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Published as doi: 10.1096/fj.06-7225com.
 (The FASEB Journal. 2007;21:1902-1914.)
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Zorro locked nucleic acid induces sequence-specific gene silencing

Rongbin Ge1, Juhana E. Heinonen, Mathias G. Svahn, Abdalla J. Mohamed, Karin E. Lundin and C. I. Edvard Smith1

Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden

1Correspondence: R. G.: Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86, Stockholm, Sweden. E-mail: ge.rongbin{at}ki.se; or C.I.E.S.: Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86, Stockholm, Sweden. E-mail: edvard.smith{at}ki.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Locked nucleic acids (LNAs) are synthetic analogs of nucleic acids that contain a bridging methylene carbon between the 2' and 4' positions of the ribose ring. In this study, we generated a novel sequence-specific antigene molecule "Zorro LNA", which simultaneously binds to both strands, and that induced effective and specific strand invasion into DNA duplexes and potent inhibition of gene transcription, also in a cellular context. By comparing the Zorro LNA with linear LNA as well as an optimized bisPNA (peptide nucleic acid) oligonucleotide directed against the same target sites, respectively, we found that the Zorro LNA construct was unique in its ability to arrest gene transcription in mammalian cells. To our knowledge, this is the first time that in mammalian cells, gene transcription was blocked by a nucleic acid analog in a sequence-specific way using low but saturated binding of a blocking agent. This offers a novel type of antigene drug that is easy to synthesize.—Ge, R., Heinonen, J. E., Svahn, M. G., Mohamed, A. J., Lundin, K. E., Smith, C. I. E. Zorro locked nucleic acid induces sequence-specific gene silencing.


Key Words: peptide nucleic acid • gene transcription • PNA/LNA • DNA binding • oligonucleotide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ABNORMAL GENE EXPRESSION is associated with many human disorders. Silencing of specific gene expression could have a tremendous effect on the treatment of these diseases. Over the years a series of oligomers has been developed that interfere with gene expression, such as various forms of antisense oligonucleotides and small interfering RNA (siRNA), including antigene strategies.

In earlier studies, it has been shown that triplex-forming oligonucleotides (TFOs) and their derivatives are able to invade the major groove of oligopyrimidineoligopurine regions in double-stranded DNA and interfere with gene transcription initiation and elongation (1 2 3) . However, this Hoogsteen-binding dependent invasion is considered slow and there is room for improvement of the intracellular efficiency of TFOs (4) . Moreover, it was recently reported that nucleotide excision repair machinery is targeted to helical distortions and altered hydrogen-bonding pattern caused by Hoogsteen binding (5) . Certain synthetic polyamides binding to the minor groove constitute an alternative to TFOs (6) . However, further exploration of other Watson-Crick binding reagents could be rewarding. Over the years, a variety of chemically modified small molecules have been developed. Among them, peptide nucleic acid (PNA) and locked nucleic acid (LNA) have shown great promise for a number of applications.

PNA is an oligonucleotide analog with a neutral pseudopeptide backbone consisting of repeating N-(2-amino-ethyl)-glycine units (7) . PNA is capable of sequence-specific recognition of DNA and RNA obeying the Watson-Crick base pairing rules (8) , and the hybrid complexes exhibit extraordinary, thermal stability and unique ionic strength dependency. PNA efficiently recognizes duplex homopurine sequences of DNA, with which they form an extremely stable PNA-DNA-PNA triplex with a looped-out DNA strand (9 10 11) . Strand invasion is most efficient when pyrimidine PNAs are connected by a flexible linker to form a bisPNA in which one PNA strand hybridizes to DNA by Watson-Crick base pairing, while the other binds to the same strand by Hoogsteen mode (10 , 12) . PNA appears to be resistant to both cellular nucleases and proteases (13) . Moreover, it has recently been reported that further optimized PNA (14) , which is extended outside the bis-region, widens the application for strand invasion and inhibits transcription in vitro (14 , 15) . These properties of PNA should be advantageous for use as antigene drugs to inhibit gene activity at the transcriptional level. Based on in vitro cell-free experiments, it is generally considered that triplex invasion complexes are effective inhibitors of transcription initiation as well as transcription elongation, especially when positioned on the template strand (14 15 16 17) . Several interesting studies by Boffa et al. (18 19 20) have reported cellular antigene effects on c-myc transcription of mixed purine-pyrimidine sequence PNAs. Given that linear PNAs show considerably reduced binding affinities as compared to bisPNAs, these results are remarkable. The exact mechanism accounting for the observed effect has, however, not been delineated and the binding stochiometry of PNA to the DNA target was not demonstrated (21) . To this end, a recent report describes the elegant use of antigene PNAs, which target transcriptional start sites, demonstrating that linear PNAs work very efficiently at high PNA to target site ratios (22) .

LNA bases contain a bridging methylene carbon between the 2' and 4' positions of the ribose ring (23 24 25 26 27) . This constraint preorganizes the oligonucleotide backbone thereby increasing the Tm values by as much as 10°C per LNA base replacement. LNA resembles natural nucleic acids with respect to Watson-Crick base pairing. Since LNA bases are introduced by standard DNA/RNA synthesis protocols, the corresponding oligonucleotide can be generated as pure LNA or as mixed LNA/DNA/RNA chimeras. Moreover, LNAs have been demonstrated to be very efficient in binding to complementary nucleic acids and to serve as active, nontoxic antisense agents in vitro and in cultured mammalian cells (28 29 30) as well as in vivo (31) . LNAs have also been used as decoys (32) , aptamers (33) , LNAzymes (34) , agents to attach functional moieties to the plasmid DNA (35) , and DNA correcting agents (36) as well as antigene reagents (4 , 37) .

Because of the favorable binding characteristics of bisPNA, we initially sought to carefully investigate the ability of bisPNA in arresting transcription elongation in cultured mammalian cells. To achieve this, we set out to block RNA polymerase II-mediated transcription of a reporter gene in cultured mammalian cells using a highly optimized PNA hybridizing to the template strand. Our results show that PNA could not efficiently block RNA polymerase II mediated transcription, unless extremely high ratios of PNA:DNA target sites were applied. To explore alternative, efficient antigene constructs, we set out to analyze the effect of LNA. We found that it was possible to generate a novel, potent antigene reagent, "Zorro LNA," that was capable of strong binding to the DNA duplex and that, more importantly, in mammalian cells, induced potent gene silencing in a sequence-specific way.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
The U-2 OS, COS-7, and NIH-3T3 cells were obtained from the American Type Culture Collection (ATCC; HTB-96, Rockville, MD, USA), and the UV4 cell line was kindly provided by Dr. Thomas Helleday (Stockholm University, Stockholm, Sweden). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum and 100 µg/ml of penicillin-streptomycin (Invitrogen, Stockholm, Sweden) at 37°C in a humidified 5% CO2 incubator. NIH-3T3 stably transfected cells carrying the PN252BS plasmid were selected with G418 (1 mg/ml; Invitrogen).

