Liver cell specific targeting of peptide nucleic acid oligomers

Chimeric molecules consisting of peptide nucleic acid (PNA) and lactose have been synthesized to test the hypothesis that lactose moieties can promote cell-specific uptake of PNAs. We find that lactose modified PNAs rapidly enter liver-derived HepG2 cells while unmodified PNAs do not and that lactose modified PNAs can inhibit cellular telomerase.     

Cellular antisense activity of peptide nucleic acid (PNAs) targeted to HIV-1 polypurine tract (PPT) containing RNA

DNA and RNA oligomers that contain stretches of guanines can associate to form stable secondary structures including G-quadruplexes. Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run. We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells. We find that PNAs can form stable complexes even with the structured PPT RNA target at neutral pH. We show that T-rich PNAs, namely the tridecamer-I PNA (C4T4CT4) forms triplex structures whereas the C-rich tridecamer-II PNA (TC6T4CT) likely forms a duplex with the target RNA. Interestingly, we find that both C-rich and T-rich PNAs arrested in vitro translation elongation specifically at the PPT target site. Finally, we show that T-rich and C-rich tridecamer PNAs that have been identified as efficient and specific blockers of translation elongation in vitro, specifically inhibit translation in streptolysin-O permeabilized cells where the PPT target sequence has been introduced upstream the reporter luciferase gene.     

Incorporation of thio-pseudoisocytosine into triplex-forming peptide nucleic acids for enhanced recognition of RNA duplexes

Peptide nucleic acids (PNAs) have been developed for applications in biotechnology and therapeutics. There is great potential in the development of chemically modified PNAs or other triplex-forming ligands that selectively bind to RNA duplexes, but not single-stranded regions, at near-physiological conditions. Here, we report on a convenient synthesis route to a modified PNA monomer, thio-pseudoisocytosine (L), and binding studies of PNAs incorporating the monomer L. Thermal melting and gel electrophoresis studies reveal that L-incorporated 8-mer PNAs have superior affinity and specificity in recognizing the duplex region of a model RNA hairpin to form a pyrimidine motif major-groove RNA₂–PNA triplex, without appreciable binding to single-stranded regions to form an RNA–PNA duplex or, via strand invasion, forming an RNA–PNA₂ triplex at near-physiological buffer condition. In addition, an L-incorporated 8-mer PNA shows essentially no binding to single-stranded or double-stranded DNA. Furthermore, an L-modified 6-mer PNA, but not pseudoisocytosine (J) modified or unmodified PNA, binds to the HIV-1 programmed −1 ribosomal frameshift stimulatory RNA hairpin at near-physiological buffer conditions. The stabilization of an RNA₂–PNA triplex by L modification is facilitated by enhanced van der Waals contacts, base stacking, hydrogen bonding and reduced dehydration energy. The destabilization of RNA–PNA and DNA–PNA duplexes by L modification is due to the steric clash and loss of two hydrogen bonds in a Watson–Crick-like G–L pair. An RNA₂–PNA triplex is significantly more stable than a DNA₂–PNA triplex, probably because the RNA duplex major groove provides geometry compatibility and favorable backbone–backbone interactions with PNA. Thus, L-modified triplex-forming PNAs may be utilized for sequence-specifically targeting duplex regions in RNAs for biological and therapeutic applications.     

Methods for assessing DNA hybridization of peptide nucleic acid-titanium dioxide nanoconjugates

