Peptide Nucleic Acid Binding Research Articles

Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA.

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA9) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA8) targeting a stem-loop. PNA9, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA9 (F-PNA13). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA9 and F-PNA13, but not PNA8 highlighting the importance of target site selection. Surprisingly, F-PNA13 which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.

Triplex-forming peptide nucleic acids as emerging ligands to modulate structure and function of complex RNAs.

Over the last three decades, our view of RNA has changed from a simple intermediate supporting protein synthesis to a major regulator of biological processes. In the expanding area of RNA research, peptide nucleic acid (PNA) is emerging as a promising ligand for triple-helical recognition of complex RNAs. As discussed in this feature article, the key advantages of PNAs are high sequence specificity and affinity for RNA (>10 fold higher than for DNA) that are difficult to achieve with small molecule ligands. Emerging studies demonstrate that triple-helical binding of PNAs can modulate biological function and control dynamic conformational equilibria of complex folded RNAs. These results suggest that PNA has a unique potential as a research tool and therapeutic compound targeting RNA. The remaining problems hampering advances in these directions are limitations of sequences that can be recognized by Hoogsteen triplexes (typically purine rich tracts), poor cellular uptake and bioavailability of PNA, and potential off-target effects in biological systems. Recent exciting studies are discussed that illustrate how synthetic nucleic acid chemistry provides innovative solutions for these problems.

Electrochemical sensor based on Fe3O4/α-Fe2O3@Au magnetic nanocomposites for sensitive determination of the TP53 gene

Considering the high cost and tedious process of gene sequencing, there is an urgent need to develop portable and efficient sensors for the TP53 gene. Here, we developed a novel electrochemical sensor that detected the TP53 gene using magnetic peptide nucleic acid (PNA)-modified Fe3O4/α-Fe2O3@Au nanocomposites. Cyclic voltammetry and electrochemical impedance spectroscopy confirmed the successful stepwise construction of the sensor, especially the high-affinity binding of PNA to DNA strands, which induced different electron transfer rates and resulted in current changes. Variations in the differential pulse voltammetry current observed during hybridization at different surface PNA probe densities, hybridization times, and hybridization temperatures were explored. The biosensing strategy obtained a limit of detection of 0.26 pM, a limit of quantification of 0.85 pM, and a wide linear range (1 pM–1 μM), confirming that the Fe3O4/α-Fe2O3@Au nanocomposites and the strategy based on magnetic separation and magnetically induced self-assembly improved the binding efficiency of nucleic acid molecules. The biosensor was a label-free and enzyme-free device with excellent reproducibility and stability that could identify single-base mismatched DNA without additional DNA amplification procedures, and the serum spiked experiments revealed the feasibility of the detection approach.

Triple-Helical Binding of Peptide Nucleic Acid Inhibits Maturation of Endogenous MicroRNA-197.

Sequence specific recognition and functional inhibition of biomedically relevant double-helical RNAs is highly desirable but remains a formidable problem. The present study demonstrates that electroporation of a triplex-forming peptide nucleic acid (PNA), modified with 2-aminopyridine (M) nucleobases, inhibited maturation of endogenous microRNA-197 in SH-SY5Y cells, while having little effect on maturation of microRNA-155 or -27a. In vitro RNA binding and Dicer inhibition assays suggested that the observed biological activity was most likely due to a sequence-specific PNA-RNA triplex formation that inhibited the activity of endonucleases responsible for microRNA maturation. The present study is the first example of modulation of activity of endogenous noncoding RNA using M-modified triplex-forming PNA.

Tertiary Base Triple Formation in the SRV-1 Frameshifting Pseudoknot Stabilizes Secondary Structure Components.