Plasmid constructs
Plasmids with 1, 2, 4, or 6 binding sites (BS), PN251BS, PN252BS, PN254BS, and PN256BS were constructed by standard molecular cloning procedures. DNA fragments were inserted into BglII and AvrII sites of the PN25 plasmid, harboring a destabilized enhanced green fluorescent protein (d2EGFP) reporter driven by the human cytomegalovirus (CMV) early promoter/enhancer elements (38) with the transcription start site located 95 bp upstream of the binding sites and the start codon located 25 bp downstream of the binding sites. The original plasmid was obtained from Dr. Piruz Nahreini (University of Colorado, Boulder, CO, USA). pIRESneo-d2EGFP was constructed by inserting a SmaI/NotI fragment containing the d2EGFP reporter gene into the EcoRV/NotI site of pIRESneo vector (Clontech, BD Biosciences, Stockholm, Sweden), and the plamids with 2 BS, pIRESneo-d2EGFP2BS1, and pIRESneo-d2EGFP2BS2 were generated by inserting 2 BS DNA fragments into SacII or MfeI of pIRESneo-d2EGFP plasmid, respectively.

Transfection
Cells were seeded in six-well plates at a density of 1 x 106 cells per well and allowed to attach for 18 h before transfection. The plasmids PN252BS and PN256BS as well as pIRESneo2BS1 and pIRESneo2BS2 (1 µg) were incubated with 10-fold molar excess of LNA oligomers or 20- or 150-fold molar excess of bisPNA (2.5 µM) in 20 mM phosphate buffer (pH 6.8) at 37°C overnight. Fugene 6 Reagent (Roche Molecular Biochemicals, Stockholm, Sweden) was used to deliver the PNA/LNA-bound plasmid DNA. Transfection solution was prepared according to the manufacturer’s protocol with serum-free medium. Cells were harvested for analysis of protein expression 48 or 72 h after transfection. In cotransfection experiments, 1 µg of plasmid PN256BS was mixed with increasing amount of PNA2582 (90-, 180-, 270-, 360-fold molar excess), or 1 µg of plasmid PN252BS was mixed with increasing amount of Zorro LNA, LNA389 (10-, 100-, 200-fold molar excess), respectively, immediately before transfection, and then the mixture of PNA/LNA and plasmid (50 µl) was transfected into U-2 OS cells (2.5x105 cells per well in 24 well plates in 450 µl of serum-containing medium) by Fugene 6. Cells were harvested for analysis of protein expression 48 h after transfection. All of the experiments were repeated at least three times. Values are presented as the mean of a triplicate ([±]SD) from a representative experiment.

Electroporation
Growing U-2 OS cells (5 x106) were trypsinized, collected by centrifugation, and resuspended in 500 µl of DMEM supplemented with 10% (v/v) heat-inactivated fetal calf serum. Five micrograms of DNA were added, and the mixture was transferred to a 0.4-cm electroporation cuvette (Bio-Rad, Hercules, CA, USA). Electroporation was performed using a Gene Pulser II apparatus and Gene Pulser II RF module (Bio-Rad) at 210 V, 960 µF. Cells were then plated in tissue culture dishes, and 30 min later, they were washed with fresh medium and cultured overnight.

Oligonucleotides, PNA, and LNA
Oligonucleotides containing bisPNA and Zorro LNA binding sites (5'-CAGCGCATGGGTGCCCCTCCTCTTTCTTCA-3' and 5'-TGAAGAAAGAGGAGGGGCACCCATGCGCTG-3') and primers used in this study were synthesized by DNA Technology A/S (Aarhus, Denmark) or Cybergene AB (Huddinge, Sweden) and purified with cartridge purification. PNA, PNA2582, shown in Fig. 1 [Acr-Lys-Lys-CCCCTCCTCTTTCTT-(eg1)3-TTJTTTJTJJ-Lys-NH2, Acr, intercalating 9-aminoacridine; Lys, Lysine] as well as a construct devoid of the aminoacridine moiety was obtained from Professor Peter Nielsen (University of Copenhagen, Denmark). PNA was synthesized as described previously (39) . PNA5171, (n-O-TCAATCTGCACCCATGCGCTGCTG-n), was purchased from BioSynthesis Inc. (Lewisville, TX, USA) and purified with HPLC. A series of Cy-3 or Cy-5 end-labeled, HPLC-purified LNA, shown in Results, as well as control LNA {alpha}SUN-LNAa (cy5-aGACTaCCCTGaGctccaCcacCacCaac; ref 40 ) were purchased from Proligo SAS (Paris, France); LNA bases are in capital letters, and DNA bases are in small letters.


Figure 1
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  		Figure 1. Schematic representation of the target site and sequence hybridized by LNA and PNA oligomers. A) Schematic representation of plasmids PN25, PN251BS, 2BS, 4BS, 6BS. B) PNA2582 and its corresponding target site. C) Zorro LNA construct, in which a 14-mer arm (LNA389) bound to coding strand, while another 16-mer arm (LNA390) bound to template strand with the two arms connected by a bridge containing seven base pairs. D) 27-mer LNA7472 and its corresponding target site, as well as 27-mer LNA7473 and its corresponding target site. Capital letters, LNA; small letters, DNA; Cy-3, Cy-5, labeled probes, Acr, intercalating 9-aminoacridine; Lys, Lysines for enhanced binding affinity; J, pseudoisocytosine and e.g., ethylene glycol (8-amino-3,6-dioxaoctanoic acid) linker units.

