©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gene Regulation by Antisense DNA Produced in Vivo(*)

(Received for publication, June 2, 1995; and in revised form, July 7, 1995)

Jau-Ren Mao Masamitsu Shimada (§) Sumiko Inouye Masayori Inouye (¶)

From the Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Antisense technology has been widely used for regulating gene expression. Single-stranded RNA or DNA complementary to a target mRNA can inhibit the translation of the mRNA. Antisense RNA is produced in vivo, while antisense DNA is chemically synthesized as an oligonucleotide, which is extracellularly added to the cells. To maintain the effect of antisense DNA, a synthetic oligonucleotide has to be constantly added to the system. An advantage of antisense DNA over antisense RNA is that the target mRNA hybridized with the antisense DNA can be specifically digested by ribonuclease H. Here, we attempted to produce in vivo short single-stranded DNAs complementary to a specific mRNA. We demonstrate that such antisense oligodeoxyribonucleotide of a desired sequence can be produced in Escherichia coli using a retron, a bacterial retroelement, as a vector and that the antisense DNA thus produced in vivo can effectively inhibit the expression of a specific E. coli gene, such as the gene for the major outer membrane lipoprotein.


INTRODUCTION

Retrons are bacterial retroelements found in Myxococcus xanthus(1, 2) , a minor population of natural isolates of Escherichia coli(3, 4, 5) , and a number of other Gram-negative bacterium (6) (see also (7) for a review). They consist of a 1.3-3.0-kb (^1)genetic unit integrated into the bacterial genome expressing a reverse transcriptase (RT) related to eukaryotic RT. The retron RTs are responsible for the production of multicopy single-stranded DNA (msDNA) consisting of a short single-stranded DNA that is attached to an internal G residue of a short RNA molecule by a 2`,5`-phosphodiester linkage. The RTs use an RNA transcript from the retrons not only as primer but also as template for msDNA synthesis. For the cDNA priming reaction the RTs require specific secondary structures for individual RTs downstream of the branching G residue (8) and a stem structure immediately upstream of the branching G residue(9) .

The requirement of the structures in the region corresponding to DNA for msDNA synthesis was also extensively investigated for msDNA-Ec107 (10) . It was demonstrated that the upper stem region consisting of 71 bases of the msDNA was not essential and could be deleted to produce a truncated msDNA consisting of only a 36-base single-stranded DNA. This result raises an interesting question of whether the upper region of an msDNA can be replaced with other unrelated sequences. In the present report, we demonstrate that msDNA indeed can be used as a vector for in vivo production of a single-stranded DNA or an oligonucleotide of a desired sequence. We further demonstrate that artificial msDNAs containing a sequence complementary to a part of the mRNA for the major outer membrane lipoprotein of E. coli effectively inhibited the lipoprotein biosynthesis upon induction of the msDNA synthesis. This is the first demonstration of in vivo synthesis of oligodeoxyribonucleotides having antisense function. Since we have previously demonstrated that bacterial retrons are functional in the eukaryotes to produce msDNAs in yeast (11) and NIH3T3 cells(12) , the present system may be also used to produce oligodeoxyribonucleotides of desired sequences inside the cells to artificially regulate eukaryotic gene expression either by duplex formation on mRNAs or by triplex formation of the chromosomal DNA.


EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids

E. coli strain JA221/F`lacI^q(13) was used in most of the experiments for the expression of msDNAs. For the experiment for the internal cleavage of an EcoRI site in msDNA-anti-lppE25, E. coli MM294 (14) was used. Strain MM294 was transformed with pJREcoRI containing the gene for endonuclease EcoRI and the gene for EcoRI methylase(14) . These two genes were obtained from pGJ440 (14) by digesting the plasmid DNA with BsaAI and ScaI. The resulting 2.1-kb fragment was inserted into the unique HincII site of a low copy plasmid, pCL1921 (spc^r)(15) . For the expression of msDNA, a pINIII (lpp) vector (16) was used.