We describe the synthesis of peptide nucleic acid (PNA)-titanium dioxide (TiO₂) nanoconjugates and several novel methods developed to investigate the DNA hybridization behaviors of these constructs. PNAs are synthetic DNA analogs resistant to degradation by cellular enzymes that hybridize to single-stranded DNA (ssDNA) with higher affinity than DNA oligonucleotides, invade double-stranded DNA (dsDNA), and form different PNA/DNA complexes. Previously, we developed a DNA-TiO₂ nanoconjugate capable of hybridizing to target DNA intracellularly in a sequence-specific manner with the ability to cleave DNA when excited by electromagnetic radiation but susceptible to degradation that may lower its intracellular targeting efficiency and retention time. PNA-TiO₂ nanoconjugates described in the current article hybridize to target ssDNA, oligonucleotide dsDNA, and supercoiled plasmid DNA under physiological-like ionic and temperature conditions, enabling rapid, inexpensive, sequence-specific concentration of nucleic acids in vitro. When modified by the addition of imaging agents or peptides, hybridization capabilities of PNA-TiO₂ nanoconjugates are enhanced, providing essential benefits for numerous in vitro and in vivo applications. The series of experiments shown here could not be done with either TiO₂-DNA nanoconjugates or PNAs alone, and the novel methods developed will benefit studies of numerous other nanoconjugate systems.     

Strand invasion by mixed base PNAs and a PNA-peptide chimera

Peptide nucleic acid oligomers (PNAs) have a remarkable ability to invade duplex DNA at polypurine–polypyrimidine target sequences. Applications for PNAs in medicine and biotechnology would increase if the rules governing their hybridization to mixed base sequences were also clear. Here we describe hybridization of PNAs to mixed base sequences and demonstrate that simple chemical modifications can enhance recognition. Easily synthesized and readily soluble eight and 10 base PNAs bind to plasmid DNA at an inverted repeat that is likely to form a cruciform structure, providing convenient tags for creating PNA–plasmid complexes. PNAs also bind to mixed base sequences that cannot form cruciforms, suggesting that recognition is a general phenomenon. Rates of strand invasion are temperature dependent and can be enhanced by attaching PNAs to positively charged peptides. Our results support use of PNAs to access the information within duplex DNA and demonstrate that simple chemical modifications can make PNAs even more powerful agents for strand invasion. Simple strategies for enhancing strand invasion should facilitate the use of PNAs: (i) as biophysical probes of double-stranded DNA; (ii) to target promoters to control gene expression; and (iii) to direct sequence-specific mutagenesis.     

Development of Peptide Nucleic Acid Probes for Detection of the HER2 Oncogene

Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes.     

Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.     

Inhibition of gene expression and growth of multidrug-resistant Acinetobacter baumannii by antisense peptide nucleic acids

Acinetobacter baumannii causes common and severe community- and hospital-acquired infections. The increasing emergence of multidrug-resistant (MDR) and pan-drug resistant A. baumannii has limited the therapeutic options, highlighting the need for new therapeutic strategies. The goal of this study was to investigate whether antisense peptide nucleic acids (PNAs) could mediate gene-specific inhibition effects in MDR A. baumannii. We described a screening strategy based on computational prediction and dot hybridization for identifying potential inhibitory PNAs, and evaluated the in vitro growth inhibition potency of two PNAs conjugated to the (KFF)₃K peptide (pPNA1 and pPNA2), both of which targeted the growth essential gene gyrA of A. baumannii. Both pPNAs showed strong inhibition effects on bacterial growth and gyrA mRNA expression in a dose-dependent manner. The lowest inhibitory and bactericidal concentration were 5 and 10 μM, respectively. Combination of the two pPNAs showed superimposed effect other than synergistic effect. Control PNAs without (KFF)₃K peptide conjugation or with mismatched antisense sequence had no inhibition effects on bacterial growth or mRNA expression. Our study suggests that anti-gyrA pPNAs can efficiently inhibit gene expression and bacterial growth, and has the potential as a new therapeutic option for MDR A. baumannii.     

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications.     

Inhibition of 5′-UTR RNA Conformational Switching in HIV-1 Using Antisense PNAs

Background

The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5′-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5′-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization.

Methods and Results

We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes.

Conclusions

Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.     

