Thermodynamic studies on PNA and PNA/DNA dendrimer formation

In this work we report a kinetic and thermodynamic study relative to the formation of gel systems based on PNA and PNA/DNA dendrimers, useful for drug delivery or diagnostic applications. We realized two kinds of systems: a PNA-based monomolecular system formed by an autoassembling PNA tridendron (A) and a PNA/DNA bimolecular system based on a PNA tridendron with a mixed sequence and a DNA crosslinker (B). Both systems have the ability to form a three-dimensional network by means of specific W-C base pairing.     

PNA technology

Peptide nucleic acids (PNA) are deoxyribonucleic acid (DNA) mimics with a pseudopeptide backbone. PNA is an extremely good structural mimic of DNA (or of ribonucleic acid [RNA]), and PNA oligomers are able to form very stable duplex structures with Watson-Crick complementary DNA and RNA (or PNA) oligomers, and they can also bind to targets in duplex DNA by helix invasion. Therefore, these molecules are of interest in many areas of chemistry, biology, and medicine, including drug discovery, genetic diagnostics, molecular recognition, and the origin of life. Recent progress in studies of PNA properties and applications is reviewed.     

PNA beacons for duplex DNA

We report here on the hybridization of peptide nucleic acid (PNA)-based molecular beacons (MB) directly to duplex DNA sites locally exposed by PNA openers. Two stemless PNA beacons were tested, both featuring the same recognition sequence and fluorophore-quencher pair (Fluorescein and DABCYL, respectively) but differing in arrangement of these groups and net electrostatic charge. It was found that one PNA beacon rapidly hybridized, with the aid of openers, to its complementary target within duplex DNA at ambient conditions via formation of a PD-like loop. In contrast, the other PNA beacon bound more slowly to preopened duplex DNA target and only at elevated temperatures, although it readily hybridized to single-stranded (ss) DNA target. Besides a higher selectivity of hybridization provided by site-specific PNA openers, we expect this approach to be very useful in those MB applications when denaturation of the duplex DNA analytes is unfavorable or undesirable. Furthermore, we show that PNA beacons are advantageous over DNA beacons for analyzing unpurified/nondeproteinized DNA samples. This feature of PNA beacons and our innovative hybridization strategy may find applications in emerging fluorescent DNA diagnostics.     

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.     

Inducing the replacement of PNA in DNA.PNA duplexes by DNA

The uncharged DNA-analogue peptide nucleic acid (PNA) can invade into dsDNA by displacing the non-complementary DNA strand. The formed strand displacement complexes can create a sterical hindrance to block access of enzymes such as nucleases and polymerases. Due to the high stability of DNA.PNA duplexes it is usually not possible to displace the PNA strand by ssDNA or ssRNA. We herein report that the polycationic, comb-type copolymer alphaPLL-g-Dex can induce such a replacement of PNA in DNA.PNA duplexes by ssDNA. The influence of the copolymer on strand exchange highly depends on the nature of the oligonucleotides. Acceleration has only been observed when both the starting duplex and the single-stranded exchanger strand were negatively charged. The presented approach should allow the withdrawal of PNA induced sterical hindrance of DNA by rehybridisation with ssDNA.     

Role of chirality and optical purity in nucleic acid recognition by PNA and PNA analogs

Peptide nucleic acids are DNA mimics able to form duplexes with complementary DNA or RNA strands of remarkable affinity and selectivity. Oligopyrimidine PNA can displace one strand of dsDNA by forming PNA(2):DNA triplexes of very high stability. Many PNA analogs have been described in recent years, in particular, chiral PNA analogs. In the present article the results obtained recently using PNA derived from N-aminoethylamino acids 7 are illustrated. In particular, the dependence of optical purity on synthetic methodologies and a rationale for the observed effects of chirality on DNA binding ability is proposed. Chirality as a tool for improving sequence selectivity is also described. PNA analogs derived from D- or L-ornithine 8 were also found to be subjected to epimerization during solid phase synthesis. Modification of the coupling conditions or the use of a submonomeric strategy greatly reduced epimerization. The optically pure oligothymine PNAs 8 were found to bind to RNA by forming triplexes of unusual CD spectra. The melting curves of these adducts presented two transitions, suggesting a conformational change followed by melting at high temperature.     