Construction of Plasmids for the Induction of Antisense msDNAs

The pINIII(lpp) vectors containing anti-lpp sequences were constructed as follows. First, to introduce a NcoI or an EcoRI site in the msd region and delete 56 bases in the upper portion of msDNA-EC73, a two-step PCR was performed using a pT7Ec73 msr-msd(17) as a template. In the first PCR, two sets of primers were used: 5`AATCTAGACAGAGCCAAACCTAG3` (oligo 5641) corresponding to bases 10411-10425 and 5`TACTTGAGCACCATGGGGCATAGCTAA3` (oligo 5790; the underlined sequence is the NcoI site) complementary to bases 10479-10489 and 10546-10556 and containing 6 bases for NcoI site in the middle and 5`TCTCTAGATCCTTATGCACCTTGA3` (oligo 5640) complementary to bases 10672-10689 and oligo 5791, which is the complementary sequence of oligo 5790. The second PCR was performed using the amplified fragments and oligos 5641 and 5640 as primers, which created an XbaI site at their 5`- and 3`-ends. The amplified fragments were cloned into the XbaI site of pINIII(lpp)A1 (16) of which the EcoRI site was eliminated. Double-stranded oligonucleotides consisting of anti-lpp sequences a and b in Fig. 1f with NcoI sites at the ends were synthesized and cloned into the NcoI site of pINIII(lpp)msDNA/NcoI, resulting in pINIII(lpp)N25 for msDNA-anti-lppN25 (Fig. 1c) and pINIII(lpp)N34 for msDNA-anti-lppN34 (Fig. 1d), respectively. In the case of construction of pINIII(lpp)E25 for the production of msDNA-anti-lppE25 (Fig. 1e), pINIII(lpp)msDNA/EcoRI was constructed with the same procedure used for pINIII(lpp)N25. To make sure EcoRI enzyme is able to digest the EcoRI site formed on a stem of msDNA, three bases were added at the 5`-end and 3`-end of EcoRI site as shown in Fig. 1e. After confirming the DNA sequence of the constructs, the 0.95-kb BamHI fragment carrying msDNA Ec73 reverse transcriptase (RT-Ec73) from pUC7Xbal73RT (17) was inserted at the BamHI site of these plasmids, and the orientation of RT-Ec73 was determined by restriction digests.


Figure 1: The proposed structures of msDNA-Ec73 and its derivatives, and the antisense sequences used in the msDNAs. a, msDNA-Ec73 isolated from clinical E. coli strain Cl-23(13) . b, msDNA-miniEc73 constructed by deleting 43 bases (from C-15 to G-57) from the DNA structure of msDNA-Ec73. c and d, msDNA-anti-lppN25 and msDNA-anti-lppN34, derivatives of msDNA-Ec73 containing anti-lpp sequences a and b (f) in the loop structure, respectively. e, msDNA-anti-lppE25, a derivative of msDNA-Ec73 containing anti-lpp sequence a (f) with an EcoRI site at the stem region. The anti-lpp sequences are circled. Boxes enclose msdRNA, and the branching G residues are circled. f, the 5`-end ribosomal binding region of the lpp mRNA and the nucleotide sequences of antisense DNA a (25 bases) and b (34 bases). The initiation codon, AUG, of the lpp gene is boxed, and the Shine-Dalgarno sequence is indicated by dots.



Other Methods

msDNA preparation was performed as described previously(3) . Membrane preparation and SDS-polyacrylamide gel electrophoresis were carried out as described previously(13) .


RESULTS AND DISCUSSION

Production of msDNA Containing Antisense DNA against the lpp mRNA

Retron-Ec73 is an E. coli retron responsible for the production of msDNA-Ec73(18) . Since msDNA-Ec73 is stable and produced at a level of approximately a few hundred copies/cell, we chose this msDNA as a vector for the in vivo production of artificial oligonucleotides. Previously we have demonstrated that the upper stem region of msDNA-Ec107 can be deleted(10) . Therefore, we first tested if a similar deletion is possible for msDNA-Ec73. By deleting a substantial central part of the msDNA-coding region (msd), a retron responsible for msDNA-miniEc73 (Fig. 1b) was constructed using a pUC vector(19) . This msDNA consists of only 30 nucleotides, 43 nucleotides shorter than msDNA-Ec73 (Fig. 1a). When this plasmid was cotransformed with pRT-73(8) , the yield of msDNA miniEc73 in the presence of 1 mM isopropyl-beta-D-thiogalactopyranoside was as high as that of msDNA-Ec73, estimated at a level of 5000 copies/cell (not shown).

Since this result indicates that at least the upper part of the stem-loop structure of msDNA-Ec73 can be replaced, we next attempted to add new sequences at the loop region of the msDNA-miniEc73. For this purpose, the entire stem-loop region was replaced with a NcoI site. This allowed insertion of sequences a and b (Fig. 1f) to produce msDNA-anti-lppN25 (Fig. 1c) and -anti-lppN34 (Fig. 1d), respectively. In these msDNAs, the loop sequences are complementary to the translation initiation region of the mRNA for the major outer membrane lipoprotein, the most abundant protein in E. coli. The protein was used as a target for antisense RNA regulation(13) . Similarly, another artificial retron was constructed to produce msDNA-anti-lppE25 (Fig. 1e). This msDNA is similar to msDNA-anti-lppN25 except that it has a longer stem so that when an EcoRI site is recreated upon the formation of the msd stem structure in the msDNA, it can be digested by EcoRI enzyme.

All the artificial retrons were constructed in the pINIII(lpp) vector (16) as in the case of msDNA-mini Ec73 so that msDNA productions were inducible by IPTG, a lac inducer. As shown in Fig. 2, the amounts of msDNA detected in the late logarithmic growth were somewhat different in three constructs, possibly due to their stabilities. msDNA-anti-lppN25 was produced at the highest level and estimated as approximately 5000 copies/cell. Multibands on nondenatured gels as shown in Fig. 2became a single band when they were analyzed on denatured gels after labeling at their 3`-ends. This result indicates that the multibands appeared due to different conformations of msDNA.