PNA binding assay
PNA binding to the target duplex was measured by using a gel mobility shift assay. Two complementary 59-mers containing the PNA binding site were synthesized. The duplex DNA was prepared by mixing both 59-mers at a ration of 1:1 in TE buffer (10 mM Tris/1mM EDTA, pH 8.0) and incubating the solution at 95°C for 5 min and then cooling it down to room temperature slowly. A fixed concentration of duplex DNA or single stranded DNA (59-mers) containing the PNA binding site (1 µM) was incubated with increasing concentration of the PNA in 20 µl of a solution containing 20 mM sodium phosphate at 37°C overnight, respectively. The reactions were analyzed by electrophoresis in a 15% native polyacrylamide gel.

LNA binding assay
Cy-3/Cy-5 labeled LNAs binding to the target sites of plasmid was detected using Molecular Imager FX equipment (Bio-Rad). One microgram of plasmid DNA PN25, PN252BS or PN256BS was mixed with 10-fold molar excess of LNA oligomers or mixed LNA/PNA oligomer in 4 µl phosphate buffer (20 mM, pH 6.8) with the final LNA concentration of 1 µM and incubated at 37°C overnight, respectively. Physiological condition of hybridization: 150 mM phosphate buffer (pH 7.4). This reaction was analyzed by electrophoresis in Novex 4–20% polyacrylamide TBE gels (Invitrogen).

Reverse transcriptase-polymerase chain reaction
Total RNA was prepared using the Qiagen RNA/DNA mini Kit (Qiagen, Stockholm, Sweden), and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using the Qiagen Onestep RT-PCR Kit (Qiagen) and gene-specific primers according to the manufacturer’s protocol. Forward and reverse primers for d2EGFP were 5'-TCAGATCGCCTGGAGACG-3' and 5'-TGTTCTGCTGGTAGTGGTC-3', respectively. GAPDH primers were 5'-GGGTGTGGGCAAGGTCATCC-3' and 5'-TCCACCACCCTGTTGCTGTA-3', respectively (41) . RT-PCR products were analyzed on Novex 4–20% polyacrylamide TBE gels (Invitrogen). The d2EGFP primers amplified a fragment of 680bp (PN25) and 738bp (PN252BS), 858bp (PN256BS), respectively. To the detection of d2EGFP and GAPDH transcripts in the exponential phase of amplification, initial experiments were performed to optimize assay conditions (i.e., number of cycles, primer concentration, and amount of RNA template).

Western blot analysis
Cells were lysed in boiling lysis buffer (2% SDS, 10 mM Tris-HCl, pH 6.8), and protein lysate was fractionated by 4–20% Novex SDS–PAGE gels (Invitrogen) and transferred to nitrocellulose membranes (Advantec MFS, Dublin, CA, USA). The nitrocellulose membrane was incubated with blocking buffer (5% dried milk in PBS and 0.1% Tween-20), probed with a 1:5000 dilution of rabbit anti-GFP antibody (BD Biosciences) followed by goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2000 dilution). Immune complexes were detected with the Supersignal West Femto Chemiluminescence Western blotting detection system (Pierce, Rockford, IL, USA). Anti-SUMO-1 antibody (Invitrogen) was used to detect a heavily 90 kDa sumoylated protein as internal control. Anti-neomycin phosphotransferase II (Biosite, Stockholm, Sweden) was applied to detect neomycin phosphotransferase.

Microinjection and immunofluorescence
The plasmid PN252BS (2 µg) was incubated with, or without, 10-fold molar excess of Zorro LNA (2.5 µM) in 20 mM phosphate buffer (pH 6.8) at 37°C overnight. The LNA-bound plasmid and mock-treated plasmid were adjusted to a concentration of 0.1 µg/µl. NIH-3T3 cells were grown to 50% confluence on 22 mm cover-slips and were coinjected with 0.25 µg/µl pDsRed2-N1 (Invitrogen) and 0.1 µg/µl LNA-bound plasmid or mock-treated plasmid, respectively, into the nucleus using an Eppendorf 5246 Microinjector. Sixteen hours after microinjection, cells were visualized by a Carl Zeiss fluorescence microscope. For microinjection of LNA into NIH-3T3 cells stably carrying the PN252BS plasmid, cells were plated on the coverslips for 16 h; 1 µg/µl Zorro LNA, LNA389 and control LNA was mixed with DsRed2-N1, respectively, and microinjected into the nucleus. Sixteen hours after microinjection, cells were analyzed for fluorescence.

FACS analysis
Cells were preseeded in 6-well plate and transfected as described in the transfection session. Forty-eight hours after transfection, cells were harvested and analyzed by FACSCalibur flow cytometer (BD Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Efficient binding of PNA to a linear DNA target sequence
PNA in the form of bisPNA is one of the most potent reagents available for efficient strand invasion recognizing homopurine sequences in the DNA duplex, thereby forming extremely stable PNA-DNA-PNA triplexes (9 10 11) . To examine PNA binding to a homopurine target sequence, a gel mobility shift assay was applied (Supplemental Fig. S1A).

Increasing amounts of PNA were incubated with fixed concentrations of duplex target DNA. As the concentration of the specific PNA, PNA2582, was augmented, the proportion of the target duplex bound increased, as manifested by the reduced mobility of the PNA-DNA complex. At a concentration of 5 µM (PNA:DNA ratio 5:1), essentially all of the 59-mer target oligos were shifted yielding two fragments with slower mobility. We do not know the exact composition of these two species, but a modest degree of stacking may exist on the lower-mobility form. From a PNA concentration of 20 µM, a blurred pattern was observed with the 59-mer target oligos displaying further reduced mobility. This result may be due to the formation of big complexes between DNA and PNA, perhaps due to stacking (42) . Very weak binding was detected to the control double-stranded 59-mer DNA oligos lacking target sequences (iDNA), only visible at the highest PNA concentration of 30 µM (Supplemental Fig. S1A). With the use of a single-stranded target oligo, all of the DNA was shifted at the expected ratio of 1:1 (Supplemental Fig. S1B).

These results suggest that PNA can induce effective strand invasion and form highly stable triplexes at modest PNA to DNA target site ratios, as manifested by the binding to a linear DNA duplex, and that PNA invasion into the DNA duplex is strictly concentration dependent. Under these conditions, which were mainly used to verify sequence-specific binding of the PNA, the PNA:DNA ratio was modest, at the maximum 30:1. We believe that formation of complexes was favored by the relatively high, final concentration applied when hybridizing to the DNA oligonucleotide together with the strong binding efficacy of this optimized PNA.

