Catalytic antisense RNAs produced by incorporating ribozyme cassettes into cDNA

Gene, 108 (1991) 175-183 0

1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved.

175

0378-l 119/91/$03.50

06145

Catalytic antisense RNAs produced by incorporating ribozyme cassettes into cDNA (Anti-viral

hammerhead

agent; gene suppression;

RNA;

plum pox virus;

self-cleavage)

Martin Tabler and Mina Tsagris Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, GR-71110 HeraklionlCrete (Greece) Received by M. Bagdasarian: 17 May 1991 Revised/Accepted: 18 June/30 August 1991 Received at publishers: 9 September 1991

SUMMARY

A simple strategy is described

for the generation

of catalytic

hammerhead-type

ribozymes

(Rz) that can be used as highly

specific endoribonucleases to cleave a particular target RNA. The technique requires that a cloned cDNA fragment is available which encodes at least a part of the target RNA. About 25 different restriction recognition sequences can be utilized to incorporate specifically designed DNA cassettes into the cDNA. Besides some nucleotides which are specific for a certain restriction site, the DNA cassettes contain a sequence corresponding to the catalytic domain of the hammerhead Rz and, optionally, selectable marker genes, that are removable. The resulting recombinant DNA constructs permit the in vitro and in vivo synthesis of novel ‘catalytic antisense RNAs’ or ‘antisense Rz (AZ)‘, which combine two features: (i) they bind like antisense RNA to their specific substrate RNA, and (ii) they cleave their target as hammerhead Rz do. The utility of the strategy to generate Rz was demonstrated experimentally by incorporating a synthetic SalI-specific DNA ribozyme (Rz) cassette into a unique Sal1 site of a cloned cDNA fragment of plum pox virus (PPV), which is a single-stranded positive sense plant RNA virus, belonging to the group of potyviruses. The resulting AZ constructs delivered AZ that were directed against the PPV ( + ) or (-) RNA, respectively, which cleaved their corresponding target RNAs in the expected manner. Besides the synthetic Rz cassette, a comparable S&-specific Rz cassette, that had been prepared from a specifically designed plasmid and that contained the tet gene inserted into the sequence of the catalytic domain of the Rz, was also incorporated into the S&I site of the PPV cDNA. The catalytic activity of the resulting AZ was maintained, though the much larger sequence of the marker gene was inserted into the catalytic domain.

INTRODUCTION

The occurrence of self-cleaving RNAs and the characterization of their reaction mechanism have facilitated the

Correspondenceto: Dr. M. Tabler,

Institute

of Molecular

Biology

Biotechnology, P.O. Box 1527, GR-7110 Tel. (30)81-238424; Fax (30)81-230469.

Heraklion/Crete

Abbreviations:

AZ, DNA (gene) encoding

AZ, antisense

bp, base pair(s);

oligo, oligodeoxyribonucleotide; DNA polymerase (gene) encoding

ribozyme(s);

IVS, intervening

sequence

(intron);

Rz, ribozyme(s);

Rz; tet,tetracycline-resistance-encoding

AZ;

nt, nucleotide(s);

PolIk, Klenow (large) fragment

I; PPV, plum pox virus;

and

(Greece)

of E. coli Rz, DNA

gene; u, unit(s).

generation of RNA enzymes (ribozymes, Rz) which can catalyze different enzymatic reactions (reviewed by Cech, 1987). One class of self-cleaving RNAs is characterized by the presence of the ‘hammerhead’ structure (Forster and Symons, 1987a,b). These RNAs undergo Mg2 + -catalyzed autolytic self-cleavage, into two products, which carry a 5’-hydroxyl and a 2’,3’-cyclic phosphodiester, respectively, at their cleaved termini (reviewed by Bruening et al., 1988; Symons, 1989; 1990). Such self-cleaving RNAs are found amongst satellite RNAs of plant viruses, one viroid and an RNA transcript of the satellite DNA of newt (reviewed by Bruening, 1989). Although the naturally occurring hammerhead RNAs cleave in cis, the reaction proceeds also in tram

176 (Uhlenbeck, 1987; Ruffner et al., 1989). Moreover, Haseloff and Gerlach (1988) have described how the catalytic domain of hammerhead-type RNAs can be used to design Rz that form with a completely unrelated substrate

A

@=a 4 5’NNNNNNNNNNN~CGACNNNNNNNNN 000Il0000 0 0 0 0 0 00000

RNA a hammerhead structure in truns, so that the target RNA is specifically cleaved at a predicted site. According to the general rules described by Haseloff and Gerlach (1988), the only requirements for a substrate RNA to be cleaved by a hammerhead-type Rz is the presence of the

Substrate

RNA

Ri RNA

c2+?=

trinucleotide sequence, hereafter called ‘cleavable motif, for which self-cleavage has been described, e.g., GUC. It was shown that Rz designated in that way can function as highly

GEC A

VG2

GU,

e

cDNA

C3

target

!j’NNNN

GTiTCGTCCTCACGGACTCATCAd

GAG

NNNN

CA

CTG

N’N’N’N’

N’N’N’N’

AAGCAGGAGTGCCTGAGTAGTC

catalytic

ribozyme

3’ 5’

domain

Rz (ribozymeRNA) e

h’z (ribozymeDNA)

transcription

strate

(Rz) directed

sequence.

