ABSTRACT
A target RNA/DNA-specific nuclease could be constructed if a specific RNA/DNA binding
domain allowing target RNA/DNA recognition was fused to a
(deoxy)ribonucleolytic domain allowing target RNA/DNA cleavage. The design and
construction of such a chimeric enzyme could be of value for both basic
research involving structure-function relationships and applied research requiring inactivation of
harmful RNA/DNA molecules of cellular or pathogenic origin. The feasibility of
this designer nuclease approach for inactivating specific RNA/DNA molecules was
assessed using human immunodeficiency virus type-1 (HIV-1) RNA as a model.
Trans
-activator of transcription (Tat) protein is one of the key regulatory
proteins encoded by HIV-1. It binds to the
trans
-activation-responsive (TAR) RNA element located within the 5
'
non-coding region of HIV-1 RNAs. The TAR RNA binding domain of this protein was fused to the
ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT). RNase H by itself lacks an RNA binding
domain. The chimeric Tat-RNase H protein was shown to specifically recognize and cleave HIV-1 TAR RNA
in vitro
. Cleavage was abolished by mutations in the Tat binding region within the TAR
RNA, indicating that it is specific to HIV-1 TAR RNA.
Inactivation of a specific RNA/DNA molecule could be achieved by directly
interfering with the structure/function of these molecules and/or of proteins
encoded by them. Several strategies have been developed which inhibit the
function of a specific DNA and/or RNA molecule. These include the antisense
RNAs/DNAs (
1
-
4
), decoy RNAs (
5
-
8
) and ribozymes (
9
-
13
). Antisense RNA/DNA molecules act via hybridization to the target RNA/DNA
molecules and therefore must be provided in excess. Decoy RNAs act as
competitive inhibitors for specific RNA-RNA or RNA-protein interactions and again should be provided in excess.
Strategies aimed at destroying the target RNAs should prove to be more
effective than those inhibiting the function but leaving the unwanted RNAs
intact. Ribozymes, used for this purpose, are small catalytic RNA molecules
that can be designed, via inclusion of specific antisense RNA sequences, to
specifically pair with and cleave the target RNAs. Although naturally occurring
ribozymes act in a catalytic manner,
in vivo
trans
-cleaving ribozymes could not be shown to act in a catalytic manner (
14
). Ribozymes with improved and/or different activities are therefore being
designed in several laboratories (
15
-
17
), including our own. An alternative novel strategy that could be employed for
inactivation of specific RNA/DNA molecules would be to confer target RNA/DNA
specificity to (deoxy)ribonucleases so that they will specifically recognize
and cleave the target RNA/DNA molecules. The feasibility of this approach was
tested against HIV-1 RNA as a model.
The 5' non-coding region of HIV-1 RNAs contains a
trans
-activation-responsive (TAR) element located between nucleotides (nt) +1 and
+59. This element forms a stem-loop structure of which a bulge at nt +23 to +25 is required for HIV-1 Tat protein binding to this element (
18
). The Tat-TAR RNA interaction is crucial for
trans
-activation of HIV-1 gene expression (
19
).
Tat is an 86 amino acid regulatory protein produced early during the virus life
cycle. The acidic N-terminal region of Tat seems to form an amphipathic [alpha]-helix required for Tat activity (
20
). This region is followed by a cluster of seven Cys residues necessary for
metal ion binding and protein-protein interactions (
21
). Another domain, consisting of a stretch of basic amino acids, directs nuclear
localization of Tat and allows Tat binding to the TAR element (
18
,
22
). The first 72 amino acids of Tat are sufficient for Tat activity. This region
was therefore used to provide HIV-1 RNA binding specificity to the HIV-1 TAR RNA-specific nuclease `TAR-RNase' engineered in this study.
The RNase domain used in `TAR-RNase' design is from the HIV-1 RT-associated RNase H domain. Electron density maps obtained by X-ray diffraction analysis of HIV-1 RNase H crystals reveal that this enzyme is
folded into a five-stranded mixed [beta]-sheet flanked by an asymmetric distribution of four [alpha]-helices (
23
). This RNase H specifically degrades the RNA moiety within a RNA-DNA hybrid (
24
) and, to a lesser degree, within a RNA-RNA hybrid (
25
). The isolated RNase H domains of HIV-1 RT either are inactive (
26
-
29
) or possess extremely low levels of activity (
30
). One possible explanation for the inactivity of the RNase H domain is that it
lacks the substrate binding domain.