Effect of bisPNA triplex on destabilized green fluorescence protein expression in mammalian cells
To determine if the optimized bisPNA could arrest transcription elongation in mammalian cells, as was previously reported for in vitro transcription (14 , 15) , PNA was bound to anchor sites generated in enhanced GFP (EGFP) reporter plasmids and transfected into cultured human U-2 OS cells. Due to the high stability of EGFP protein, we used a destabilized form, d2EGFP, with a half-life of only 2 h (43) .

Plasmids were generated to contain a single, or multiples, of 2, 4, or 6 homopurine stretches of 15 nucleotides each, introduced at BglII and AvrII restriction sites located between a CMV promoter and the d2EGFP reporter gene (Fig. 1A ). The homopurine stretches served as anchor sites for the aminoacridine-containing, optimized PNA2582. The constructed PN25, PN252BS, PN256BS plasmids (1 µg), which are depicted in Fig. 1A , were mixed with 20-fold molar excess of PNA2582 per binding site and incubated at 37°C overnight to form the PNA-DNA-PNA triplex (Fig. 1B ). PNA-bound plasmid and mock-treated plasmid were transfected into the U-2 OS cells. The intensity of the d2EGFP protein expression was monitored by fluorescent microscopic analysis after 2 days of culture (Fig. 2 A). No decrease in GFP expression was detected in U-2 OS cell transfected with plasmids containing 1, 2, or 6 anchor sites that were pretreated with 20-fold molar excess of PNA2582 (Fig. 2A and data not shown). Interestingly, the lack of an effect was observed despite that the gel mobility shifts of restriction enzyme (HindIII) digested plasmids proved that essentially all target sites on the plasmid were occupied by PNA2582 (Supplemental Fig. S1C, right panel, and data not shown).


Figure 2
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  		Figure 2. Effect of PNA triplex on destabilized green fluorescence protein expression. A) Intensity of the d2EGFP protein expression after transfection of PNA-bound plasmid or mock-treated plasmid, respectively. 1) Mock-treated PN252BS; 2) prehybridized PN252BS with 20-fold molar excess of PNA; 3) PN256BS; 4) prehybridized PN256BS with 20-fold molar excess of PNA; 5) prehybridized PN256BS with 150-fold molar excess of PNA; 6) (Mock-treated PN25, 7) prehybridized PN25 with 150-fold molar excess of PNA. B) Detection of expression of d2EGFP and NeoR gene by Western blot. Sumoylated RanGAP was used as internal control. C) d2EGFP mRNA level was determined by RT-PCR, (20) 20-fold molar excess, (150) 150-fold molar excess. D) FACS analysis of d2EGFP expression after transfection of PNA-bound plasmid PN256BS and mock-treated plasmid into cells, respectively. Bars show inhibition in d2EGFP expression after transfection of PNA-bound plasmids PN25, PN251BS, 2BS, 4BS, and 6BS into cells, respectively. E) Densitometer analysis of inhibition in d2EGFP expression after cotransfection of plasmid PN256BS and increasing amount of PNA.

In contrast, significant down-regulation in d2EGFP protein expression was observed in U-2 OS cells transfected with the PN256BS plasmid, which was pretreated with 150-fold molar excess of PNA2582 per binding site (Fig. 2A , panel 5). In addition, the level of d2EGFP mRNA was assessed by RT-PCR. The mRNA level was equally reduced by ~90% in cells transfected with the PN256BS plasmid, which was pretreated with 150-fold molar excess of PNA2582 per binding site, whereas there was no significant decrease in level of d2EGFP mRNA detected in cells transfected with the PN256BS plasmid, which was pretreated with 20-fold molar excess of PNA2582 per binding site. The level of GAPDH mRNA indicated equal RNA loading (Fig. 2C ). Since the molar ratio of PNA:plasmid in this case was 900:1, this phenomenon is likely due to formation of big complexes around the multiple binding sites caused by the presence of a very high excess of PNA. To investigate the influence of the number of binding sites for the inhibition of gene expression, we studied plasmids with 1, 2, 4, or 6 PNA anchor sites to observe the effect of the optimized PNA, PNA2582. All plasmids were prehybridized with 150-fold molar excess of PNA2582 per binding site, and then the PNA-bound plasmids were transfected into U-2 OS cells. After 48 h of culture, the intensity of the d2EGFP protein expression was monitored by FACS analysis (Fig. 2D and data not shown). Densitometer measurements revealed that the plasmid carrying 6 binding sites was inhibited by 85% in d2EGFP expression. As the number of binding sites decreased, the degree of inhibition became reduced (4BS plasmid 65% inhibition, 2BS plasmid 40% inhibition, 1BS plasmid ≤20% inhibition; no decrease in expression of d2EGFP observed on control plasmid, PN25) (Fig. 2D ). Subsequently, we cotransfected plasmid PN256BS with increasing amounts of PNA2582. After 48 h of culture, the d2EGFP protein expression was monitored by Western blot analysis. Densitometer measurements revealed that a 360-fold molar excess of PNA caused maximal inhibition of d2EGFP (90%), whereas as the amount of PNA decreased, the efficiency of inhibition was reduced (Fig. 2E ). Similar results were seen in NIH-3T3 and COS-7 cell lines as well (data not shown).

To define the specific effect of PNA on transcription, we also measured the expression level of the neomycin resistance gene located in cis and driven by the long terminal repeat (LTR) promoter (Fig. 1A ). This analysis demonstrated that PNA did not affect transcription of the adjacent neomycin resistance gene (Fig. 2B ).