Figure 2: Production of msDNAs containing anti-lpp sequences. msDNAs were isolated from 5-ml cultures of JA/F`lacI^q(13) harboring pINIII(lpp)N25 (lanes2 and 3), pINIII(lpp)N34 (lanes4 and 5), and pINIII(lpp)E25 (lanes7 and 8). Lanes1 and 2 were JA/F`lacI^q without a plasmid. MW is the HaeIII-digested pBR322 DNA, and numbers on the left indicate the number of bases. msDNAs were isolated by the alkali-SDS method described by Lampson et al.(3) , treated with RNase A (25 µg/ml) for 10 min at 37 °C, and then analyzed by 8% polyacrylamide gel electrophoresis. Cells growing in M9 medium were induced with 1 mM IPTG at a Klett unit of 30 and harvested at a Klett unit of 150 to isolate msDNA.



Effects of Antisense DNA on the lpp Expression

We next examined their effects as antisense DNA on the production of the E. coli major outer membrane lipoprotein. In the presence of 1 mM IPTG, significant inhibition was detected in all the constructs (75% for N25, lane4; 70% for N34, lane6; and 77% for E25, lane8 in Fig. 3). Note that some inhibitory effects were also observed even in the absence of IPTG. This is probably due to leaky expression of msDNA in the pIN vector as observed previously(13) .


Figure 3: Inhibition of lipoprotein production by antisense DNA. One ml of the same cultures used in Fig. 2was labeled with 5 µCi of TranS-label (Amersham Corp.) for 10 min at a Klett unit of 150. Membrane fractions were isolated by the method described previously (13) and analyzed by 17.5% SDS-polyacrylamide gel electrophoresis. Lanes1 and 2, JA221/F`lacI^q; lanes3 and 4, JA221/F`lacI^q harboring pINIII(lpp)N25; lanes5 and 6, harboring pINIII(lpp)N34; and lanes7 and 8, harboring pINIII(lpp)E25. Lanes 2, 4, 6, and 8 were treated with 1 mM IPTG. The lipoprotein was quantitated using an imaging densitometer model GS-670 (Bio-Rad) by comparing the density of the lipoprotein to the density of OmpA indicated by an arrow with the letter A.



EcoRI Digestion of msDNA Inside the Cell

In msDNA anti-lppE25, an EcoRI site was designed to be present in the stem region of the msDNA. If the proposed structure in Fig. 1e is formed, the msDNA should be digested by EcoRI. When the msDNA extracted from the cells and purified by polyacrylamide gel electrophoresis was digested by EcoRI, three single-stranded DNA fragments were obtained in expected sizes: 46 bases, from 12 to 48; 16 bases, from 49 to 65, and 11 bases, from 1 to 11 of msDNA-anti-lppE25 (Fig. 1e) (data not shown). The result indicates that the msDNA molecules indeed form the secondary structure shown in Fig. 1e. Next, to generate a short single-stranded DNA inside cells, msDNA anti-lppE25 was transformed into E. coli strain MM294, which was expressing the EcoRI enzyme as well as the EcoRI methylase. In this cell, the chromosomal DNA is protected from EcoRI digest because of the methylation at the EcoRI sites(14) . However, since the EcoRI site on the msDNA is formed as a result of annealing of the unmethylated single-stranded DNA synthesized by RT, the EcoRI site should be still susceptible to EcoRI cleavage. No intact msDNA was detected indicating that the msDNA was indeed digested by EcoRI. However, we were unable to detect the fragments generated by EcoRI digestion (data not shown) most likely due to their sensitivities to nucleases. This result indicates that msDNA is a potential vector to release shorter single-stranded oligodeoxyribonucleotides.

In the present report we demonstrated that msDNA can be used as a vector for the in vivo production of a short single-stranded DNA fragment. Antisense DNA thus produced in vivo effectively blocked a specific gene expression. Earlier we have shown that msDNA can be produced in yeast (11) as well as mammalian cells (12) by introducing a retron into these eukaryotic cells. Therefore, the bacterial retrons may also be used in the eukaryotes including plants as a vector to produce artificial single-stranded DNAs inside the cell. Such DNAs may be designed to target not only specific mRNAs, their precursors, and other functional RNAs but also chromosomal double-stranded DNA to form triple helices(20) .


FOOTNOTES

*
This work was supported by United States Public Health Service Grant GM44012. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Takara Shuzo Co., Ltd., Central Research Laboratories, Sita 3-4-1, Otsu, Shiga 520-21, Japan.

To whom all correspondence should be addressed.

(^1)
The abbreviations used are: kb, kilobase(s); RT, reverse transcriptase; msDNA, multicopy single-stranded DNA; PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-beta-D-galactopyranoside.


ACKNOWLEDGEMENTS

We thank Dr. Sue Harlocker for critical reading of this manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.