The design, engineering and cleavage specificity of `TAR-RNase', also referred to as Tat-RNase H, in cleaving HIV-1 RNA are reported.
A Tat-RNase H gene was constructed containing sequences from the HIV-1
tat
gene encoding amino acids 1-72 (
31
) and from the HIV-1
pol
gene encoding the RNase H domain located within amino acids 432-560 of HIV-1 RT. Primers B (5'-CTCTATCAAAGCAACCCATAGTAGGAGCAGAAACC-3') and C (5'-CTAAGGATCCCTATAGTACTTTCCTGATTCC-3') were used
during the first round of polymerase chain reaction (PCR) using pACRT DNA to
amplify the RNase H sequences. This PCR product, along with primer A (5'-GTCTGCAGCATATGATGGAGCCAGTAGATCC-3'), were then used in a second PCR using pTev DNA to
amplify Tat sequences (
31
). PCRs were performed in 50 [mu]l reaction mixtures containing 20 mM Tris-HCl, pH 8.75, 10 mM KCl, 10 mM (NH
4
)
2
SO
4
, 2 mM MgSO
4
, 0.1% Triton X-100, 0.1 mg/ml BSA, 100 ng primers and 2.5 U Pfu DNA polymerase
(Stratagene) for 1 min at 95oC, 1 min at 55oC and 2 min at 72oC for a total of 30 cycles. The resulting 597 bp PCR product
contained an
Nde
I restriction site, Tat-RNase H coding sequences and a
Bam
HI restriction site. This DNA was digested with
Nde
I and
Bam
HI restriction enzymes and cloned within the pET-15b (Novagen) vector at the same sites located downstream of a T7 promoter
and in-frame with the region encoding 20 amino acids containing six His residues
and a thrombin cleavage site. The correct clone was screened by restriction
enzyme analysis and was referred to as pET-TH. pET-T encoding the first 72 amino acids of Tat and pET-H encoding amino acids 433-560 of reverse transcriptase (RT) were constructed in a
similar fashion and served as controls.
Tat-RNase H protein was overexpressed in
Escherichia coli
strain BL21 (DE3; Novagen) transformed with the pET-TH vector. This protein contains six His residues within the N-terminal region. It was purified by non-denaturing immobilized metal affinity chromatography (
32
) as follows. A 100 ml culture of
E.coli
cells expressing pET-TH vector was grown at 37oC until an optical density of 0.6 at 600 nm was reached. The culture
was then induced with 1 mM isopropyl-[beta]-D-thiogalactopyranoside (IPTG) and the incubation
continued for a further 2 h. The cells were harvested and disrupted by
sonication (six cycles of 50 s with a 1 min pause in between on ice). The
lysate was centrifuged at 4oC for 30 min at 40 000
g
. The supernatant was filtered through a 0.45 [mu]m membrane (Millipore) and was applied to a 1 ml nickel nitrilotriacetate
affinity column (Novagen). The fusion protein was eluted with 6 ml of native
buffer containing 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl and 500 mM imidazole. The protein fraction was
dialyzed against 50 mM Tris-HCl, pH 7.8, 100 mM KCl, 1 mM EDTA, 1 mM DTT and 50% glycerol and stored
at -70oC. The various protein fractions were analyzed by 0.1% SDS-15% polyacrylamide gel electrophoresis followed by Coomassie
blue staining (
33
). Tat and RNase H proteins were purified in a similar fashion.
The N-terminal 20 amino acids containing the six His were cleaved by treating 10
[mu]g Tat-RNase H protein with 4 U thrombin (Sigma) in 200 [mu]l of reaction mixture containing 20 mM Tris-HCl, pH 8.4, 150 mM NaCl and 2.5 mM CaCl
2
for 2 h at 25oC. Unless indicated otherwise, the uncleaved protein was used in the
experiments described below.