Minor-groove base triples formed between stem 1 and loop 2 of the simian retrovirus type 1 (SRV-1) mRNA frameshifting pseudoknot are essential in stimulating -1 ribosomal frameshifting. How tertiary base triple formation affects the local stabilities of secondary structures (stem 1 and stem 2) and thus ribosomal frameshifting efficiency is not well understood. We made a short peptide nucleic acid (PNA) that is expected to invade stem 1 of the SRV-1 pseudoknot by PNA-RNA duplex formation to mimic the stem 1 unwinding process by a translating ribosome. In addition, we used a PNA for invading stem 2 in the SRV-1 pseudoknot. Our nondenaturing polyacrylamide gel electrophoresis data for the binding of PNA to the SRV-1 pseudoknot and mutants reveal that mutations in loop 2 disrupting base triple formation between loop 2 and stem 1 in the SRV-1 pseudoknot result in enhanced invasion by both PNAs. Our data suggest that tertiary stem 1-loop 2 base triple interactions in the SRV-1 pseudoknot can stabilize both of the secondary structural components, stem 1 and stem 2. Stem 2 stability is thus coupled to the structural stability of stem 1-loop 2 base triples, mediated through a long-range effect. The apparent dissociation constants of both PNAs are positively correlated with the pseudoknot mechanical stabilities and frameshifting efficiencies. The relatively simple PNA local invasion experiment may be used to characterize the energetic contribution of tertiary interactions and ligand binding in many other RNA and DNA structures.

Impact of Chirality and Position of Lysine Conjugation in Triplex-Forming Peptide Nucleic Acids.

Conjugation with cationic lysine residues improves the biophysical and biological properties of peptide nucleic acids (PNAs). A single lysine is routinely used to improve the solubility and prevent aggregation of the neutral and hydrophobic amide backbone of PNA. Literature precedents include the attachment of lysine at either the N- or the C-terminus. Moreover, conjugation with short lysine peptides (four to eight residues) improves the cellular uptake of PNA akin to more complex cell-penetrating peptides. Herein, we report a systematic study of the effect of lysine location (N- vs C-terminus) and chirality (d- vs l-) on triple-helical binding of PNA to double-stranded RNA and DNA (dsRNA and dsDNA). The results confirmed our earlier findings that conjugation with lysine significantly increased the stability of PNA-dsRNA and PNA-dsDNA triplexes and that PNA affinity for dsRNA was about an order of magnitude higher than for the same sequence of dsDNA. In contrast, conjugation of PNA with noncharged amino acids decreased the affinity of PNA. Surprisingly, neither the location nor the chirality of lysine had significant impact on PNA affinity for either dsRNA or dsDNA. The results are consistent with the lack of chiral preorganization of single-stranded PNAs, even after conjugation with four d- or l-amino acids. Instead, the positive charge of lysine appears to be the main driving force behind the increased affinity.

Nucleobase-Modified Triplex-Forming Peptide Nucleic Acids for Sequence-Specific Recognition of Double-Stranded RNA.

Because of the important roles noncoding RNAs play in gene expression, their sequence-specific recognition is important for both fundamental science and the pharmaceutical industry. However, most noncoding RNAs fold in complex helical structures that are challenging problems for molecular recognition. Herein, we describe a method for sequence-specific recognition of double-stranded RNA using peptide nucleic acids (PNAs) that form triple helices in the major grove of RNA under physiologically relevant conditions. We also outline methods for solid-phase conjugation of PNA with cell-penetrating peptides and fluorescent dyes. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

Cooperative Cellular Uptake and Activity of Octaarginine Antisense Peptide Nucleic acid (PNA) Conjugates.

Cellular uptake and antisense activity of d-octaarginine conjugated peptide nucleic acids (PNAs) is shown to exhibit pronounced cooperativity in serum-containing medium, in particular by being enhanced by analogous mis-match PNA–cell-penetrating peptide (PNA–CPP) conjugates without inherent antisense activity. This cooperativity does not show cell or PNA sequence dependency, suggesting that it is a common effect in cationic CPP conjugated PNA delivery. Interestingly, our results also indicate that Deca-r8-PNA and r8-PNA could assist each other and even other non-CPP PNAs as an uptake enhancer agent. However, the peptide itself (without being attached to the PNA) failed to enhance uptake and antisense activity. These results are compatible with an endosomal uptake mechanism in which the endocytosis event is induced by multiple CPP–PNA binding to the cell surface requiring a certain CPP density, possibly in terms of nanoparticle number and/or size, to be triggered. In particular the finding that the number of endosomal events is dependent on the total CPP–PNA concentration supports such a model. It is not possible from the present results to conclude whether endosomal escape is also cooperatively induced by CPP–PNA.

A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system

The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription.

Ultrasound‑targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal mediate transfection of exogenous genes by improving cytoplasmic and nuclear import.