RNA

(G/UCGAC)

against

the GUC

(A) Hammerhead

(target

RNA)

containing

and its corresponding

RNA of tobacco

ringspot

et al., 1986; Haseloff substrate

a Sal1

derived

virus (sTobRV)

which are assumed domain

nt as marked (AGG/CCU)

(Buzayan

from

a sub-

sequence domain’

the satellite

et al., 1986; Prody

1989). The sequence

created.

was modified

(modified

GUC

directions

nt marked

of the

indicated

pair, corresponding has to be replaced

by superscript nt marked

are synthesized

by arrows.

reaction

the sequence sequence

are

of the

ways by replacing

a recognition

some

for

StuI

2) or an additional

one

by superscripts

of the double-stranded

and Rz RNA

For conversion

in the cleavage

in Figs. 2-5,

in two different Thus,

(modified

(B) Sequence target

to participate

described

by arrows.

for XhoI (C/UCGAG)

parts.

recognition

Rz RNA with the ‘catalytic

and Gerlach,

For experiments

catalytic

which

a Sal1

between

RNA is the ‘cleavable motif and the site of cleavage is indicated.

Residues boxed.

motif within

conformation

5’-CUGAUGAGUCCGUGAGGACGAA

(a) The utility of the Sal1 recognition sequence for inserting a DNA ribozyme cassette So far, Rz were produced from a corresponding oligo that had to be newly synthesized and that always contained, beside the actual catalytic domain, sequences which cause the specific binding of the Rz to its target RNA. An alternative way to obtain Rz more conveniently and in a more generalized manner is to incorporate precisely just the catalytic domain of the hammerhead RNAs into the DNA that encodes the target RNA (cDNA). In that case, the ‘flanking parts’ of the Rz RNA are derived from the cDNA and have not to be synthesized. Several restriction sites that overlap with certain cleavable motifs can be utilized for this purpose. For example, the sequence GUC, that forms a cleavable motif, is also part of the Sal1 restriction sequence (G/TCGAC). An RNA containing this hexamer sequence can be targeted by an Rz in the manner shown in Fig. 1A. In the resulting hammerhead complex, the sequence GUC of the Sal1 recognition sequence becomes a functional catalytic part since it represents the cleavable motif, whereas the succeeding sequence GAC is only part of the complementary regions which cause binding of substrate and Rz RNA. It is apparent from Fig. 1B that the two double-

RNA

3’

Fig. 1. Ribozyme

AND DISCUSSION

_

transcription

recognition RESULTS

catalytic domain

GM’

sequence-specific endoribonucleases in vitro and in vivo (Cotten and Birnstiel, 1989; Cameron and Jennings, 1989; Sarver et al., 1990; Saxena and Ackerman, 1990). In order to simplify the generation of the hammerhead-type Rz and to avoid the individual design of an Rz according to each particular cleavable motif and its corresponding unique sequence context in a certain target RNA, a strategy was developed that allows to insert different, but universally applicable DNA cassettes into cDNA. The insertion technique has also the advantage that in the resulting AZ RNA, the regions flanking the actual catalytic domain are quite long. The length of these regions is quite important, since these parts of the Rz are responsible for sequence-specific and efficient binding of the catalytic RNA to its target.

3’

DNA

2 and 3) was

templates

upon transcription

from in the

The DNAs differ only in the two boxed

of a cDNA

into an AZ construct,

the boxed C/G

to the third position within the GUC cleavable by the boxed 22 bp representing

the catalytic

motif,

domain.

stranded DNA templates, from which the two RNAs in Fig. 1A are derived, are identical outside the Sal1 recognition sequence. However, the DNA which encodes the AZ accommodates, in lieu of the C/G pair of the Sal1 recognition sequence within the substrate RNA, an insert representing the catalytic domain of the hammerhead Rz. Transcription of the two constructs - from appropriate promoters and in different directions - generates substrate and Rz RNA.

