Composites of peptide nucleic acids with ttitanium dioxide nanoparticles. I. Construction of nanocomposites containing DNA/PNA duplexes and their delivery into HeLa cells

In order to study the possibility of using titanium dioxide (TiO₂) nanoparticles to deliver peptide nucleic acids (PNA) in eukaryotic cells, a PNA oligomer was synthesized, and a method of PNA immobilization in the form of hybrid DNA/PNA duplexes on the surface of TiO₂ nanoparticles covered with polylysine (PL) was developed. The attachment of a DNA/PNA duplex to TiO₂ · PL nanoparticles occurs due to electrostatic interactions between the negatively charged DNA chain and the positively charged amino groups of PL. The binding of the PNA to the nanocomposite is achieved through noncovalent Watson-Crick interactions between PNA and complementary DNA. The capacity of the obtained TiO₂ · PL · DNA/PNA nano-composites depending on immobilization conditions was 10?C30 nmol PNA per 1 mg of TiO₂ particles, which corresponds to ??1?C3 PNA molecules per one TiO₂ particle with a size of 4?C6 nm. It was shown by confocal laser scanning microscopy that fluorescently-labeled PNA molecules in the TiO₂ · PL · DNA/^FluPNA nano-composites effectively penetrate into HeLa cells without transfection agents, electroporation, or other auxiliary procedures.     

Comparison of thermodynamic stabilities between PNA (peptide nucleic acid)/DNA hybrid duplexes and DNA/DNA duplexes.

We have examined quantitatively stabilities of PNA/DNA hybrid duplexes with identical nearest-neighbor base pairs and compared stabilities between PNA/DNA and DNA/DNA. The average difference of stabilization energy of the short PNA/DNA was 0.9 kcal mol(-1), which suggests that the stability of the hybrids with identical nearest-neighbor base pairs can be predicted with the nearest-neighbor model as well as those of nucleic acid duplexes.     

Modulation of remote DNA oxidation by hybridization with peptide nucleic acids (PNA)

We have examined the efficiency of DNA photooxidation in DNA/PNA duplex and DNA/(PNA)(2) triplex for the first time. DNA/PNA duplex was cleaved at GG steps by external riboflavin with high efficiency like specific GG cleavage in DNA/DNA duplex. However, the 5'G selectivity of the GG oxidation in DNA/PNA duplex was much lower than that observed in DNA/DNA duplex. Remote DNA oxidation of oxidant-tethered DNA/PNA duplex was considerably suppressed. In contrast, the formation of DNA/(PNA)(2) triplex by hybridization with two PNA strands completely inhibited the remote GG oxidation, indicating that PNA acts as an inhibition for remote oxidative DNA damage.     

Unique properties of purine/pyrimidine asymmetric PNA.DNA duplexes: differential stabilization of PNA.DNA duplexes by purines in the PNA strand

PNA.DNA duplexes are significantly stabilized by purine nucleobases in the PNA strand. To elucidate and understand the effect of switching the backbone in a nucleic acid duplex, we now report a thermodynamics study along with a solution conformations study of two purine/pyrimidine strand asymmetric duplexes and a strand symmetrical control by comparing the behavior of all four possible PNA/DNA combinations. In essence, we are comparing an identical basepair stack connected by either an aminoethyl glycine PNA or a deoxyribose DNA backbone. We show that the PNA.DNA duplexes containing purine-rich PNA strands are stabilized with regard to the thermal melting temperature and free energy as well as enthalpy (and concomitantly relatively less entropically disfavored). Based on our data, we find it unlikely that differences in counterion binding (identical ionic-strength dependence was observed), hydration (identical and insignificant water release was observed), or single-strand conformation can be responsible for the difference in duplex stability. The only consistent difference observed between the purine-rich PNA versus the pyrimidine-rich PNA in isosequential PNA.DNA duplexes is the significant increase in both binding enthalpy and entropy for the PNA.DNA duplexes containing pyrimidine-rich PNA in organic solvent, which would indicate that these duplexes are relatively enthalpically disfavored in water. Although our results so far do not allow us to identify the origin of the different stabilities of homopurine/homopyrimidine PNA.DNA duplexes, the evidence does point to a significant structural component, which involves enthalpic contributions both within the duplex structure and also from bound water molecules.     

Control of electron transfer in DNA by peptide nucleic acids (PNA).

The one-electron oxidation of PNA-DNA hybrid containing G-triplet sequence was examined. In DNA duplex G-triplet was selectively cleaved by oxidation, whereas in PNA-DNA hybrid cleavage efficiency was extremely lowered. These result suggested that cleavage efficiency of PNA-DNA hybrid was different from that of B-form DNA duplex.     

End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes

Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine–homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA–DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection.     

Construction of peptide conjugates with peptide nucleic acids containing an anthracene probe and their interactions with DNA

We designed and synthesized the peptide nucleic acid (PNA)-peptide conjugates having anthracene chromophores and investigated their interactions with calf thymus DNA, [d(AT)(10)](2), [d(GC)(10)](2), and [d(AT)(10)dA(6)](2). Considering the synthesis compatibility and expecting that a novel DNA analogue, PNA, can improve DNA binding properties of alpha-helix peptides, we attempted to attach thymine PNA oligomers at the C-terminus of a 14 amino acid alpha-helix peptide that contained a pair of artificial intercalators, anthracene, as a probe, and to examine their interactions with DNA using anthracene UV, fluorescence and circular dichroism properties. The results observed in this study showed that the designed peptide folded in an alpha-helix structure in the presence of calf thymus DNA, [d(AT)(10)](2), and [d(AT)(10)dA(6)](2) with the chromophores at the side-chain being fixed with a left-handed chiral-sense orientation. The alpha-helix and the anthracene signals were not observed for [d(GC)(10)](2). Incorporation of thymine PNA oligomers into the designed alpha-helix peptide increased the DNA binding ability to [d(AT)(10)dA(6)](2) with increasing the length of the PNA without changing the conformations of the peptide backbone and the anthracene side-chains.     