Effect of binding of Zorro LNA to DNA
Because PNA did not block transcription of reporter genes when target site binding was saturated at PNA:DNA ratios of 20:1, we set out to analyze the effect of another nucleic acid analog, namely LNA. Our previous experience using LNA had taught us that, compared to bisPNA, LNA binds less well to double-stranded DNA (44 ; unpublished data). For this reason, we initially tested a novel type of construct, which simultaneously binds both strands of a DNA duplex (Fig. 1C ). Due to the shape of this oligomer, forming a Z-like structure, and to avoid any confusion with the left-handed, Z-form of DNA, we designated these constructs "Zorro LNA." Moreover, it is our experience that hybridization of LNA to DNA causes less of retardation in gel mobility shift assays as compared to the noncharged PNA. For this reason, all of the LNAs, which we used in this study, were end-labeled with Cy3 or Cy5 fluorophores to simplify their detection. The plasmid PN25 and PN252BS (5 µg) were mixed with 10-fold molar excess of Zorro LNA, formed by the 14-mer LNA389 together with the 16-mer LNA390. As controls, 27-mer linear LNAs, designated LNA7472 and LNA7473 (Fig. 1D ), were included. LNA-bound plasmids and control plasmids were run on 4–20% polyacrylamide TBE gels (Fig. 3A ). Supercoiled plasmid was stained by SYBR Gold (Fig. 3 A, panel 1). Exposure for Cy5 revealed that binding induced by Zorro LNA was much more powerful than that induced by one of the linear Zorro arms, LNA390, (16-mer, lower half of Zorro LNA), whereas on control plasmid PN25, lacking binding sites, only very weak unspecific binding by Zorro LNA was seen (Fig. 3A , panel 2). Exposure for Cy3 revealed that binding induced by Zorro LNA was also stronger than that induced by the other arm, LNA389, (14-mer, upper half of Zorro LNA). Likewise, only very weak unspecific binding of Zorro LNA to the control plasmid was seen when Cy3 was analyzed (Fig. 3A , panel 3).


Figure 3
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  		Figure 3. DNA duplex binding of Zorro LNA compared to other constructs. A) Comparison of binding efficiency of Zorro LNA and other different LNA constructs. 1) Signals from supercoiled plasmid stained with SYBR Gold (2). Signals from LNA labeled by Cy5 probe (3). Signals from LNA labeled by Cy3 probe. Densitometer analysis of binding efficiency of Zorro LNA and LNA389/390. B) Densitometer analysis of binding efficiency induced by Zorro LNA and PNA/LNA hybrid. Right panel, schematic representation of Zorro PNA5171/LNA390. C) Densitometer analysis of binding efficiency of Zorro LNA and Blocked Zorro LNA. Right panel, schematic representation of Blocked Zorro LNA. D) Comparison of binding efficiency of Zorro LNA on plasmid PN251BS and PN252BS, (10) 10-fold molar excess; (40) 40-fold molar excess.

To distinguish between the possibilities that the enhanced signal in duplex DNA binding by Zorro LNA is due to the characteristic Zorro structure or caused by the longer hybridization region, to which both halves of the Zorro contribute, we used two linear 27-mer control LNAs, antisense LNA7472 and sense LNA7473, respectively (Fig. 1D ). We found that binding of these extended, single-stranded LNA 27-mers, is also very effective. Although, as seen in Fig. 3A , we noticed that antisense 27-mer linear LNA7472 gave a stronger signal, as compared to sense 27-mer linear LNA7473 signal (Fig. 3A , panels 2, 3), this is most likely because the LNA7473 contained a lower amount of fluorophore label (data not shown).

Binding of Zorro PNA-LNA hybrids to a DNA duplex
To compare the hybridizing ability of Zorro LNA to that of similarly shaped PNA, we synthesized the same two arms based on PNA chemistry. To promote solubility, we added 2 asparagine residues in the N terminus (5' end). Asparagine rather than lysine was used to avoid the influence of charge interactions, which may favor hybridization to the plasmid thereby violating the comparison with LNA, which is negatively charged. Conversely, the putative interaction between the positively charged lysine residues and the negatively charged LNA may instead impair its strand-invading capacity. However, despite that each PNA-arm was soluble, when mixed to allow for the bridge to form by hybridization, we observed that the solubility decreased considerably, a phenomenon not infrequently seen when using long PNA sequences (21) .

We therefore investigated a hybrid form of LNA-PNA Zorro construct for binding in which one arm (LNA389) in Zorro LNA was substituted by a PNA arm (PNA5171) with identical sequence (Fig. 3B , right panel). The analysis showed that both Cy5 and Cy3 signals became much weaker, if one arm of Zorro LNA (LNA389) was replaced by PNA5171 (Fig. 3B ). This result could be because the melting temperature of DNA/PNA hybrids is lower than that of DNA/LNA (45) .

To understand the importance of the bridge structure between the two LNA arms for binding, two 7-mer LNAs (LNA71 and LNA72; Fig. 3C , right panel) were used to block the mutual binding of two LNA arms of Zorro LNA. This experiment also showed that both Cy5 and Cy3 signals became much weaker, if the bridge structure of Zorro LNA was blocked (Fig. 3C ).

The hybridization kinetics of Zorro LNAs to plasmids was also investigated (Supplemental Fig. S2). Eight µg of plasmid PN252BS (corresponding to 3.2 pmol LNA binding sites) was hybridized at 1:1 (red dots) or with 10-fold (blue dots) molar excess of Zorro LNA and incubated at 37°C for increasing time periods (0, 5, 15 min and 1, 3, 5, 8, 12, 16, and 30 h). The LNA binding was analyzed by electrophoresis as described in Materials and Methods, and the amount of bound LNA was estimated against a standard curve. The data showed that short-time incubation with 10-fold molar excess of LNA, or at the 1:1 ratio at any time point, was not enough to saturate the binding sites; overnight incubation (16 h) at the 10:1 ratio was required to saturate the binding. We believe that a potential explanation for this is that there is a need for two adjacent Zorro LNAs to bind in order to obtain facilitated strand invasion and stable hybridization.

To evaluate the binding efficiency of Zorro LNA in vivo, we also tried to mimic physiological conditions during hybridization. One microgram of plasmid PN252BS or control plasmid PN25 was hybridized with 10-fold molar excess of Zorro LNA, LNA389, LNA390, respectively, in 150 mM sodium phosphate buffer (pH 7.4) and incubated at 37°C overnight. The reaction was analyzed by electrophoresis in 4–20% polyacrylamide TBE gels (Supplemental Fig. S3). Exposure of Cy5 and Cy3 signals revealed that binding to Zorro LNA was not significantly different under physiological conditions.

We also attempted to analyze hybridization of plasmids carrying a single anchor site with Zorro LNA. The exposure of Cy5 and Cy3 showed that binding induced by 10-fold molar excess of Zorro LNA was rather weak. Instead, 40-fold molar excess of Zorro LNA saturated the single anchor site, whereas it did not significantly induce unspecific binding on the control plasmid PN25 (Fig. 3D ).