177 5’ NNNN

cDNA

ha,,

GTCGAC

NNNN

m,an,,

I III

3’ N’N’N’N’ CAGCTG

3’

N’NN’N’ 5’

sense strand

(b) Generation of an AZ RNA by inserting a synthetic SalI-

antisense strand

specific Rz cassette To examine the applicability of the strategy outlined in Fig. 2 experimentally, we introduced an Rz cassette into the

4

Sal I digestion

TCGAC ;

~‘NNNN G ,118 8 3’ N’N’N’N’ CAGCT

Trimming

NNNN 3’ I;,;r;l;.5.

is ~‘NNNN III, 3’ N’N’N’N’

G I C

C

NNNN3’

;;

;.;u;,;,

~‘NNNN

G[TITTCGGCCTCAAGGCCTCATCAGIGA~CNNNN

I I I , 1111,10111111,,1111111,1

II

3’ N’N’N’N’ C ~A~~~~,(@ff$~~$~~C (=I

Fig. 2. Strategy

the Sal1 site are removed the protruding ing the catalytic tional terminal

by Sal1 digestion

domain (hatched

removed

ing on the orientation

of

and subsequent

trimming

of

Rz cassette

is inserted

nt with superscript

compris-

2), plus addi-

Sal1 ends by trimming.

an RNA can be transcribed

DNA, which is able to cleave the sense strand

RNA (sense-directed

the GUC

nt TCGA

three of the four substrate-specific

from the protruding of insertion,

against

The four internal

part), which here is modified to contain

Fig. lA, modified

nt, which replace

(Rz)

(Rzl Q

of an Rz directed

sequence.

ends. Then, a MI-specific

a StuI site, (compare

ribozyme

ribozyme

for the generation

motif within a S&I recognition

3’

IIIII

T] G N’N’N’N’5’

sense-directed

antisense-directed

recombinant

sized, which can anneal to each other to form a SalIspecific Rz cassette. Compared to the previously described Rz (Haseloff and Gerlach, 1988; Cotten and Birnstiel,

5’

4

Liga tion

previously

Sal1 site located in a cDNA fragment of a plant RNA virus, the PPV, which is a member of the potyviruses. For this purpose, the two oligos Rz-Sal1 and Rz-Sal2 were synthe-

AZ) or its antisense

strand

nt,

Dependfrom the

of the target

(antisense-directed

AZ).

Fig. 2 summarizes the strategy for converting a cDNA, which encodes a target RNA (as described in Fig. l), into an AZ construct by manipulating the Sal1 site. After digesting the cDNA with Sal1 and successive trimming of the protruding ends, an Rz cassette is inserted by blunt-end ligation. The Rz cassette contains the sequence of the catalytic domain plus 3 of the 4 nt that had been removed from the protruding Sal1 ends. These manipulations ensure that the C/G pair of the Sal1 site is precisely replaced by the catalytic domain of the hammerhead Rz. In the presence of an appropriate promoter, the resulting DNA constructs allow the synthesis of RNA transcripts containing the catalytic Rz domain that will cleave their corresponding unmanipulated complementary RNA transcripts. Depending on the orientation in which the Rz cassette is inserted in the cleaved and trimmed Sal1 site, the resulting Rz will be directed to cleave RNA of sense or of antisense polarity. Usually only the sense-directed Rz is desired, but in the case of RNA viruses, that replicate via the RNA-RNA route, both types of Rz are desirable, especially in case of (-) RNA viruses where Rz can be directed against the viral genomic (-) RNA and the viral ( + ) mRNA.

1989; Cameron and Jennings, 1989; Sarver et al., 1990; Saxena and Ackerman, 1990), the catalytic sequence within the DNA cassette was modified at 2 nt to include a StuI (AGG/CCT) site (see also Figs. 1A and 2), and a compensatory third nt change was made to conserve the RNA base-pairing structure of the catalytic domain. The presence of the StuI site, which does not cut in most of the cloning vectors used, simplifies screening for recombinant plasmids containing the desired cassette. Moreover, the StuI site can be used to remove redundant cassettes when two (or more) cassettes have been inserted accidentally. The removal is, however, only useful when the two cassettes are head-to-tail connected. The synthetic Rz cassette composed of Rz-Sal1 and Rz-Sal2 was inserted into the Sal1 sites of pPV1 and pPV2 (Fig. 3A) which contain the PPV cDNA fragment. The resulting recombinant constructs encoded two AZ that were directed against the viral ( + ) and (-) RNAs (Fig. 3). Despite the sequence modification within the catalytic domain, the catalytic RNA transcripts cleaved their target RNAs in the expected manner (Fig. 3B). Therefore, the synthetic SalI-specific Rz cassette described here is universally applicable to create AZ starting from any cDNA fragment which contains a Sal1 restriction site. (c) Generation of a selectable Rz cassette In order to prepare a S&I-specific Rz cassette which could carry a selectable marker gene, the oligo AZ-Sal1 was synthesized and cloned, generating the plasmid pAzSal1 (Fig. 4). The oligo was designed to contain two sites for EarI, a class-IIS restriction enzyme which cleaves one and 4 nt away from its actual asymmetric recognition sequence (CTCTTCN,/N,; see review of Szybalski et al., 1991). After Ear1 digestion and filling-in the protruding ends, the cleaved out fragment corresponds to a S&-specific Rz cassette (Fig. 4). As compared to the synthetic cassette that we used before, this cloned cassette had a further nt modification (U --t C) in the loop region of the Rz core sequence and, therefore, contained in addition to the StuI site, an X/z01 recognition sequence (C/TCGAG) (compare Fig. 1A).