TAR RNA, containing the first 75 nt of HIV-1 RNA, was produced as follows. A DNA template containing a T7 promoter driving TAR RNA expression was
generated by PCR amplification of pHXB[Delta]SVCat DNA as described above using T7-TAR (5'-AAATTAATACGACTCACTATAGGGTCTCTCTGGTTAG-A-3') as 5' primer and TAR-3' (5'-TTGAGGCTTAAGCAGAGTGG-3')
as 3' primer. This PCR product was then transcribed
in vitro
for 2 h at 37oC in a 100 [mu]l reaction mixture containing 1 mM each rNTPs, 40 mM Tris-HCl, pH 8.0, 8 mM MgCl
2
, 5 mM dithiothreitol (DTT), 1 mM spermidine, 0.001% Triton X-100, 50 [mu]g/ml BSA and 100 U T7 RNA polymerase (BRL) (
34
). RNA was analyzed by 10% polyacrylamide-8 M urea gel electrophoresis (
35
) followed by methylene blue staining (
36
). The band containing the TAR RNA was excised and eluted overnight in a buffer
containing 0.5 M ammonium acetate, 10 mM Tris-HCl, pH 8.0, 10 mM EDTA and 0.5% SDS. The RNA was extracted with phenol/chloroform and then recovered by
ethanol precipitation.
5'-End-labeling of TAR RNAs was performed using a modification of
the procedure described in Donis-Keller
et al
. (
37
). RNA transcripts were dephosphorylated at 37oC for 60 min using 0.5 U calf intestinal alkaline phosphatase (Boehringer
Mannheim) in a 50 [mu]l reaction mixture containing 20 mM Tris-HCl, pH 8.0, and 0.1% SDS. After adding 5 [mu]l of 3 M sodium acetate, pH 5.5, the RNA was extracted with
phenol/chloroform saturated with 300 mM sodium acetate, pH 5.5, 10 mM EDTA and
then precipitated with ethanol. The RNA pellet (25 pmol) was resuspended in 10 [mu]l buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM spermidine, 0.1 mM EDTA and heated at 50oC for 5 min. To this were added 0.1 vol. 500 mM Tris-HCl, pH 9.0, 100 mM MgCl
2
, 50 mM DTT, 3 [mu]l [[gamma]-
32
P]ATP (3000 Ci/mmol; Amersham) and 5 U T4 polynucleotide kinase (Boehringer
Mannheim) and the reaction mixture was incubated at 37oC for 60 min. EDTA (50 mM) was then added to stop the reaction.
Unless specified otherwise, cleavage reactions were performed for 30 min at 37oC using
32
P-labeled TAR RNA (30 000 c.p.m.) and affinity-purified Tat-RNase H (25 ng) in 30 [mu]l reaction mixtures containing 50 mM Tris-HCl, pH 7.8, 8 mM MnCl
2
and 50 mM KCl. The cleavage products were then analyzed by 8 M urea-15 or 20% polyacrylamide gel electrophoresis (
2
). The
32
P-labeled TAR RNA was also digested by alkali or partial RNase T1 (
23
) to produce molecular weight markers.
In order to confer HIV-1 TAR RNA specificity to RNase H, amino acids 1-72 of Tat containing the TAR RNA binding domain and amino acids 433-560 of HIV-1 RT containing the RNase H domain (between amino
acids 440 and 560) were fused (Fig.
1
A). The Tat-RNase H gene was constructed by two-step PCR (
39
) and cloned downstream of the T7 promoter in an
E.coli
expression vector, pET-15b, in-frame with the 20 amino acids encoded by the vector, which include
six His residues to facilitate purification and a thrombin cleavage site to
remove the His tag, if required. The resulting vector, pET-TH, allows IPTG-inducible expression of Tat-RNase H protein which can be easily purified by metal
affinity chromatography (Fig..
1
B). Plasmids pET-T expressing the first 72 amino acids of Tat and pET-H expressing amino acids 432-560 of RT containing the RNase H domain were constructed in
parallel. Tat and RNase H proteins expressed from these vectors were purified
in a similar manner and used as controls.
In order to demonstrate Tat-RNase H activity, it was incubated with 5'-end-labeled HIV-1 TAR RNA containing the first 75 nt of HIV-1 RNA.
In vitro
cleavage was assessed in the presence of Mg
2+
and Mn
2+
. While no cleavage product could be detected in the absence of Tat-RNase H (control), several bands corresponding to specific TAR RNA
cleavage products could be detected when the cleavage reaction was performed in
the presence of Mn
2+
(Fig.