The development of an efficient delivery system is critical for the successful treatment of cardiovascular diseases using non-viral gene therapies. Cytoplasmic and nuclear membrane barriers reduce delivery efficiency by impeding the transfection of foreign genes. Thus, a gene delivery system capable of transporting exogenous genes may improve gene therapy. The present study used a novel strategy involving ultrasound-targeted microbubbles and peptide nucleic acid (PNA)-binding nuclear localization signals (NLS). Ultrasound-targeted microbubble destruction (UTMD) and PNA-binding NLS were used to improve the cytoplasmic and nuclear importation of the plasmid, respectively. Experiments were performed using antibody-targeted microbubbles (AT-MCB) that specifically recognize the SV40T antigen receptor expressed on the membranes of 293T cells, resulting in the localization of ultrasound microbubbles to 293T cell membranes. Furthermore, PNA containing NLS was inserted into the enhanced green fluorescent protein (EGFP)-N3 plasmid DNA (NLS-PNA-DNA), which increased nuclear localization. The nuclear import and gene expression efficiency of the AT-MCB with PNA-binding NLS were compared with AT-MCB alone or a PNA-binding NLS. The effect of the AT-MCB containing PNA-binding NLS on transfection was investigated. The ultrasound and AT-MCB delivery significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability. The nuclear import and gene expression of combined microbubble- and PNA-transfected cells were significantly greater compared with cells that were transfected with AT-MCB or DNA with only PNA-binding NLS. The quantity of EGFP-N3 plasmids in the nuclei was increased by >5.0-fold compared with control microbubbles (CMCB) and NLS-free plasmids. The gene expression was ~1.7-fold greater compared with NLS-free plasmids and 1.3-fold greater compared with control microbubbles. In conclusion, UTMD combined with AT-MCB and a PNA-binding NLS plasmid significantly improved transfection efficiency by increasing cytoplasmic and nuclear DNA import. This method is a promising strategy for the noninvasive and effective delivery of target genes or drugs for the treatment of cardiovascular diseases.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

Experiment study on the transfection of exogenous genes promoted by ultrasound-targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal

Objective To increase the transfection of EGFP-N3 plasmids into 293T cells using ultrasound-targeted microbubbles delivery(UTMD) mediated a peptide nucleic acid (PNA) binding nuclear localization signal (NLS). Methods Antibody-targeted microbubbles were used in the experiments which can specifically recognize the SV40T antigen receptor. The SV40T antigen receptors were expressed on the membranes of 293T cells. The PNA containing the NLS were inserted in the EGFP-N3 plasmid DNA, which increased nuclear localization. Ultrasound-targeted microbubble delivery (UTMD) and the PNA binding NLS were utilized to improve the cytoplasmic import of plasmids and the nuclear intake of the plasmid from the cytoplasm, respectively. The study was divided into five groups: Contrast (group A), Common microbubble+ DNA (group B), Antibody-targeted microbubbles+ DNA (group C), Common microbubbles+ NLS-PNA-DNA (group D), Antibody-targeted microbubbles+ NLS-PNA-DNA (group E). Fluorescence microscope was used to observe the fluorescent light in each group; flow cytometry to test the transfection; RT-PCR and Western blot to detect genes′ mRNA and protein expression level. Results Ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability(>80%). Fluorescent microscope showed that the quantities of green fluorescence in cells were increased successfully.The transfection results of flow cytometry were 0, (9.30±0.46)%, (26.46±2.01)%, (29.54±0.62)%, (45.72±1.86)%, respectively, and the differences were statistically significant(P<0.05). The relative mRNA and protein expression in group E were greater than those in group C and D respectively (P<0.05). Conclusions UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid can significantly improve transfection efficiency of exogenous genes by enhancing both cytoplasmic and nuclear DNA import. Key words: Sonication; Microbubbles; Peptide nucleic acid; Nuclear localization signal; Gene transfection