178

3631

6

pPV1

GTCGAC

T7

&sii;

13

0”

: PPV (+I RNA : PPV t-b RNA

60°C

SafI S’aJI

Eco RI

EcoRI

pPV2

GTCGAC i;bibib

: PPV (-1 RNA : PPV (+I RNA

T7 T3

3631

-

,-,!!sJ:-

pPV11

: (+I directed

T3

PPV 3409

EcoRI

pPV12

3831

G/WC ~=IAGI .

Eco RI

Eco RI

pPV21

Eco RI

Fig. 3. Antisense

3409

qyqm

Rz (AZ) directed

against

PPV

PPV

RNA of PPV. (Map A) From

unpublished), an EcoRI fragment, containing a unique Sal1 site was isolated. (Techeney et al., 1989), was inserted into pT3T7-Sal [a modification ofpT3T7 trimming

and religation]

mung-bean

nuclease

cassette

in Fig. 2 (hatched

were screened

of the DNA cassette

T3 and T7 RNA transcripts transcripts polymerase serum

DNA synthesizer,

for the presence

DNA cassette

were identified. (BRL)/40

Mannheim,

and Rz-Sal2: The DNA cassette

u of human

RNase

inhibitor

(Promega,

plasmids

the reaction

of phenol.

Tris

HCljl

was stopped

by addition

mM EDTA pH 8.0 to separate

30 fmol) of the RNA transcripts incubated,

from pPV2 and pPVl2,

revealed

whether

and re-ligation.

through

one, two

The orientation

respectively,

and the

and T3 and T7 RNA

mM dithiothreitol/lOO

ATP, CTP, GTP,

(RNase-free,

RNA transcripts Boehringer)

a 2 ml gel-filtration

with T7 RNA pg per ml bovine

and 20/tM

UTP plus IOpCi

were prepared,

no [a-32P]UTP

were added and after 2 min at 37°C.

column

ng of RNA transcript

as substrate

DNA

Recombinant

pPV11, pPV12, pPV21 and pPV22 (map A middle and

WI)/5OOpM

About 50-100

which served

plasmids.

were cut with PvuII and HindIII,

When unlabeled

6 u of DNaseI

was passed

EcoRI fragment

by StuI digestion

products

of 1 pg of tRNA and Na . acetate

were separated

on a denaturing

by autoradiography.

In lane 6, unlabeled

from %/I-linearized

plasmid

corresponds

to the 5’-terminal

two smaller

RNAs.

5 y0 polyacrylamide

(Biogel A0.5, BioRad)

was synthesized.

(S) and antisense-ribozyme

product

comprises

obtained

of 200 mM, the RNAs were collected

gel (0.125 y0 bisacrylamide),

AZ RNA was used. Lane 7 contains

pPV2. This transcript cleavage

to a final concentration

the PPV (-)

by AZ cleavage.

a truncated

containing

marker

RNA between

with

10 pMol of which

with Hind111 and used for in vitro transcription

Madison,

[a-32P]UTP.

with SalI, treated

et al., 1989). About

in 10 mM

About 4 ng (approx.

RNA (AZ), respectively

alone at 0°C or 60°C (lanes 1,3 and 2,4) or in mixture at 60°C (lane 5) for 30 min in a final volume of 20 ~1 in 20 mM MgCl,/SO mM Tris

pH 8.0. After the addition reaction

derived

The supernatant

the unincorporated

of PPV (M.Ts.,

so that they could form a double-stranded

HCl pH 8.0/20 mM MgCl,/S

[G(-~‘P]UTP (10 Ci/mmol, NEN), and was started by adding 25 u ofT7 RNA polymerase. was 500 FM. After 90 min at 37°C

Sambrook

labeled PPV ( + ) or (-) RNA, respectively,

So, the AZ derivatives

40 mM Tris

was added and the UTP concentration

‘Larissa’

was ligated into the pre-treated

were removed

1 peg of pPV2 and pPV12 was linearized

placental

isolate

5’-TCCTGATGAGGCCTTGAGGCCGAAA,

The size of the resulting

with radioactively

mixture contained

F.R.G.;

and annealed

Excess cassettes

( + ) RNA, respectively.

with (-) and

(Panel B) About

clone of the Greek

pPV1 and pPV2 (2 pg of each) were digested

of the DNA cassette.

were incubated

a cDNA

(Boehringer

was tested by Rz assay (see below). The recombinant

in a tinal volume of 20 ~1. The reaction

albumin

Plasmids

were phosphorylated

had been inserted.

from pPV1 derivatives

from pPV2 derivatives

lower parts)

part).