In summary, the binding activity to the DNA duplex of a 14-mer, or a 16-mer, linear LNA is quite limited, as compared to Zorro LNA or 27-mer linear LNAs under both low salt and physiological conditions. Densitometer measurements revealed that binding induced by Zorro LNA was 10-fold more efficient than that induced by LNA390 and 7-fold more efficient than that induced by LNA389 (Fig. 3A ). Moreover, if one arm of Zorro LNA was substituted by a PNA oligonucleotide, the effect of binding to a DNA duplex decreased significantly. In addition, if the linkage of the bridge structure between the two LNAs of Zorro LNA was blocked, binding decreased significantly. Finally, high molar excess of Zorro LNA was required to saturate plasmids carrying a single anchor site.

Inhibition of destabilized green fluorescence protein caused by Zorro LNA
To investigate if LNA could serve as an antigene reagent to block gene transcription in a sequence-specific way, we further studied the effect of the novel Zorro LNA construct on the expression of d2EGFP protein in human osteosarcoma cells. The PN252BS plasmid (1 µg) was prehybridized with 10-fold molar excess of Zorro LNA or with LNA389, LNA390, LNA7472, or LNA7473, respectively, in 20 mM phosphate buffer at 37°C overnight. The Zorro LNA-bound plasmid, mock-treated plasmid and other control samples were transfected into preseeded U-2 OS cells. After 48 h, cells were harvested and d2EGFP protein expression was measured by Western blot of cellular lysates. Densitometer measurements revealed a ≥90% specific inhibition of d2EGFP protein expression in the Zorro LNA-bound plasmid. No significant decrease in d2EGFP protein expression was seen using mock-treated plasmid or any of the other LNA constructs (Fig. 4 A). The same results were achieved when analyzing transfected cells by a fluorescence microscope (Fig. 4B ). To define the specific effect of LNA on transcription, we also measured the expression level of the neomycin resistance gene, located in cis and driven by LTR promoter (Fig. 1A ). Zorro LNA did not affect transcription of this gene (Fig. 4C ). In addition, the level of d2EGFP mRNA was assessed by RT-PCR. The mRNA level was equally reduced by ≥90% in cells transfected by Zorro LNA bound PN252BS plasmid, whereas there was no significant decrease in level of d2EGFP mRNA detected in cells transfected by PN25 control plasmid treated with Zorro LNA. The level of GAPDH mRNA indicated equal RNA loading (Fig. 4D ). Similar results were seen in the fibroblast cell line NIH-3T3 (data not shown).


Figure 4
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  		Figure 4. Inhibition of destabilized EGFP expression caused by Zorro LNA. A) Expression of d2EGFP protein after transfection of prehybridized plasmids PN25 and PN252BS with different LNA constructs and mock-treated plasmids into U-2 OS, respectively. Densitometer analysis of inhibition in d2EGFP protein expression. B) Imaging of cells transfected as described in A. C) Detection of NeoR protein expression. D) d2EGFP mRNA level as determined by RT-PCR. E) Densitometer analysis of inhibition of d2EGFP expression induced by 10-fold molar excess of Zorro LNA and Zorro PNA/LNA hybrids, respectively. F) Densitometer analysis of inhibition of d2EGFP expression induced by 10-fold molar excess of Zorro LNA and Blocked Zorro LNA, respectively. G) Densitometer analysis of inhibition of d2EGFP expression on the plasmids PN252BS and PN251BS induced by Zorro LNA; (10) 10-fold molar excess, (40) 40-fold molar excess.

We also compared the ability of Zorro LNA with that of a Zorro PNA-LNA hybrid (Fig. 3B , right panel) for inhibition of gene transcription in osteosarcoma cells. The plasmids PN25 and PN252BS were prehybridized with Zorro LNA or Zorro PNA-LNA hybrids, respectively, and subsequently transfected into U-2 OS cells. After 48 h, cells were harvested and analyzed by Western blot. Densitometer measurements revealed that a pronounced decrease in expression of d2EGFP protein was induced by Zorro LNA, whereas Zorro PNA-LNA hybrids only induced 30% decrease in expression of d2EGFP (Fig. 4E ). No significant decrease in protein expression was observed on the mock-treated plasmid.

Densitometer measurements revealed a pronounced decrease in expression of d2EGFP protein by Zorro LNA, whereas Zorro LNA in which the formation of a bridge was inhibited (Fig. 3C , right panel) only induced 20–30% decrease in expression of d2EGFP (Fig. 4F ), in concordance with the results from the binding analysis.

Moreover, to investigate the effect of Zorro LNA on plasmids carrying a single binding site, we also compared the effect of Zorro LNA on the plasmids PN252BS (two binding sites) with the effect on PN251BS (a single binding site) for inhibition of gene transcription in U-2 OS cells. We found that the inhibitory effect of Zorro LNA on the plasmid PN251BS was quite limited. Although we found that binding to plasmid PN251BS can be saturated by 40-fold molar excess of Zorro LNA (Fig. 3D ), there was no evident down-regulation detected on plasmid PN251BS, whereas the effect on the plasmid PN252BS was significant (Fig. 4G ).

Subsequently, we cotransfected unhybridized plasmid PN252BS with increasing amounts of Zorro LNA or LNA389, respectively. After 48 h of culture, the d2EGFP protein expression was monitored by Western blot analysis (Fig. 5 A). Densitometer measurements revealed that whereas 10-fold molar excess of Zorro LNA, or LNA389, did not cause inhibition of d2EGFP expression, 100-fold molar excess of Zorro LNA induced 70% down-regulation while antisense LNA389 only induced 25% down-regulation. If the amount of Zorro LNA, or LNA389, was increased up to 200-fold molar excess, 90% down-regulation of d2EGFP protein was induced by Zorro LNA, whereas only 40% down-regulation was measured on exposure to antisense LNA389. On the control plasmid PN25, there was no significant unspecific down-regulation detected (Fig. 5B ). Importantly, in contrast to the case for PNA, we found no evidence for the formation of supramolecular complexes using 200-fold excess of Zorro LNA (Fig. 5C ). This further supports the notion that PNA and LNA oligomers interact with dsDNA in different ways.


Figure 5
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  		Figure 5. In vivo effect of Zorro LNA on d2EGFP gene expression. A) Expression of d2EGFP protein after cotransfection of Zorro LNA, LNA389 and plasmids PN25, PN252BS, as well as mock-treated plasmids into U-2 OS cells, respectively (10). 10-fold molar excess, (100) 100-fold molar excess, (200) 200-fold molar excess. B) Densitometer analysis of inhibition of d2EGFP expression induced by LNA389 and Zorro LNA, respectively. C) Gel mobility experiment showing plasmid PN252BS untreated (left lane), incubated with 200-fold molar excess of PNA (middle lane) or 200-fold molar excess of LNA (right lane).