Information transfer from DNA to peptide nucleic acids by template-directed syntheses.

Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.     

A formula for thermal stability (Tm) prediction of PNA/DNA duplexes.

An empirical formula for thermal stability (T m) prediction of PNA/DNA duplexes has been derived. The model is based on the T m as calculated for the corresponding DNA/DNA duplex employing a nearest neighbour approach, by including terms for the pyrimidine content and length of the PNA to take into account the increased thermostability of PNA/DNA hybrids and the asymmetry of the PNA-DNA heteroduplex. The predictive power of the T m prediction formula was challenged with an independent data set not used for model building. The T m of >90% of the sequences was predicted within 5 K; 98% of the predicted T ms differ by not more than 10 K from the experimentally determined T m.     

Interaction of antisense DNA with nucleic acids/proteins.

In order to study interaction of various types of labeled antisense DNAs were prepared. Fluorescein and 2,2,6,6-tetramethypiperidine-N-oxyl were the label molecules, which were introduced to 5'-end of oligonucleotides and their analogs. Interactions of labeled antisense DNAs with nucleic acids or proteins such as HSA, HIG and TF, were studied by UV, fluorescence depolarization spectroscopy, and ESR spectroscopy. Hybrid formation of antisense DNAs with oligonucleotides in solution could be monitored by the increase in fluorescence anisotropy (r) and by intensity change in ESR spectra. When phosphorothioate type antisense molecules anchoring fluorescein (F-OPT) were mixed with proteins, r drastically increased, whereas ODN slightly increased. These results suggest that OPTs have much more affinity for proteins than ODNs.     

Positional effect of single bulge nucleotide on PNA(peptide nucleic acid)/DNA hybrid stability.

We report positional effect of bulge nucleotide on PNA/DNA hybrid stability. CD spectra showed that PNA/DNA hybrids required at least seven base pairings at a stem region to form a bulged structure. On the other hand, DNA/DNA could form bulged structure when there are only four base pairings adjacent to the bulge nucleotide. We discuss why PNA requests such a many base pairings to form bulged structure from a nearest neighbor standpoint.     

Synthesis of a monocharged peptide nucleic acid (PNA) analog and its recognition as substrate by DNA polymerases.

The preparation of a novel phosphoramidite monomer based on thyminyl acetic acid coupled to the secondary nitrogen of 2-(2-amino-ethylamino)ethanol is described. This monomer can be used to attach a deoxynucleotide to the carboxy terminus of a PNA oligomer by solid-phase synthesis. The resulting PNA primer is recognized as a substrate by various DNA polymerases.     

Interaction of phenosafranine with nucleic acids and model polyphosphates. III. Heterogeneity in phenosafranine interactions with DNA base pairs.

Fluorescence and circular dichroism spectral measurements, thermal denaturation studies and binding competition experiments with netropsin and actinomycin D were carried out in systems containing phenosafranine bound to DNA's differing in base composition. The investigated properties exhibit a heterogeneity related to the content of A.T and G.C pairs in DNA and to the nature of phenosafranine binding modes. At low level of saturation of binding sites (r less than 0.1) phenosafranine does not show strong preference for any of the DNA base pairs in the overall binding. However, the strong monomer non-cooperative binding outside the helix (mode I1) occurs predominantly, even though not exclusively in G.C rich regions. The strong binding modes involving intercalated dye molecules (mode I2 and eventually mode II1) prevail in A.T rich regions. These binding modes become the principal types of strong phenosafranine interaction with DNA when the level of saturation of binding sites increases, i.e. at r greater than 0.1.20     

Derivation of nearest-neighbor properties from data on nucleic acid oligomers. II. Thermodynamic parameters of DNA.RNA hybrids and DNA duplexes

Using nearest-neighbor models consisting of independent short sequence combinations of nearest neighbors (ISS models), values of thermodynamic parameters for sets of independent sequences are derived from published oligomer data for DNA.RNA hybrids [N. Sugimoto, S. Nakano, M. Katoh, A. Matsumura, H. Nakamuta, T. Ohmichi, M. Yoneyama, and M. Sasaki (1995) Biochemistry, Vol. 34, pp. 11211-11216] and dsDNA duplexes [J. SantaLucia, Jr., H. T. Allawi, and P. A. Seneviratne (1996) Biochemistry, Vol. 35, pp. 3555-3562]. The results are compared with those from models that assign values of thermodynamic parameters to individual nearest neighbors (INN models). Differences in the use of ISS and INN models are also illustrated in an appendix, which shows examples of analyses for values of a fictitious nearest-neighbor property. INN models that include an initiation parameter contain an implicit assumption that combinations of end neighbors have the same value of a property. It is found that combinations of end neighbors (e.g., base pairs neighboring solvent) in oligomers can have significant and different apparent values of thermodynamic properties, so that the assumption inherent in INN models is not always correct. Even though ISS models do not allow the assignment of values to individual nearest neighbors, except for the like neighbors [such as d(AA)/r(UU), etc., for hybrids and d(AA)/d(TT) and d(GG)/d(CC) for DNA duplexes], they do provide physically meaningful values for the like neighbors, for sequence combinations, and for specified combinations of end neighbors.     

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly from poly(dG-dC) than from poly(dA-dT) but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC).poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a single exponential. Dissociation of daunomycin, adriamycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibiotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.     

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly poly(dG-dC) than from poly(dA-dT), but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC)·poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a sinigle exponential. Dissociation of daunomycin, adrianmycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

Summary In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to functionalize their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented