be used as building

blocks for the construction of twin ribozymes Even

though HPTWRJ is not as efficient as its

tandem-configured relative [16], its successful construction and

the demonstration of its functionality supports the idea

of creating novel RNA catalysts by rational design

Furthermore, reverse-joined hairpin ribozymes can act

on RNA substrates that are not readily accepted by

conventional hairpin ribozymes For example, a

hair-pin ribozyme variant with the terminal base pair of the

ribozyme–substrate duplex at the hinge changed from

3 0 -CU-5 0

5 0 -GA-3 0 to 3

0 -GA-5 0

5 0 -CU-3 0 displayed 10-fold lower cleavage

activity compared with the wild-type ribozyme [15] By

contrast, corresponding variants of the reverse-joined

hairpin ribozyme showed no significant difference in

cleavage behaviour (C Schmidt & S Mu¨ller,

unpub-lished results) We attributed this result to enhanced

coaxial stacking of the two domains in the relevant

variant of the hinged conventional hairpin ribozyme

leading to enrichment of an extended inactive

confor-mation [34,35] Because of the single-stranded linker,

joining the two domains in reverse-joined hairpin ribo-zymes, coaxial stacking is less probable and therefore not sensitive to the sequence at the hinge point

A useful application for twin ribozymes is

site-speci-fic labelling or functionalization of transcripts in vitro and possibly in vivo as we have recently demonstrated with tandem configured twin ribozymes [18] The twin ribozyme accepts RNA fragments that are conjugated with a dye (here Cy5) A number of other dyes and modifications are incorporated equally well even in rather long and structured RNA molecules [18] Thus, the twin-ribozyme strategy paves the way for

site-speci-fic labelling⁄ modification of RNA molecules that are too long for chemical synthesis Furthermore, apart from fragment-exchange reactions, simple clipping of a fragment of desired length and sequence from a nat-ural RNA may be a useful application For example, RNA fragments that involve modified nucleobases are easily obtainable from naturally occurring RNA by the use of twin ribozymes Subsequently, these fragments can be investigated using various analytical methods Another potential application is genotyping of single nucleotide polymorphisms in human genomes [36] Subsequent analysis of appropriate RNA fragments by

MS can reveal if a certain nucleotide in the target gene has been altered [37,38] Thus, the development and application of twin ribozymes may lead to a number

of interesting strategies in molecular biology, genome analysis and possibly molecular medicine It is cer-tainly advantageous having a number of twin-ribozyme variants that can be adapted to and optimized for a specific target It has been shown previously that the sequence of the substrate-binding domain of the hairpin ribozyme can be adapted to cleave a desired RNA substrate, just a few conserved nucleobases are required [39] The sequence requirements for conven-tional and reverse-joined hairpin ribozyme substrates are virtually the same [29,30] However, the distinct mode of joining the two ribozyme domains in both variants leads to distinct acceptance of specific sequences particularly in the region of the domain hinge The new designed twin ribozyme HP–TWRJ is thus a valuable addition to the existing variants of a tandem configured hairpin ribozyme

Experimental procedures

Synthesis of ribozymes and substrates Reverse-joined hairpin ribozymes HP–RJWTAn (n¼ 6, 7,