To explore the mechanism of inhibition induced by Zorro LNA, we transferred the binding sites either into an intron or upstream of the CMV promoter (Supplemental Fig. S4A). The Zorro LNA-bound plasmids pIRESneo-d2EGFP2BS1 and pIRESneo-d2EGFP2BS2 were tranfected into U-2 OS cells, respectively. We found that the d2EGFP expression was significantly reduced by Zorro LNA, if the LNA binding sites were located in the intron, whereas the d2EGFP expression was unaffected, if the binding sites were located upstream of the promoter (Supplemental Fig. S4B). This demonstrates that Zorro LNA inhibited transcription elongation when bound to an intron, whereas, as expected, an upstream location did not influence expression.

Microinjection or electroporation yields similar results as cationic lipid transfection
To rule out the possibility that either cellular uptake or nuclear delivery could be affected by binding of Zorro LNA, we made intranuclear microinjection of prehybridized PN252BS with Zorro LNA into NIH-3T3 cells. Sixteen hours after injection of LNA-bound PN252BS plasmid and mock-treated plasmid, d2EGFP gene expression was determined (Fig. 6 A–D). The intensity of d2EGFP gene expression was significantly decreased in the cells, which were injected with Zorro LNA-bound plasmid (Fig. 6C ) as compared to mock-treated plasmid (Fig. 6A ). In all cases, control plasmid, pDsRed2-N1, was coinjected as a marker (Fig. 6B, D ).


Figure 6
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  		Figure 6. Zorro LNA-mediated block of d2EGFP gene expression following microinjection. Detection of d2EGFP expression after intranuclear injection of LNA-bound PN252BS plasmid and mock-treated plasmid. A, B) NIH-3T3 cells injected with mock-treated plasmid PN252BS. C, D) NIH-3T3 injected with Zorro LNA-bound PN252BS. Zorro LNA induced inhibition of stably integrated d2EGFP gene expression. Detection of d2EGFP expression in stable NIH-3T3 after intranuclear injection of Zorro-LNA; E, F) single-stranded antisense LNA389; G, H) or a control LNA, I, J), respectively. In all cases, pDsRed2-N1 was coadministered to identify injected cells. d2EGFP fluorescence in a few representative cells is shown to the left and DsRed2 fluorescence is shown to the right.

To further rule out the possibility that cationic lipid transfection reagent could interfere with transfection efficacy of PNA-bound plasmid or destabilize the PNA/DNA triplex, we also attempted an alternative transfection method, namely electroporation (Supplemental Fig. S5). The intensity of d2EGFP gene expression was significantly decreased in the cells transfected with Zorro LNA-bound plasmid (Supplemental Fig. S5C), whereas there was no significant inhibition detected in the cells transfected by PNA-bound plasmid (Supplemental Fig. S5B), comparing with the intensity of d2EGFP expression in the cells transfected by mock-treated plasmid (Supplemental Fig. S5A). The results were consistent with previous transfection data made by cationic lipid transfection reagent.

DNA repair is not removing DNA-bound PNA
A possible explanation for the failure of saturated PNA binding from blocking transcription in living cells is that the PNA is removed during transcription, and another possibility is that the DNA repair machinery recognizes the altered structure and excises this portion of the plasmid. To investigate if PNA forming triplexes could activate DNA repair mechanism in the cells, PNA or Zorro LNA bound reporter gene plamids were transfected into UV4 cells, respectively (Supplemental Fig. S6). No decrease in d2EGFP protein expression was detected using plasmids prehybridized with 20-fold molar excess of bisPNA 2582, whereas gene silencing was readily seen in the sample using the Zorro LNA-bound plasmid. According to gel shift analysis, 20-fold molar excess of PNA was enough to saturate target sites on the plasmid (Supplemental Fig. S1C).

Thus, no inhibition of protein expression could be observed in several mammalian cell lines of different origins, including UV4 cells, which are defective in nucleotide excision repair. Although inhibition could be seen in cells transfected with PN256BS when prehybridized in 150-fold molar excess of PNA, this phenomenon could be because supramolecular complexes are formed around the multiple binding sites in this plasmid.

Zorro LNA induces inhibition of stably integrated d2EGFP gene expression
To investigate the possibility of inhibiting endogenous gene expression in mammalian cells with Zorro LNA, we established NIH-3T3 cells stably carrying the PN252BS plasmid. The NIH-3T3 stable cell lines were microinjected with Zorro LNA, antisense LNA389, or control LNA, respectively. Sixteen hours after microinjection, the fluorescence was measured (Fig. 6E –J). In a majority of microinjected cells (88%), expression from a stably transfected destabilized EGFP gene was inhibited by Zorro LNA (Fig. 6E ), as compared to 28% of cells injected with antisense LNA389 (Fig. 6G ), whereas no cells were affected by control LNA (Fig. 6I ). In all cases, pDsRed2-N1 was coinjected as a marker (Fig. 6F, H , J).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we generated a novel sequence-specific antigene reagent "Zorro LNA" in which a 14-mer LNA oligonucleotide binds to the coding strand, while a connected 16-mer LNA binds to the opposite template strand. We have verified that this takes place in a sequence specific manner and that potent gene down-regulation (≥90%) in cultured mammalian cells was induced. When comparing the Zorro LNA to 16-mer or 27-mer linear LNA oligomers, or to the extremely stable PNA:DNA-PNA triplex, we found that the Zorro LNA construct was unique in efficiently blocking RNA polymerase II-dependent gene transcription in mammalian cells in a sequence-specific way. We also found that the linear LNA7473 oligonucleotide did not induce inhibition of d2EGFP expression, even at 200-fold molar excess (Supplemental Fig. S7). This suggests that the formation of a duplex structure on the transcribed strand is not sufficient to induce gene down-regulation. Moreover, we also showed that Zorro LNA inhibited transcription elongation when bound to an intron. Moreover, the inhibition of expression induced by Zorro LNA was specific, since it only affected the gene expression that is in the same expression cassette (Fig. 4C ; Supplemental Fig. S4).