Beverly, MA) and phosphatase

parts in Figs. 2 and 3 are identical).

by SruI digestion

or three copies of the SalI-specific of insertion

Biolabs,

with the aid of an automated

as outlined

plasmids

in pPV1 and pPV2 (upper

A2

This fragment, which corresponds to nt 3409-3831 of another PPV isolate (Boehringer), from which the Sal1 site was removed by cleavage with SalI,

oligos Rz-Sall: 5’-TTTCGGCCTCAAGGCCTCATCAGGA

the complementary were synthesized

resulting

(New England

: (+I direected

T7 3409

( + ) and (-)

A2

pPV22

Eco RI

AC ITli;

ClAGl

3831

: (4 directed

T3

LC-TJG

A2

PPV

91-F

Eco RI

: t-b directed

T7

ITjk

A2

transcript

nt 3831-3631

Lanes 5 and 6 demonstrate

by ethanol

precipitation.

were HCI The

8 M urea (Tsagris et al., 1987) and visualized (T), synthesized

by T7 RNA polymerase

(see map of pPV2 in upper

part A), and

that the AZ RNA cleaves the PPV target

into

179

x>AzSall I

Ear1

XhoI

5’--CTCTTCG

TTTCGGCCTCGACGCCTCATCAGGA

TGAAGAG--

3’--GAGAAGCAAAGCCGGAGCTCCGGAGTAGTCCTACTTCTC--

3’

Sd

5’

between

I-R2

cassette

Earl

sites

Ear1

StUI

Ear I digestion XhoI

Ear1

GGA

TTTCGGCCTCGAGGCCTCATCA

5’--CTCTTCG ~‘--GAGAAGC

GCCGGAGCTCCGGAGTAGTCCT

AAA

TGAACAG--

3’

ACTTCTC--

5’

Ear1

stu1

Fill-in

with

Pal/k

XhOI 5

TTTCCCCCTCGAGGCCTCATCAGGA

3’

3’

AAAGCCCGAGCTCCGCAGTAGTCCT

5’

desired Sellcassette

Rz

stu1

let gene

6

5’--CTCTTCG

pAzSall-

TTTCGGCCTCCAGGCCTCATCAGCA

3’--CAGAAGCAAAGCCGGACCTCCGGACiTAGTCCT

tet

TGAAGAG--

3’

Sal I-RI

ACTTCTC--

5’

including

tet

gene

between

Earl

sites

Ear I

stu1

cassette

desired 5’

TTTCGCCCTCGAGCCCTCATCAGGA

3’

Rz

3’

AAAGCCGGAGCTCCCCAGTAGTCCT

5’

with

S&l-

cassette

let

gene

stu1

Fig. 4. Creation A&all

described protruding

of a Sell-specific

(Haseloff

and Gerlach,

ringspot

1988), yielding pAzSal1

also an XhoI site (compare

nt marked

the ret gene. Plasmid

pT3T7 was cut with BamHI

by arrows

(top drawing). convenient

Plasmid

pAzSal1

for the strategy

of the catalytic

and superscripts

was annealed

domain

delivers

outlined

2 and 3 in Fig. 1). Plasmid

Rz cassette

XhoI fragment, including

carrying

the fet gene, was inserted

ret gene by Ear1 digestion

The XhoI site of pAZSal1 was used to insert the tet gene, creating pAzSall-tet (Fig. 4). After Ear1 digestion, followed by filling-in the protruding ends, a S&I-specific Rz cassette was obtained which was about 1450 bp in size, due to the insertion of the tet gene. The presence of the tet gene allows direct selection during AZ construction and expedites the screening of clones that have the inserted cassette in an appropriate orientation by restriction mapping. Subsequently, the tet gene can be removed by XhoI digestion, so that only the catalytic Rz domain remains. The WI-specific Rz cassette derived from pAzSall-tet was inserted into the same PPV cDNA fragment as