Composites of peptide nucleic acids with titanium dioxide nanoparticles. III. Kinetics of PNA dissociation from nanocomposites containing DNA/PNA duplexes

When delivering peptide nucleic acids (PNA) into cells in the TiO₂ · PL · DNA/PNA nanocomposites consisting of titanium dioxide nanoparticles coated with polylysine (PL) and immobilized DNA/PNA duplexes, it is important to control the rate of the release of PNA from the carrier due to dissociation of the immobilized DNA/PNA duplex, followed by the desorption of PNA to solution while the DNA remains on the carrier. It was found that the rate constant of dissociation of the DNA/PNA duplex in the TiO₂ · PL · DNA/PNA nanocomposites depended on the number of complementary bases in the duplex. The half-retention time values for PNA in the studied nanocomposites containing the duplexes with 10, 12, 14, and 16 overlapping complementary base pairs were 10, 14, 22, and 70 min, respectively. Thus, it was shown that the rate of the release of PNA from the proposed nanocomposites can be controlled by varying the number of overlapping complementary base pairs in the immobilized DNA/PNA duplex. The method of the PNA immobilization may be used for designing nanocomposites having the optimum time value of the PNA release. The proposed TiO₂ · PL · DNA/PNA nanocomposites can be used to efficiently deliver therapeutically significant PNA drugs for their selective effect on pathogenic nucleic acids in cells.     

Thermodynamic comparison of PNA/DNA and DNA/DNA hybridization reactions at ambient temperature

The thermodynamics of 13 hybridization reactions between 10 base DNA sequences of design 5'-ATGCXYATGC-3' with X, Y = A, C, G, T and their complementary PNA and DNA sequences were determined from isothermal titration calorimetry (ITC) measurements at ambient temperature. For the PNA/DNA hybridization reactions, the binding constants range from 1.8 x 10(6)M(-1)for PNA(TT)/DNA to 4.15 x 10(7)M(-1)for PNA(GA)/DNA and the binding enthalpies range from -194 kJ mol(-1)for PNA(CG)/DNA to -77 kJ mol(-1)for PNA(GT)/DNA. For the corresponding DNA/DNA binding reactions, the binding constants range from 2.9 x 10(5)M(-1)for DNA(GT)/DNA to 1.9 x 10(7)M(-1)for DNA(CC)/DNA and the binding enthalpies range from -223 kJ mol(-1)for DNA(CG)/DNA to -124 kJ mol(-1)for DNA(TT)/DNA. Most of the PNA sequences exhibited tighter binding affinities than their corresponding DNA sequences resulting from smaller entropy changes in the PNA/DNA hybridization reactions. van't Hoff enthalpies and extrapolated Delta G values determined from UV melting studies on the duplexes exhibited closer agreement with the ITC binding enthalpies and Delta G values for the DNA/DNA duplexes than for the PNA/DNA duplexes.     

Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences

Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this 'double-duplex invasion', a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the alpha-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G-C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.     

Evaluation of transfection protocols for unmodified and modified peptide nucleic acid (PNA) oligomers

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.     

Modulation of DNA and RNA by PNA

Peptide nucleic acid (PNA), a synthetic DNA mimic that is devoid of the (deoxy)ribose-phosphate backbone yet still perfectly retains the ability to recognize natural nucleic acids in a sequence-specific fashion, can be employed as a tool to modulate gene expressions via several different mechanisms. The unique strength of PNA compared to other oligonucleotide analogs is its ability to bind to nucleic acid targets with secondary structures such as double-stranded and quadruplex DNA as well as RNA. This digest aims to introduce general readers to the advancement in the area of modulation of DNA/RNA functions by PNA, its current status and future research opportunities, with emphasis on recent progress in new targeting modes of structured DNA/RNA by PNA and PNA-mediated gene editing.     

CONSTRAINED FLEXIBILITY IN PNA: DNA BINDING STUDIES WITH BRIDGED AMINOPROPYLGLYCYL PNA

Introduction of methylene bridges in aegPNA and apgPNA molecules give rise to cyclic five and six membered ring structures. Synthesis of a new six membered cyclic PNA monomer, aminopipecolyl PNA (pipPNA) is reported. Incorporation of pipPNA into PNA oligomers and comparative binding with target DNA sequences is studied.     