When replacing one of the arms of Zorro LNA with PNA, we found that the Zorro PNA-LNA hybrid only induced 30% inhibition in gene transcription, which is consistent with reduced ability for plasmid hybridization. Thus, binding induced by Zorro LNA oligomer was much more efficient (Fig. 3B ). This result could be because the ability of strand invasion induced by linear PNA is weaker than that induced by linear LNA (45) . To this end, it may well be that Zorro bisPNA constructs could be even more powerful in strand invasion and inhibition of gene transcription than Zorro LNA. However, bisPNA only efficiently recognizes homopurine sequence in the DNA duplex, strongly restricting its versatility. In contrast, LNA binds very well to mixed DNA sequences, so even if two adjacent target sites are needed for efficient inhibition finding suitable genomic anchor sequences does not impose any limitation. When a homopurine target sequence is available, a Zorro bisPNA hybrid might also be highly efficacious, provided that the solubility of the construct is adequate.

We also attempted to analyze hybridization of plasmids carrying a single anchor site with Zorro LNA. We found that high molar excess of Zorro LNA was required to saturate a single anchor site (Fig. 3D ). Interestingly, after transfection of Zorro LNA-bound plasmid PN251BS into cells, there was no significant inhibition detected (Fig. 4G ). To this end, we and others have previously reported on facilitated hybridization when accurately spaced PNA peptides are combined (46 47 48) and since LNA can enhance the binding of PNA (44) , it seems likely that adjacently hybridized LNA species could facilitate each other’s binding. Moreover, although we currently do not fully understand the reason for this result, it provides clues for further experimentation and could prove to be significant for the general understanding of transcriptional repression by oligonucleotides. In a chromosomal context, where two adjacent, identical binding sites may be lacking, this limitation can easily be overcome by generating two different Zorro constructs, which bind to proximal target sites.

It was also investigated whether Zorro LNAs could inhibit DNA replication either in bacteria or in mammalian cells harboring plasmids containing origins of replication. We found no evidence for any major effect (unpublished observations).

We also evaluated the biological effect of the novel Zorro LNA in the cellular context in the absence of prehybridization. From our studies, we found that the 100-fold molar excess of Zorro LNA constructs inhibited d2EGFP expression by 75%, 200-fold molar excess (0.16 µM) inhibited d2EGFP expression by 90%, whereas antisense LNA389 only inhibited gene expression by 25 and 40%, respectively (Fig. 5) . A maximal transcriptional inhibition of 75% was reported by Brunet et al., using 0.1 µM of acridine-conjugated TFOs (4 , 37) , whereas increasing the dose further did not enhance the effect.

Most interestingly, in our studies, we found that in a majority of microinjected cells (88%), expression from a stably transfected destabilized EGFP gene was inhibited by Zorro LNA (Fig. 6E ), as compared to 28% of cells injected with antisense LNA389 (Fig. 6G ), whereas no cells were affected by control LNA (Fig. 6I ). This argues in favor of that Zorro-LNA can find its way to an endogenous target (in vivo).

Moreover, from our results, we conclude that although PNA oligomers are potent in strand invasion, and albeit they are known to form extremely stable PNA:DNA:PNA triplexes with a looped-out DNA strand, perturbing gene transcription in vitro in cell-free experiments (14 15 16) , a highly optimized PNA oligonucleotide could not efficiently block RNA polymerase II dependent expression in cultured mammalian cells. Only when extremely high ratios of PNA:DNA target sites were applied, suppressed expression could be observed. We do not know the underlying mechanism, but we have ruled out the possibility that one form of DNA excision repair mechanism interferes with PNA induced gene silencing. Furthermore, from in vitro cell-free experiments, it was reported that PNA could induce gene transcription by strand displacement, thus mimicking an artificial promoter (49) . However, to date, we are not aware of any convincing data showing that PNA induces gene transcription by simply increasing the accessibility of RNA polymerases, through the displacement of the two DNA strands as an artificial promoter. One study reported that PNA induced human gammaglobin gene expression in vivo (50) , but we noticed that the anchor site for PNA binding in that paper overlapped with a binding site for a transcriptional repressor, Oct-1, thus offering an alternative explanation.

We thus suggest that high molar excess of PNA induces inhibition of expression due to the formation of a supramolecular complex restricting access to the transcriptional machinery, and as the number of binding sites decreased or the amount of PNA decreased, the degree of inhibition became reduced (Fig. 2D, E ). In previous studies, Boffa et al., showed cellular antigene effects on c-myc transcription of mixed purine-pyrimidine sequence PNA (19) . These authors observed 75% inhibition of gene expression, when cells were exposed to high dosage of PNA (10 µM), whereas as the amount of PNA decreased, the efficiency of inhibition was reduced. Similarly, Diviacco et al., demonstrated that a PNA triplex inhibited DNA replication efficiently (51) . Although these are highly interesting findings, also this phenomenon only occurred when large excess of PNA was applied and this was also the case in the recent report by Janowski et al. (22) . These results are thus consistent with our data.

Thus, if supramolecular complexes are formed, large aggregates of PNA bound to DNA may restrict access to both the replicative and transcriptional machineries. Since we have found that this phenomenon does not take place in control plasmids, devoid of anchor sites for PNA, it suggests that the specific PNA anchor sequence acts as a nucleation site, which initiates this process. However, irrespective of the details of this phenomenon we have been able to develop a novel form of antigene drug, designated "Zorro LNA" that is easy to synthesize and that could hopefully serve as a versatile tool not only for gene silencing, but also for other aspects of efficient targeting to DNA. We are presently working on the generation of constructs, which more efficiently inhibit the expression of endogenous genes in chromosomal DNA. Although they are based on the novel findings presented in this study they are constructed to have additional features in order to enhance their targeting capability.


   ACKNOWLEDGMENTS
 
This work was supported by the European Union grants "Novel gene drug strategy based on hybridization of novel functional entities to plasmids" (QLRT-2001–01997) and "Nucleic acid based nanostructures" (NMP4-CT-2004–013775). Moreover, we also appreciate the support from the Wallenberg Foundation, the Swedish Science Council, Aroseniusfonden, Sigurd, Elsa Golje Memorial Foundation, and the Swedish Foundation of Strategic Research Bio-X grant. We are indebted to Professor Peter E. Nielsen for synthesizing the PNA oligomers used in this study and Dr. H. Jose Arteaga for critical reading of the manuscript.

Received for publication August 28, 2006. Accepted for publication January 11, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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