+ KpnI, and the phosphorylated to the cohesive

after Ear1 digestion

in Fig. 2. Compared

was modified

to contain

pAzSall-tet

tetgene flanked by two XhoI sites, an XhoI linker was successively

et al., 1977). The resulting a SalI-specific

Rz cassette

virus given in Fig. IA, the sequence

into the XhoI site. To obtain the to obtain

accommodating

ends with PolIk a 25-bp SalI-specific

of the tobacco

(Bolivar

Rz cassette

(5’-p-GATCCTCTTCATCCTGATGAGGCCTCGAGGCCGAAACGAAGAGGTAC)

and successive

to the naturally

as

filling-in of the

occurring

sequence

a SruI site (like in Fig. 2) but, in addition,

(bottom

introduced

drawing)

contains

a ret gene inserted

into the EcoRI and AvaI sites of pBR322

into the XhoI site of pAzSal1,

and subsequent

oligo

ends and inserted

creating

pAzSall-tet,

which allows

filling-in with PolIk.

described before, and pPV12-tet was obtained (Fig. 5A). Before the tet gene was excised by XhoI digestion, an AZ derived from this plasmid, which contained the tet sequence within the catalytic domain, was tested for catalytic activity. Fig. 5B demonstrates that this RNA molecule is able to cleave the substrate RNA despite the insertion of about 1430 bp within the catalytic domain. (d) Restriction sites that can be used for insertion of Rz cassettes The hammerhead-type Rz can cleave substrate RNAs with different trinucleotide target motifs (cleavable motifs).

180

pPV12-tet Fig. 5. An AZ construct tilling-in the protruding of the fragment cassette arrows

the desired

Fig. 4, bottom

and superscripts

pPVl2

with mung-bean

for cleaving PPV(-) (HindIII-linearized)

were incubated incubated

as described

a catalytic

including domain

the tet gene. This Rz with the nt marked

nuclease. cassette

By selecting for ampicillin detailed)

was identified,

(XbaI-linearized),

of pPVl2-tet

by

and tetracycline

which is related was prepared

labeled T7 RNA transcripts representing

the PPV(-)

to and

from pPV2, RNA sub-

AZ RNA without (AZ) and with the ret gene (AZ-t), respectively,

for Fig. 3B alone at 0°C (lanes l-3) or 60°C (lanes 4-6). RNA S was

at 60°C with the labeled AZ RNA (lane 7) or the labeled and unlabeled

8 and 9). The reaction

(Fig. 4),

gel for purification

in Fig. 1A, was ligated into pPV1, which had been

RNA. (Panel B) Radioactively

strate (S), the PPV( -) RNA-directed

pAzSall-tet

on an agarose

with XbaI, a T7 RNA transcript

and pPV12-tet

c==)

of plasmid

Rz cassette

that contains

(map A, with inserted

(Fig. 3). After linearization

assayed pPVl2

pPVl2-tet

MI-specific

drawing),

2 and 3 as indicated

cleaved with Sal1 and treated resistance,

the let gene. After Ear1 digestion

ends with PolIk, the DNA was separated

representing

(compare

tet gene

carrying

products

were analyzed

on gels as described

shows that Az-t cleaves the PPV (-) target RNA in the same manner

Az-t RNA (lanes

for Fig. 3B. The autoradiograph as RNA derived from pPVl2.

So far, cleavage has been reported for AUC, CUA, CUC, GUA, GUC, GUU and UUC motifs (Forster and Symons, 1987a; Haseloff and Gerlach, 1988; Koizumi et al., 1988a,b) and the motif GUG was cleaved in one case (Sheldon and Symons, 1989), whereas it was not cleaved in others (Haseloff and Gerlach, 1988; Koizumi et al., 1988a). For most of these motifs it is possible to generate AZ by incorporating specific Rz cassettes into certain restriction sites as it was outlined for the GUC motif within Sal1 sites. The requirement is that the cleavable motif overlaps, at least in part, with a particular restriction enzyme recognition sequence. An additional requirement concerns the third nt of the cleavable motif within the substrate RNA. In the hammerhead complex formed between substrate and Rz RNA, this nt, at the 3’ site of which cleavage occurs, is the only residue of the substrate RNA which is unpaired. During generation of the AZ construct, this residue is removed from the cDNA and replaced by the catalytic domain (compare Fig. 1, A and B). To displace this nt from the cDNA it is a necessity that this residue is located within the protruding ends created by a suitable restriction enzyme, SO that it can be eliminated by a single-strand-specific exonuclease like mung-bean nuclease. Since more nt, than the actual one desired, are removed by trimming the protruding ends, the Rz cassette that is needed for insertion has to be designed to replace these superfluously erased nt. In addition to these ‘replacement nt’, the different Rz cassettes contain the catalytic domain of the Rz sequence, with or without modifications such as the introduction of the StuI and X/z01 sites or the tet gene.