Helix 69 of Escherichia coli 23S ribosomal RNA as a peptide nucleic acid target

A fragment of 23S ribosomal RNA (nucleotides 1906–1924 in E. coli), termed Helix 69, forms a hairpin that is essential for ribosome function. Helix 69 forms a conformationally flexible inter-subunit connection with helix 44 of 16S ribosomal RNA, and the nucleotide A1913 of Helix 69 influences decoding accuracy. Nucleotides U1911 and U1917 are post-transcriptionally modified with pseudouridines (Ψ) and U1915 with 3-methyl-Ψ.We investigated Helix 69 as a target for a complementary synthetic oligonucleotide – peptide nucleic acid (PNA). We determined thermodynamic properties of Helix 69 and its complexes with PNA and tested the performance of PNA targeted at Helix 69 in inhibiting translation in cell-free extracts and growth of E. coli cells.First, we examined the interactions of a PNA oligomer complementary to the G1907–A1919 fragment of Helix 69 with the sequences corresponding to human and bacterial species (with or without pseudouridine modifications). PNA invades the Helix 69 hairpin creating stable complexes and PNA binding to the pseudouridylated bacterial sequence is stronger than to Helix 69 without any modifications. Second, we confirmed the binding of PNA to 23S rRNA and 70S ribosomes. Third, we verified the efficiency of translation inhibition of these PNA oligomers in the cell-free translation/transcription E. coli system, which were in a similar range as tetracycline. Next, we confirmed that PNA conjugated to the (KFF)3K transporter peptide inhibited E. coli growth in micromolar concentrations. Overall, targeting Helix 69 with PNA or other sequence-specific oligomers could be a promising way to inhibit bacterial translation.

Ultrasound-Targeted Microbubbles Combined with a Peptide Nucleic Acid Binding Nuclear Localization Signal Mediates the Transfection of Exogenous Genes by Improving both Cytoplasmic and Nuclear Import

The development of an efficient delivery systems is critical for non-viral gene therapies used to treat cardiovascular diseases. The cytoplasmic and nuclear membrane barriers reduce delivery efficiency by impeding the import of foreign genes. Thus, a gene delivery system capable of transporting exogenous genes could improve gene therapies. We have employed a novel strategy involving ultrasound-targeted microbubbles and a PNA (Peptide Nucleic Acid) binding NLS (Nuclear Localization Signal). Ultrasound-targeted microbubble delivery (UTMD) and the PNA binding NLS were utilized to improve the cytoplasmic import of plasmids and the nuclear intake of the plasmid from the cytoplasm, respectively. We conducted experiments using antibody-targeted microbubbles that specifically recognize the SV40T antigen receptor expressed on the membranes of 293t cells so that ultrasound microbubbles could be gathered around the 293t cell membranes. Furthermore, we inserted the PNA containing the nuclear localization signal (NLS) in the egfp-n3 plasmid DNA, which increased nuclear localization. The nuclear import and gene expression efficiency of the antibody-microbubbles with the PNA binding NLS were then compared with antibody-targeted microbubbles alone or a PNA binding NLS. We specifically investigated the effect of the antibody-targeted microbubbles containing the PNA binding NLS on transfection. The results indicate that ultrasound and antibody-targeted microbubble delivery (UTMD) significantly enhanced the cytoplasmic intake of exogenous genes and maintained high cell viability. We found the nuclear import and gene expression of combined microbubble and PNA transfected cells were significantly higher than cells transfected with antibody-targeted microbubbles or DNA with only the PNA binding NLS.The quantity of pEGFP-N3 plasmids present in the nucleus increased by more than 3.2-fold relative to the control mircrobubbles and NLS-free plasmids.The data indicate that the gene expression was 2.0-fold greater. These results demonstrate that UTMD combined with antibody-targeted microbubbles and a PNA binding NLS plasmid significantly improved transfection efficiency by enhancing both cytoplasmic and nuclear DNA import. It's a very promising method for noninvasive and effective delivering target genes or drugs to the targeted to treat cardiovascular diseases.

GW27-e0210 Ultrasound-targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal mediates the transfection of exogenous genes by improving both cytoplasmic and nuclear import

GW27-e0210 Ultrasound-targeted microbubbles combined with a peptide nucleic acid binding nuclear localization signal mediates the transfection of exogenous genes by improving both cytoplasmic and nuclear import

24 Interference of PNA binding to the non-template strand with transcription supports the general model for transcription blockage by R-loop formation