2
). The optimal Mn
2+
concentration required for TAR RNA cleavage ranged between 6 and 20 mM. The Tat-RNase H protein showed no detectable activity in the presence of Mg
2+
. Increasing concentrations of Tat-RNase H protein resulted in only a slight increase in cleavage efficiency
(results not shown). The limiting factor could be the lack of a correct
conformation of TAR RNA and/or of Tat-RNase H protein. It is difficult to be sure that all TAR RNA forms the
tertiary structure that can be recognized by Tat-RNase H. Once cleaved, 5' cleavage products containing the TAR element could also act as a
competitive inhibitor preventing 100% cleavage. Furthermore, removal of the N-terminal six His residues by thrombin did not affect Tat-RNase H activity (results not shown). These results demonstrate
that Tat-RNase H can recognize and cleave HIV-1 TAR RNA
in vitro
at several specific sites and that this cleavage requires the presence of Mn
2+
.
That Tat-RNase H cleaves TAR RNA in a specific manner following Tat-TAR RNA interaction was confirmed by comparing cleavage specificity of 5'-end-labeled wild-type and mutant TAR RNAs. The mutant TAR
RNA contains U
23
-> C
23
, G
26
-> A
26
and C
39
-> U
39
substitutions within the bulge region responsible for Tat protein binding onto
the TAR RNA (
19
,
40
,
41
). This mutant TAR RNA displayed no cleavage at all upon incubation with Tat-RNase H; only the wild-type TAR RNA could be cleaved (Fig.
3
). This result confirms that Tat-RNase H specifically cleaves TAR RNA and that Tat-TAR interaction is required for this cleavage.
Purified Tat and RNase H proteins were tested, either separately or in
combination, for 3'-end-labeled TAR RNA cleavage
in vitro.
Neither one of these two proteins displayed any cleavage of the TAR RNA (Fig.
4
). Cleavage occurred only when the Tat-RNase H fusion protein was used. These results confirm that the observed
cleavage is solely due to the presence and activity of the Tat-RNase H fusion protein and not due to the presence of RNase H by itself
or other contaminating
E.coli
RNases.
Cleavage products generated after various time intervals were analyzed to assess
whether, following Tat-RNase H binding to the 3'-end-labeled TAR RNA, the RNA is cleaved only once or
several times. If multiple cleavages occurred, with time the intensity of some
of the bands should have decreased in favor of others. The results indicate
that the intensity of bands corresponding to all cleavage products increases
with time (Fig.
5
), suggesting that under the experimental conditions used TAR RNA was cleaved
only once. It is conceivable that, once cleaved, the TAR stem structure is
disrupted, preventing Tat-RNase H-TAR RNA interaction and/or further cleavage from taking place.
The location of various cleavage sites was determined by assessing the size of
various 5'- and 3'-end-labeled TAR RNA cleavage products (Fig.
6
A). Cleavage occurs at three different locations, after nt +37, after nt +46 to
+49 and after nt +56 to +58; for the latter two locations, maximum cleavage
takes place at the center after nt +47 and +48 and after nt +57. Of the three
different locations, cleavage is maximal at nt +56 to +58 (Fig.
6
A). No other band corresponding to exonucleolytic cleavage was observed,
confirming that the TAR RNA is only cleaved in an endonucleolytic manner.
The TAR RNA secondary structure with various cleavage sites is shown in Figure
6
B. It is obvious from this figure that the cleavage sites are all located on one
side of the TAR RNA stem-loop structure and that the three cleavage regions are distanced by some
10 nt.
Taken together, the data presented here clearly indicate that the Tat-RNase H fusion protein has acquired a new structure-specific endonucleolytic cleavage activity. It specifically
recognizes the TAR RNA via Tat-RNase H-TAR RNA interaction and cleaves one side of the stem-loop structure at three different locations. Design and
engineering of other fusion proteins containing a nucleic acid binding domain
and a nucleolytic domain is in progress to confer cleavage specificity to this
and other nucleases against different cellular and viral target RNAs/DNAs. Such
target nucleic acid-specific nucleases may be ideal in studying structure/function of specific
nucleic acids. Also, and more importantly, they should have a great therapeutic
value.
This work was supported by grants from the National Health and Research
Development Program and Medical Research Council of Canada. Plasmid pHXB[Delta]SVCat was obtained from Dr E.A.Cohen, plasmid pTev was received from Dr
Barbara Felber and plasmid pACRT was constructed by W.Marhin in our laboratory.
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