Table I reviews the currently known cleavage motifs and corresponding recognition sequences of commercially available restriction enzymes (New England Biolabs catalogue, 1990), which are suitable for incorporating the specified Rz cassettes to obtain an AZ construct. In order to insert the Rz cassette, recombinant plasmids should, ideally, contain the suitable restriction site only once, namely located within the cDNA insert. Therefore, only those restriction enzymes are listed in Table I for which at least one appropriate cloning vector is available. Enzymes that cut once in cloning vectors are listed, provided their single cleavage site can be destroyed or deleted. It should be noted that some Rz cassettes listed in Table I can be used for several restriction recognition sequences, e.g., the SalIspecific Rz cassette can be used for the GUC motif within the Sal1 site and also for the CUC motif of the X/z01 site. A limitation of the insertion strategy occurs when the cDNA contains the restriction site of interest more than once. Usually, it is, however, possible to subclone a cDNA fragment that contains only one site. The utility of the incorporation technique to create AZ was successfully tested in our laboratory also for CUA, GUU and UUC motifs within cDNAs containing X&I, BstEII and BstBI sites, respectively (M. Zoumadakis and M.Ta., unpublished data). We are currently testing the suitability of several other cassettes summarized in Table I in combination with the restriction enzymes indicated. The applicability of AZ (with and without selectable genes) to suppress the expression of specific target RNAs in vivo is under investigation and preliminary data suggest that AZ

181 TABLE

I

Rz cassettes

for the insertion

into different

restriction

Target

sites

motif

Number

DNA cassettee

Sequence

of cases’

Suitable

Cleavable

Restriction

motif

enzyme”

AUC

BumHI

-G/GATCC-

100

-G

c-

GAT(cd)-

- -

pUC”’

Bell

-T/GATCA-

100

-T

A-

GAT(cd)-

- -

pUC’O’

BglII

-A/GATCT-

100

-A

T-

GAT(cd)-

- -

pUC”’

100

-R

Y-

GAT(cd)-

- -

qlxl74’u’

100

-AT

AT-

- - -(cd)G

- -

pUC’”

BstYI

-RGATC/Y-

Cla I CUA

cut CUNh GUA

-AT/CGAT-C/CTAGG-

100

- CT(cd)G

- -

pUC””

-G/CTAGC-

100

-c -G

G-

NheI

c-

- CT(cd)G

- -

pUC’“’

SpeI

-A/CTAGT-

100

-A

T-

- CT(cd)G

- -

Xba I”

-T/CTAGA-

100

-T

A-

- CT(cd)G

- -

pUC”” pUC’“l

100

-c

G-

- - T(cd)GA

-

pUC’”

50

-c

G-

- -T(cd)GR

-

pUC”’

XhoI

-C/

AvaI

-C/YCGRG-

TCGAG-

Bsu361

-CC

/ TNAGG-

50

-cc

GG

- - T(cd)A - -

pUC””

EspI

-GC

/ TNAGC-

50

-GC

GC

- - T(cd)A - -

pUC”” pUC’“‘)

Asp718

-G/GTACC-

100

-G

c-

BsiW 1’

-C/GTACG-

100

-c

G-

100

-G

c-

25

-GT

25

-GT

100

-G

-GGTAC/C-

AccIk

-GT

/ ATAC-

-GT

/ AGAC/ TCGAC-

-

-GT

AC-

- - -(cd)G

- -

pUC”’

-GT

/ CTAC-

25

-GT

AC-

- - -(cd)T

- -

pUC’

-G/GWCC-

50

-G

PpuMI

-RG/GWCCY-

50

-RG

fir11

-CG

50

-CG

Eco01091

-RG/GNCCY-

25

-RG

/ GWCCG-

-G/GTNACC-

100

-G

100

BspHI

-(N)T/CATGA-

BspEI XbaP

enzymes

-G

- -

PUC””

CY-

- GT(cd)-

- -

pUC’

c-

- GT(cd)A

C -

pUC’”

’’

- - T(cd)C A -

M13”” pACY 184’”

-(N)T/CCGGA-

-(N)

T

A-

- - -(cd)CGG

-(N)T/CTAGA-

100

-(N)

T

A-

- - -(cd)TAG

puc’n) pUC’“”

-TT

AA-

- - -(cd)G

pUC””

/ CGAA-

100

Biolabs

Catalogue,

1990), which can be used for insertion

vector

is available

that has none or only one cleavage

cloning

of restriction

which is present

put?