Transcription blockage can strongly affect various DNA and RNA transactions (reviewed in Hanawalt & Spivak, 2008; Belotserkovskii, Mirkin, & Hanawalt, 2013). Thus, it is of interest to study the various factors that can cause transcription blockage and to elucidate mechanisms of their action. Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. The effect of PNA binding to the (GAA/CTT)n sequence within the transcribed DNA region upon T7 RNA polymerase transcription was studied in vitro. In the case of the PNA binding to the template strand, the blockage signals concentrated primarily in the narrow area close to the upstream flank of the PNA-bound sequence, consistent with the blockage being caused by RNA polymerase “bumping” into the PNA/DNA hybrid (Belotserkovskii, Liu, & Hanawalt, 2009). In contrast, for PNA binding to the non-template strand, a characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site (Belotserkovskii and Hanawalt, 2014), similar to that produced by G-rich homopurine-homopyrimidine sequences (Belotserkovskii et al, 2010, 2013). This striking similarity between transcription blockage patterns caused by two seemingly unrelated factors suggests a common mechanism of blockage. This common mechanism likely involves R-loop formation, which is facilitated both by PNA binding to the non-template strand and by G-rich homopurine-homopyrimidine sequences, due to sequestration of the non-template strand or due to formation of an extra-stable RNA/DNA hybrid, respectively. We suggest that there is a general mechanism of transcription blockage by R-loop formation, which presumably involves destabilization of the transcription complex, making it more prone to spontaneous pausing or termination.

G-quadruplex formation between G-rich PNA and homologous sequences in oligonucleotides and supercoiled plasmid DNA.

Guanine (G)-rich DNA sequences can adopt four-stranded quadruplex conformations that may play a role in the regulation of genetic processes. To explore the possibility of targeted molecular recognition of DNA sequences with short G-rich peptide nucleic acids (PNA) and to assess the strand arrangement in such complexes, we used PNA and DNA with the Oxytricha nova telomeric sequence d(G4T4G4) as a model. PNA probes were complexed with DNA targets in the following forms: single-stranded oligonucleotides, a loop of DNA in a hairpin conformation, and as supercoiled plasmid with the (G4T4G4)/(C4A4C4) insert. Gel-shift mobility assays demonstrated formation of stable hybrid complexes between the homologous G4T4G4 PNA and DNA with multiple modes of binding. Chemical and enzymatic probing revealed sequence-specific and G-quadruplex dependent binding of G4T4G4 PNA to dsDNA. Spectroscopic and electrophoretic analysis of the complex formed between PNA and the synthetic DNA hairpin containing the G4T4G4 loop showed that the stoichiometry of a prevailing complex is three PNA strands per one DNA strand. We speculate how this new PNA-DNA complex architecture can help to design more selective, quadruplex-specific PNA probes.

PNA binding to the non-template DNA strand interferes with transcription, suggesting a blockage mechanism mediated by R-loop formation.

Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. T7 RNA polymerase (T7 RNAP) transcription upon encountering PNA bound to the non-template DNA strand was studied in vitro. A characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site, similar to that produced by G-rich homopurine-homopyrimidine (hPu-hPy) sequences and likely caused by R-loop formation. Since blocked transcription complexes in association with stable R-loops may interfere with replication and in some cases trigger apoptosis, targeted R-loop formation might be employed to inactivate selected cells, such as those in tumors, based upon their unique complement of expressed genes.

Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni

ObjectivesCmeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically important antibiotics in Campylobacter. This study aimed to identify the optimal target sites for the inhibition of CmeABC with antisense peptide nucleic acid (PNA).MethodsEighteen PNAs were designed to bind to the translational initiation regions of cmeABC, spanning the ribosome-binding site (RBS) and the start codon of the cmeABC genes. Campylobacter jejuni was treated with CmeABC-specific PNAs (CmeABC-PNAs) at various concentrations and subjected to western blotting to measure changes in the level of CmeABC expression. The MICs of ciprofloxacin and erythromycin were measured to evaluate the impact of CmeABC knockdown on antibiotic susceptibility.ResultsWhile antisense PNA significantly affected CmeA and CmeB expression, interestingly, CmeC expression was not altered by any of the CmeC-PNAs used in this study. A CmeA-PNA targeting the RBS of cmeA and its upstream region reduced CmeA expression most efficiently, and CmeB expression was most significantly decreased by PNA binding to the RBS of cmeB and its downstream region. CmeA- and CmeB-PNAs increased the susceptibility of C. jejuni to ciprofloxacin and erythromycin in proportion to the inhibition levels observed in western blotting.ConclusionsThe cmeA gene is the best target to knockdown CmeABC with antisense PNA. The RBS is the major target for the PNA-mediated antisense inhibition of CmeABC. However, regions in its vicinity also significantly influence the effectiveness of the PNA-based knockdown of CmeABC.