8, 12) and the twin ribozyme HP–TWRJ were transcribed

in vitro from oligonucleotide templates using T7 RNA

polymerase essentially as described previously [15] Briefly,

double-stranded DNA templates were generated from two

synthetic primers (BioTez, Berlin, Germany) overlapping by

15 complementary bases (for generation of templates for

transcription of HP–RJWTAn) or 32 complementary bases

(for generation of the template for transcription of

HP–TWRJ) After primer annealing, DNA templates were

completed with DNA polymerase I, Klenow fragment exo–

(Fermentas, St Leon-Rot, Germany) Transcription was

carried out with T7 RNA polymerase at 37
C in standard

transcription buffer (40 mm Tris⁄ HCl pH 7.9, 6 mm MgCl2,

10 mm dithiothritol, 10 mm NaCl, 2 mm spermidine) over

3 h (HP–RJWTAn), or in Hepes buffer (20 mm Hepes

pH 8.0, 10 mm Mg(OAc)2, 10 mm NaOAc, 1 mm

dithio-threitol, 25 lgÆmL)1bovine serum albumin) for 4.5 h (HP–

TWRJ) Proteins were removed by phenol⁄ chloroform

extraction and RNA was precipitated from ethanol

Ribo-zymes were purified by denaturing gel electrophoresis (7 m

urea, acrylamide⁄ bis-acrylamide 19 : 1) on a 10%

poly-acrylamide gel (HP–TWRJ) or a 20% polypoly-acrylamide gel

(HP–RJWTAn) Product-containing bands were eluted with

2 m LiClO4overnight at room temperature and precipitated

from acetone

Substrate oligoribonucleotides SU9, SG9 and S40F3F5

were synthesized using the phosphoramidite method on a

1 lmole scale using an automated DNA⁄ RNA synthesizer

(Gene Assembler Special, Pharmacia Biotech, Freiburg,

Germany) 5¢-Fluorescein labelling was achieved by

solid-phase coupling of ‘fluoreprime’ phosphoramidite

(Amer-sham Biosciences, Freiburg, Germany) For labelling at the

3¢-end, controlled pore glass was used to which fluorescein

was attached via a thiourea functionality as a succinate

linkage (ChemGenes Corp., Wilmington, USA) It

con-tained a dimethoxytrityl-protected hydroxyl group which

after deprotection was used for chain elongation All RNA

substrates were purified by electrophoresis on 15–20%

denaturing polyacrylamide gels, eluted with 2 m LiClO4

and precipitated from acetone

For internal Cy5 labelling of the 20-mer fragment

S20 cp(Cy5), a deoxythymidine carrying an amino function

(amino modifier C6dT phosphoramidite, ChemGenes

Corp.) was built into a 29-mer oligonucleotide during

chemical synthesis (5¢-GUCCAGAAA-NH2

C6dT-CUCC-CUCACAGUCCUCUUU-3¢) After standard deprotection

and gel purification the amino function was coupled with

Cy5-NHS ester (Amersham Biosciences) To this end,

30 nmol of the oligonucleotide were solved in 500 lL

car-bonate buffer (0.1 m pH 8.5) and mixed with 1 mg dye

The reaction was allowed to proceed for 1 h at room

tem-perature in the dark with occasional shaking The reaction

was stopped by ethanol precipitation The side product

N-hydroxysuccinimid was removed by washing the resolved

precipitate over a NAP column; the labelled oligonucleotide

was separated from nonlabelled species by gel

electrophor-esis and then cleaved with a conventional hairpin ribozyme

HP–WTTL [27] to yield the 20-mer fragment S20 cp(Cy5) with 2¢,3¢-cyclic phosphate In the same way RNA frag-ments S17F5 cp, and S20 cp containing a 2¢,3¢-cyclic phos-phate group (cp) were obtained from cleavage of appropriate chemically synthesized RNA molecules with HP–WTTL [27] Extinction coefficients of ribozymes and substrates were calculated with oligoanalyzer 3.0 (http:// www.idtdna.com) taking into account the absorption of fluorophores

Ligation experiments Individual ribozymes were mixed with the respective sub-strate, SU9 or SG9 in Tris⁄ HCl (pH 7.5) buffer, heated

at 90
C for 1 min followed by incubation at 32
C for

15 min The second ligation substrate S17F5 cp was added and the mixture was incubated at 32
C for another

10 min Reactions were started by the addition of MgCl2

and spermine The final volume of the reaction mixture was

10 lL, final concentrations were: 200 nm ribozyme, 20 or

200 nm SU9 and SG9, 200 nm S17F5 cp, 10 mm MgCl2,

2 mm spermine, 15 mm Tris⁄ HCl, pH 7.5 Ligation was allowed to proceed at 32
C for 30 min Aliquots (2.5 lL) were removed at suitable intervals and reactions were quenched by addition to 3 lL stop-mix (10 mm EDTA in 90% formamide) followed by intensive vortexing, heating

at 90
C for 1 min, and immediate cooling on ice Each experiment was repeated at least once Samples were ana-lysed by PAGE on 8% denaturing gels (7 m urea) using an ALF DNA sequencer (Pharmacia Biotech) as described pre-viously [15] Data were processed and analysed using dna fragment analyzer1.2 Software (Pharmacia Biotech) Kinetic constants of ligation reactions were determined under single turnover conditions Reactions were carried out essentially as described above However, owing to very fast ligation the required volumes of MgCl2 stock solution

as well as of spermine stock solution were placed on the inner wall of the Eppendorf tube and reaction was started

by intensive vortexing The final volume of the reaction mixture was 15 lL; final concentrations were: 80–500 nm ribozyme, 20 nm SU9 or SG9, respectively, 88–550 nm S17F5 cp, 10 mm MgCl2, 2 mm spermine, 15 mm Tris⁄ HCl,

pH 7.5 Reaction analysis was as described above Appar-ent first-order rate constants (kobs) were obtained from a plot of product formation against time in the linear phase

of reaction Rate and dissociation constants (klig and KD) were calculated from linear curve fitting using plots of kobs

against kobs⁄ [E], where [E]¼ ribozyme concentration (Eaddie–Hofstee plot) The margin of error was < 10% in all measurements