Vitamin B12-peptide nucleic acids use the BtuB receptor to pass through the Escherichia coli outer membrane

Short modified oligonucleotides that bind in a sequence-specific way to messenger RNA essential for bacterial growth could be useful to fight bacterial infections. One such promising oligonucleotide is peptide nucleic acid (PNA), a synthetic DNA analog with a peptide-like backbone. However, the limitation precluding the use of oligonucleotides, including PNA, is that bacteria do not import them from the environment. We have shown that vitamin B₁₂, which most bacteria need to take up for growth, delivers PNAs to Escherichia coli cells when covalently linked with PNAs. Vitamin B₁₂ enters E. coli via a TonB-dependent transport system and is recognized by the outer-membrane vitamin B₁₂-specific BtuB receptor. We engineered the E. coli ΔbtuB mutant and found that transport of the vitamin B₁₂-PNA conjugate requires BtuB. Thus, the conjugate follows the same route through the outer membrane as taken by free vitamin B₁₂. From enhanced sampling all-atom molecular dynamics simulations, we determined the mechanism of conjugate permeation through BtuB. BtuB is a β-barrel occluded by its luminal domain. The potential of mean force shows that conjugate passage is unidirectional and its movement into the BtuB β-barrel is energetically favorable upon luminal domain unfolding. Inside BtuB, PNA extends making its permeation mechanically feasible. BtuB extracellular loops are actively involved in transport through an induced-fit mechanism. We prove that the vitamin B₁₂ transport system can be hijacked to enable PNA delivery to E. coli cells.     

Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or ‘antispacer’, PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.     

Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.     

Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates

Peptide nucleic acids (PNAs) offer a distinct option for silencing gene expression in mammalian cells. However, the full value of PNAs has not been realized, and the rules governing the recognition of cellular targets by PNAs remain obscure. Here we examine the uptake of PNAs and PNA-peptide conjugates by immortal and primary human cells and compare peptide-mediated and DNA/lipid-mediated delivery strategies. We find that both peptide-mediated and lipid-mediated delivery strategies promote entry of PNA and PNA-peptide conjugates into cells. Confocal microscopy reveals a punctate distribution of PNA and PNA-peptide conjugates regardless of the delivery strategy used. Peptide D(AAKK)(4) and a peptide containing a nuclear localization sequence (NLS) promote the spontaneous delivery of antisense PNAs into cultured cells. The PNA-D(AAKK)(4) conjugate inhibits expression of human caveolin 1 (hCav-1) in both HeLa and primary endothelial cells. DNA/lipid-mediated delivery requires less PNA, while peptide-mediated delivery is simpler and is less toxic to primary cells. The ability of PNA-peptide conjugates to enter primary and immortal human cells and inhibit gene expression supports the use of PNAs as antisense agents for investigating the roles of proteins in cells. Both DNA/lipid-mediated and peptide-mediated delivery strategies are efficient, but the compartmentalized localization of PNAs suggests that improving the cellular distribution may lead to increased efficacy.     

Delivery of antisense peptide nucleic acids (PNAs) to the cytosol by disulphide conjugation to a lipophilic cation

Peptide nucleic acids (PNAs) are effective antisense reagents that bind specific mRNAs preventing their translation. However, PNAs cannot cross cell membranes, hampering delivery to cells. To overcome this problem we made PNAs membrane-permeant by conjugation to the lipophilic triphenylphosphonium (TPP) cation through a disulphide bond. The TPP cation led to efficient PNA uptake into the cytoplasm where the disulphide bond was reduced, releasing the antisense PNA to block expression of its target gene. This method of directing PNAs into cells is a significant improvement on current procedures and will facilitate in vitro and pharmacological applications of PNAs.     

PNA/DNA interstrand cross-links from a modified PNA base upon photolysis or oxidative conditions

PNA/DNA interstrand cross-links (ICLs) were observed when peptide nucleic acids (PNAs) containing modified thymine derivatives were hybridized with the complementary or one-base mismatched DNA upon photolysis or treatments of oxidative agent. PNA/DNA ICL formation provides a useful method for biological applications such as antisense technologies or PNA chips.