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.     

New thiazane and thiazolidine PNA monomers: synthesis, incorporation into PNAs and hybridization studies

New constrained PNA monomers containing a substituted thiazolidine or a thiazane ring were synthesized and incorporated in the center of a 9-mer homothymine PNA. The PNA/DNA hybrids stability was studied by UV-melting experiments which showed that the presence of the modified unit destabilizes the PNA/DNA triplexes.     

PNA monomers fully compatible with standard Fmoc-based solid-phase synthesis of pseudocomplementary PNA

Here we report the synthesis of new PNA monomers for pseudocomplementary PNA (pcPNA) that are fully compatible with standard Fmoc chemistry. The thiocarbonyl group of the 2-thiouracil (sU) monomer was protected with the 4-methoxy-2-methybenzyl group (MMPM), while the exocyclic amino groups of diaminopurine (D) were protected with Boc groups. The newly synthesized monomers were incorporated into a 10-mer PNA oligomer using standard Fmoc chemistry for solid-phase synthesis. Oligomerization proceeded smoothly and the HPLC and MALDI-TOF MS analyses indicated that there was no remaining MMPM on the sU nucleobase. The new PNA monomers reported here would facilitate a wide range of applications, such as antigene PNAs and DNA nanotechnologies.     

Optimization of label-free DNA detection with electrochemical impedance spectroscopy using PNA probes

DNA biosensors, especially those based upon detection of the intrinsic negative charge of target DNA, can be greatly improved by the use of uncharged peptide nucleic acid (PNA) probes. Hybridization causes an increased electrostatic barrier for the negatively charged ferri/ferrocyanide redox couple, resulting in an increase in charge transfer resistance R(ct) that is measured using electrochemical impedance spectroscopy. We report on the optimization of PNA probe surface density by the simultaneous co-immobilization of thiol-modified probes and mercaptohexanol, with the PNA surface density controlled by the thiol mole ratio in solution. Maximum R(ct) change upon hybridization is obtained with 10% PNA mole fraction. The effect of the measurement buffer ionic strength is investigated. The electrostatic barrier for charge transfer to the ferri/ferrocyanide redox couple is approximately independent of ionic strength with PNA probes, but greatly increases with decreasing ionic strength, after hybridization with target DNA. This significantly enhances the R(ct) change upon hybridization. The optimization of PNA surface density and measurement buffer ionic strength leads to a 385-fold increase in R(ct) upon hybridization, a factor of 100 larger than previously reported results using either PNA or DNA probes.     

Unique chromosome identification and sequence-specific structural analysis with short PNA oligomers

We have extended our earlier work to show that individual 14–20mer peptide nucleic acid probes directed against interspersed α-satellite sequences can specifically identify chromosomes. Peptide nucleic acid (PNA) probes were used to detect chromosomal abnormalities and repeat structure in the human genome by fluorescence in situ hybridization (FISH). The hybridization of a single PNA probe species directed against a highly abundant α-satellite DNA repeat sequence was sufficient to absolutely identify a chromosome. Selection of highly repetitive or region-specific DNA repeats involved DNA database analysis. Distribution of a specific repeat sequence in human genome was estimated through two means: a computer program ``whole genome' approach based on ∼400 Mb (12%) human genomic sequence. The other method involved directed search for alpha satellite sequences. In total, ∼240 unique DNA repeat candidates were found. Forty-two PNA probes were designed for screening chromosome-specific probes. Ten chromosome-specific PNA probes for human Chromosomes (Chrs) 1, 2, 7, 9, 11, 17, 18, X, and Y have been identified. Interphase and metaphase results demonstrate that chromosome-specific PNA probes are capable of detecting simple aneuploidies (trisomies) in human. Another set of PNA probes showed distinct banding-like patterns and could be used as sequence-specific stains for chromosome ``bar coding'. Potential application of PNA probes for investigating repeat structure and function is also discussed. Received: 2 September 1999 / Accepted: 16 December 1999