Determination of kinetic parameters Kinetic parameters of twin ribozyme cleavage reactions using individual substrates S40F5dA15 and S40F5dA31

were determined under single turnover conditions Substrate

was mixed with ribozyme in Tris⁄ HCl (pH 7.5) buffer,

hea-ted at 90
C for 1 min and incubated at 37
C for 15 min

Reactions were started by addition of MgCl2and spermine,

portions of which were placed before on the Eppendorf

tube wall as described in ligation experiments The final

volume of the reaction mixture was 15 lL; final

concentra-tions were: 40–200 nm ribozyme, 20 nm substrate, 10 mm

MgCl2, 2 mm spermine, 15 mm Tris⁄ HCl, pH 7.5

Kinetic parameters of twin ribozyme ligation reactions

were determined under single turnover conditions, using the

following protocol Ribozyme was mixed with the respective

substrate, SU9 or SG9 in Tris⁄ HCl (pH 7.5) buffer, heated

at 90
C for 1 min followed by incubation at 37
C for

15 min The second ligation substrate S17F5 cp was added

and the mixture was incubated at 37
C for another 10 min

Reactions were started by addition of MgCl2and spermine

The final volume of the reaction mixtures was 20 lL; final

concentrations were: 40–200 nm ribozyme, 20 nm short

substrate, 44–220 nm long substrate, 10 mm MgCl2, 2 mm

spermine, 15 mm Tris⁄ HCl Samples were processed as

des-cribed above; parameters of cleavage as well as of ligation

were determined from Eaddie–Hofstee plots

Sequence exchange reaction

A mixture of ribozyme and substrate in Tris⁄ HCl (pH 7.5)

buffer was heated at 90
C for 1 min followed by

incuba-tion at 37
C for 15 min The cleavage reaction was started

by addition of MgCl2 and spermine The final volume of

the reaction mixtures was 20 lL; final concentrations were:

220 nm ribozyme, 220 nm substrate, 11 mm MgCl2, 2.2 mm

spermine, 16.5 mm Tris⁄ HCl, pH 7.5

After 30 min, an aliquot (2 lL) was removed from the

reaction mixture and substituted by an equal volume

con-taining the 20-mer fragment S20 cp (2 lL), such that

ori-ginal RNA fragments (ribozyme, initial substrate and

added 20-mer) had a final concentration of 200 nm

Aliqu-ots (2.5 lL) were taken at suitable time intervals and added

to 3 lL of stop-mix on ice Samples were analysed using an

ALF DNA sequencer, and data were processed using alf

fragment manager as described previously [15] When

the reaction was carried out with the Cy5-labelled 20-mer

S20 cp(Cy5), samples were also analysed using a DNA

Sequencer 4200 (LI-COR Biosciences, Bad Homburg,

Ger-many) and the data were processed with gene imagir 4.05

(LI-COR)

Melting temperatures and thermodynamic

parameters

To generate duplexes individual amounts of oligonucleotides

(for sequence see Fig 2B) were mixed in reaction buffer

con-taining 10 mm Tris⁄ HCl, pH 7.5 and 10 mm MgCl2, heated

at 90
C for 5 min followed by slow cooling to 20
C

Individual samples with duplex concentrations of 500 nm,

750 nm, 1 lm, 2.5 lm and 5 lm have been prepared Melting profiles were measured at 260 nm in 1 mL quartz cells using a UV visible spectrometer CARY 1E (VARIAN INC., Palo Alto, USA) equipped with a temperature control device Thermostat temperature was varied from 20 to

90
C with a heating rate of 0.2
CÆmin)1 To avoid solvent evaporation 100 lL mineral oil was placed on the sample surface Melting points Tm(
C) were defined as the maxima

of the first derivation Individual values for DH and DS were obtained by plotting R ln[C] against 1⁄ Tm (K)1) The free enthalpy of the exchange of the short fragment (Fig 2, red) for the longer fragment (Fig 2, green) on the ribozyme at

298 K (DG) was defined as DG(298 K)¼ DGproduct duplex(298 K) – DGeduct duplex(298 K)

Acknowledgements

Financial support by DFG and Ju¨rgen Manchot Foundation as well as a PhD studentship to SV by the Ju¨rgen Manchot Foundation is gratefully acknow-ledged

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