(Received for publication, June 2, 1995; and in revised form, July 7, 1995)
From the
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.
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 ()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.
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.
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
(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
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.
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
; lanes3 and 4, JA221/F`lacI
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.
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) .