- GT(cd)-

- --(cd)ATG

of the restriction

of the Rz cassette

- GT(cd) - - -

CG-

C-

-G

enzymes,

which are part of the DNA encoding

sequence

sites which provide GGWCC.

recognition

sequence

in each cassette

a target motif, e.g., the sequence

after digestion

is indicated

and removing

into the DNA encoding

RNA, are given; the cleavage

GGTCC

the protruding

represents

to contain

only approx.

the target

single-stranded

a target RNA; only one strand

by (cd), only the ‘replacement

only restriction

enzymes

site is indicated

motifs are underlined.

Only 50% of the AvaII sites can be expected

which is used for insertion

of an Rz cassette;

- -

site for this enzyme.

the target

by a slash; the nt N are A, C, G or T; W are A or T; Y are C or T; R are A or G; the target

nt of the restriction

CY-

A-

are listed (New England

’ The % gives the proportion

M13”’

T

are listed for which at least one suitable

AvaII sites with the recognition

’’

- GT(cd) - - -

c-

-(N)

/ TGCAC-

-TT

sequences

c-

100 100

BsrBI

domain

- -

- - T(cd)GA

25

ApaLI”

catalytic

- - -(cd)G

IJ pUC’’ ’ put ’’ pUC’ ’ )

/ CGAC-

NUC

d Remaining

AC-

1

pUC”’ pUC’”

-GT

GUG

’ The sequence

- - -

AccIk

BstEII

’ The recognition

AC-

- GT(cd)C - - -(cd)T

-G

GUN

a Restriction

GT(cd)C - - GT(cd)C - -

Sal1

AvaII

UUG

vector‘

AvrII

Kpn I

GUC

after trimmingd

(%)

nt’ are given (compare

50% of all possible

motif GTC.

ends. is given, the part representing with the SalI-specific

the

Rz cassette

in Fig. 2). r At least one suitable

cloning vector (New England

Biolabs Catalogue,

‘pUc’ and ‘M 13’ refer to the pUC and M 13 series of cloning vectors; pUC”“)

indicates

g XbaI contains ’ So far, cleavage ’ New England

that there are vectors two target

of the pUC series having

motifs: -(N)T/CTAGA-

has only been described Biolabs

Product

1990) is listed in which the cDNA can be cloned for insertion

the superscripts

(‘i and (‘i indicate either none or one cleavage site.

the number

of cleavage

of the Rz cassette;

sites within that vector;

and -T/CTAGA-.

for CUA and CUC motifs.

News Vol. 3, Issue 1.

Ir There are four possible AccI sites GT/ATAC, GT/AGAC, “’ Not every GUG motif is cleavable; compare section d.

GTjCGAC

work more efftciently than antisense RNAs which is not surprising since AZ inhibition is due to irreversible cleavage, whereas the antisense complex can dissociate. Considering the Rz character of these RNA transcripts, it should be noted that the long flanking sequences ensure accurate and

and GTjCTAC

which contain

a GTA and GTC motif, respectively.

efficient binding of the catalytic RNA to their targets. This seems to be essential for Rz action, since it was found in an in vitro processing system, monitoring the maturation of histone pre-mRNA, that Rz directed against U7 RNA with only eight complementary nt flanking the catalytic domain

182 were effective only in a lOOO-fold molar excess (Cotten et al., 1989) whereas inhibition by antisense RNA was detected

already

Bruening,

at a sixfold molar excess.

and other

G., Buzayan,

of small satellite enzymatic

(e) Conclusions

(Eds.),

The incorporation of a catalytic domain could extend the application of the antisense RNA technology for gene suppression (reviewed by Van der Krol et al., 1988a,b; Rothstein and Lagrimini, 1989; Helene and Toulmt, 1990). AZ could be produced also by other techniques, e.g., insertion mutagenesis, but the strategy of inserting an Rz cassette combines several advantages. (I) The technique is easy to perform, used in conjunction with a selectable

satellite RNAs 546-558.

Cameron,

especially when and removable

marker. (2) A given Rz cassette can be used in a universal way for insertion into its specific restriction site in any cDNA encoding a target RNA, without the need to design and synthesize an additional oligo. (3) It delivers sense- and antisense-directed Rz in one cloning step. (4) A restriction map of the target is sufficient, sequence information is not necessary. (5) The length of the flanking sequences which are responsible for the binding of the Rz to its target can be as long as the RNA transcript, so that insufficient binding due to secondary structures within target and/or Rz RNA is eliminated. (6) It allows the insertion of selectable markers that can be also used for the in vivo expression of the Rz. (7) Several cassettes can be incorporated into a cDNA, creating an AZ with multiple catalytic domains.

and

Jennings,

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of a virusoid (1987a)

gene

suppression

RNA

and RNA enzymes.

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destruction

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R.H.: Self-cleavage

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R.H.: Self-cleavage

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