Table 1. Plasmids and PNAs used in this study Plasmids pOU82 pPR95 PNAs Anti Sok PNA-1 Anti Sok PNA-2 Anti Sok PNA-3 Control SP69 Features bla+, hok/sok , mini-R1 bla+, hok/sok+, mini-R1 (N)-(KFF)3K-eg1-TATGTCTAGTC (N)-(KFF)3K-eg1-TATGTCTAG (N)-(KFF)3K-eg1-TATGTCTAGT (N)-(KFF)3K-eg1-GTTACATGAT References (42) (9) This study This study This study (21) were probed using an oligonucleotide that is complementary to the 30-terminal region of the hok open reading frame (Figure 4A): (50-CTACTTACCGGATTCGTAAGCCATGA- AAGCCGCCACCTCCCTGTGTCCGTCTCTG-30); labelled using cross-linking alkaline phosphatase and chemiﬂuorescent detection systems (Amersham Biosciences). Complex formation and binding kinetics analyses in vitro Sok-RNA and the mature, processed form of hok mRNA were synthesised in vitro using T7 polymerase (Fermentas) and PCR fragment templates generated using sok and hok pri- mers: sok F, (50-AAGAAGATAGCCCCGTAGTAAG-30); sok R, (50-TGTAATACGACTCACTATAGACTAGACAT- AGGGATGCCTC-30) T7 promoter region is underlined; hok F, (50-TGTAATACGACTCACTATAGGGCGCTTGA- GGCTTTCTGC-30) T7 promoter region is underlined; hok R, (50-AAGGCGGGCCTGCG-30). To determine binding kinetics, radioactively labelled Sok-RNA was generated by incorporation of [a-32P]CTP (800 Ci/mmol; Amersham Bio- science) during T7 transcription. Small amounts of labelled RNA ( 3000 c.p.m.) were used to spike non-radioactive Sok-RNA. For binding reactions we mixed hok mRNA (30 nM) with Sok-RNA (2 nM) or PNA (500 nM) with Sok-RNA (38 nM) in TMN buffer [20 mM Tris–OAc pH 7.5, 10 mM Mg(OAc)2, 100 mM NaCl]. Bound and unbound fractions were separated on an 8% polyacrylamide gel containing 7 M urea at 5 V/cm. The radioactive bands were analysed by autoradiography and densitometry using ImageQuant software (Molecular Dynamics). To minimise complex formation during handling prior to electrophoresis, aliquots were immediately diluted into two volumes of load- ing buffer (92% formamide, 17 mM Na2EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue) and loaded onto gels with current applied at each time point. The second- order binding-rate constant (k2) was calculated as described previously (24). Annotation of antisense RNA-regulated toxin genes The fully sequenced genomes of 218 prokaryote organisms were downloaded from the NCBI Web-site and searched for Hok and Ldr protein-encoding genes using the TBLASTN program (25). Hok and Ldr sequences of E.coli K-12 were used as seed sequences (Supplementary Table S1). The coordinates for each candidate and the encoded protein sequences are listed in Supplementary Table S1. The toxin genes were annotated on the genomes using Vector NTI (version 7.0). Nucleic Acids Research, 2006, Vol. 34, No. 20 5917 RESULTS Design of anti Sok PNAs The secondary structure of Sok-RNA has been established experimentally (12,14,26,27). The 64 nt long RNA consists of a stable stem–loop structure and a single-stranded 50-tail (Figure 1). Initially, the 11 nucleotide-long 50-tail of Sok- RNA recognises a single-stranded loop in the truncated, refolded isoform of hok mRNA (12). Therefore, we decided to design antisense PNAs complementary to the 50 end single- stranded tail of Sok-RNA in the anticipation that they could out-compete Sok-RNA binding to hok mRNA and thus induce hok mRNA translation. We designed and synthesized PNAs that have 9 (anti Sok PNA-2), 10 (anti Sok PNA-3) or 11 (anti Sok PNA-1) bases complementary to 50-tail of Sok-RNA. Anti Sok PNAs inhibit cell growth and are bactericidal To test whether competitive inhibition of hok mRNA::Sok- RNA interaction could trigger Hok synthesis in bacteria, anti Sok PNAs were added to cultures of E.coli cells carrying the hok/sok locus. Growth was assessed by monitoring the growth curve as an indicator for cell survival. All three of the anti Sok PNAs inhibited the growth of a hok/sok-carrying strain (CSH50/pPR95). Anti Sok PNA-3 was the most potent of the three anti Sok PNAs (Figure 2A) and no growth was detected in the culture with 10 mM PNA after 20 h, indicating an MIC of 10 mM for PNA-3. To control for possible non-speciﬁc effects of PNAs, such as reduced plasmid copy number, two negative control experiments were included. First the hok/sok-deﬁcient strain (CSH50/pOU82) was grown together with anti Sok PNA and no changes in cell growth were observed, showing that hok/sok genes are needed for inhibition of cell growth. Second, a scrambled PNA was introduced to both strains for detection of any non-speciﬁc effect of PNA chemistry. As expected, cell growth was not affected by these negative controls. Also, previous studies using targeted and non- targeted oligomers indicate that PNAs do not reduce plasmid copy number (22,28). Therefore, the anti Sok PNAs act in a sequence-speciﬁc manner to sequester Sok-RNA and activate Hok toxin synthesis. To assess the extent of cell killing by Hok activity, we treated cells with anti Sok PNAs and determined the number of viable cells over time by scoring the number of c.f.u. The results show that Hok induction using anti Sok PNA almost completely eliminated c.f.u. (Figure 2B). Therefore, hok induction by competitive inhibition of Sok-RNA binding can eliminate the vast majority of viable bacteria within a large E.coli population. For comparison, rifampicin treatment was included in the experiment. Rifampicin was signiﬁcantly less inhibitory than the anti Sok PNAs, even at much higher concentrations (100–200 mM) (Figure 2B). We cannot explain this efﬁciency; however, the results are similar to those reported by two other laboratories (28,29), which show that PNA and morpholino (PMO) antisense oligomers are more potent than ampicillin in E.coli killing assays, both in vitro and in vivo. Also, unpublished and published data (30) indicate that PNA oligomers accumulate in E.coli and are not removed by transporters which efﬂux