High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA–dsDNA triplexes—mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7.2, 150 mM Na⁺). Binding affinity can be modulated more than 1000-fold by changes in pH, PNA oligomer length, PNA net charge and/or by substitution of pseudoisocytosine for cytosine, and conjugation of the DNA intercalator 9-aminoacridine. Furthermore, 9-aminoacridine conjugation also strongly enhanced triplex invasion. Specificity for the fully matched target versus one containing single centrally located mismatches was more than 150-fold. Together the data support the use of homopyrimidine PNAs as efficient and sequence selective tools in triplex targeting strategies under physiological relevant conditions.     

Combined triplex/duplex invasion of double-stranded DNA by "tail-clamp" peptide nucleic acid

"Tail-clamp" PNAs composed of a short (hexamer) homopyrimidine triplex forming domain and a (decamer) mixed sequence duplex forming extension have been designed. Tail-clamp PNAs display significantly increased binding to single-stranded DNA compared with PNAs lacking a duplex-forming extension as determined by T(m) measurements. Binding to double-stranded (ds) DNA occurred by combined triplex and duplex invasion as analyzed by permanganate probing. Furthermore, C(50) measurements revealed that tail-clamp PNAs consistently bound the dsDNA target more efficiently, and kinetics experiments revealed that this was due to a dramatically reduced dissociation rate of such complexes. Increasing the PNA net charge also increased binding efficiency, but unexpectedly, this increase was much more pronounced for tailless-clamp PNAs than for tail-clamp PNAs. Finally, shortening the tail-clamp PNA triplex invasion moiety to five residues was feasible, but four bases were not sufficient to yield detectable dsDNA binding. The results validate the tail-clamp PNA concept and expand the applications of the P-loop technology.     

Targeting duplex DNA with DNA‐PNA chimeras? Physico‐chemical characterization of a triplex DNA‐PNA/DNA/DNA

Targeting double-stranded DNA with homopyrimidine PNAs results in strand displacement complexes PNA/DNA/PNA rather than PNA/DNA/DNA triplex structures. Not much is known about the binding properties of DNA-PNA chimeras. A 16-mer 5'-DNA-3'-p-(N)PNA(C) has been investigated for its ability to hybridize a complementary duplex DNA by DSC, CD, and molecular modeling studies. The obtained results showed the formation of a triplex structure having similar, if not slightly higher, stability compared to the same all-DNA complex.     

Electron microscopy mapping of oligopurine tracts in duplex DNA by peptide nucleic acid targeting.

Biotinylated homopyrimidine decamer peptide nucleic acids (PNAs) are shown to form sequence-specific and stable complexes with complementary oligopurine targets in linear double-stranded DNA. The noncovalent complexes are visualized by electron microscopy (EM) without chemical fixation using streptavidin as an EM marker. The triplex stoichiometry of the PNA-DNA complexes (two PNA molecules presumably binding by Watson-Crick and Hoogsteen pairing with one of the strands of the duplex DNA) is indicated by the appearance of two streptavidin 'beads' per target site in some micrographs, and is also supported by the formation of two retardation bands in a gel shift assay. Quantitative analysis of the positions of the streptavidin 'beads' revealed that under optimized conditions PNA-DNA complexes are preferably formed with the fully complementary target. An increase in either the PNA concentration or the incubation time leads to binding at sites containing one or two mismatches. Our results demonstrate that biotinylated PNAs can be used for EM mapping of short targets in duplex DNA.     

Strand displacement recognition of mixed adenine-cytosine sequences in double stranded DNA by thymine-guanine PNA (peptide nucleic acid).

Mixed pyrimidine-purine peptide nucleic acids (PNAs) composed of thymines and guanines are shown to form a PNA(2)-DNA triplex with Watson-Crick complementary adenine-cytosine oligonucleotides and to bind complementary adenine-cytosine targets in double stranded DNA by helix invasion. These results for the first time demonstrate binding of an unmodified PNA oligomer to a mixed pyrimidine-purine target in double stranded DNA and illustrate a novel binding mode of PNA.     

An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA

We investigated the mechanism and kinetic specificity of binding of peptide nucleic acid clamps (bis-PNAs) to double-stranded DNA (dsDNA). Kinetic specificity is defined as a ratio of initial rates of PNA binding to matched and mismatched targets on dsDNA. Bis-PNAs consist of two homopyrimidine PNA oligomers connected by a flexible linker. While complexing with dsDNA, they are known to form P-loops, which consist of a [PNA]2-DNA triplex and the displaced DNA strand. We report here a very strong pH-dependence, within the neutral pH range, of binding rates and kinetic specificity for a bis-PNA consisting of only C and T bases. The specificity of binding reaches a very sharp and high maximum at pH 6.9. In contrast, if all the cytosine bases in one of the two PNA oligomers within the bis-PNA are replaced by pseudoisocytosine bases (J bases), which do not require protonation to form triplexes, a weak dependence on pH of the rates and specificity of the P-loop formation is observed. A theoretical analysis of the data suggests that for (C+T)-containing bis-PNA the first, intermediate step of PNA binding to dsDNA occurs via Hoogsteen pairing between the duplex target and one oligomer of bis-PNA. After that, the strand invasion occurs via Watson-Crick pairing between the second bis-PNA oligomer and the homopurine strand of the target DNA, thus resulting in the ultimate formation of the P-loop. The data for the (C/J+T)-containing bis-PNA show that its high affinity to dsDNA at neutral pH does not seriously compromise the kinetic specificity of binding. These findings support the earlier expectation that (C/J+T)-containing PNA constructions may be advantageous for use in vivo.     

Structural isomers of bis-PNA bound to a target in duplex DNA

Upon binding of a decamer bis-PNA (H-Lys-TTCCTCTCTT-(eg1)(3)-TTCTCTCCTT-LysNH(2)) to a complementary target in a double-stranded DNA fragment, three distinct complexes were detected by gel mobility shift analysis. Using in situ chemical probing techniques (KMnO(4) and DMS) it was found that all three complexes represent bona fide sequence-specific PNA binding to the designated target, but the complexes were structurally different. One complex that preferentially formed at higher PNA concentrations contains two bis-PNA molecules per DNA target, whereas the other two complexes are genuine triplex invasion clamped structures. However, these two latter complexes differ by the path relative to the DNA target of the flexible ethylene-glycol linker connecting the two PNA oligomers that comprise a bis-PNA. We distinguish between one in which the linker wraps around the non-target DNA strand, thus making this strand part of the triplex invasion complex and another complex that encompass the target strand only. The implications of these results are discussed in terms of DNA targeting by synthetic ligands.     

Peptide nucleic acid-anthraquinone conjugates: strand invasion and photoinduced cleavage of duplex DNA.

A bis-peptide nucleic acid (PNA)-anthraquinone imide (AQI) conjugate has been synthesized and shown to form strand invasion complexes with a duplex DNA target. The two arms of the bis-PNA each consist of five consecutive thymine residues and are linked by a flexible, hydrophilic spacer. Probing with potassium permanganate reveals that the bis-PNA complexes to duplex DNA at A5.T5sites with local displacement of the T5DNA strand. The 5 bp sequence targeted by the PNA is the shortest strand invasion complex reported to date. Irradiation of the strand invasion complex results in asymmetric cleavage of the displaced strand, with more efficient cleavage at the 3'-end of the loop. This result indicates that the bis-PNA binds to the DNA such that the C-terminal T5sequence forms the strand invasion complex, leaving the N-terminal T5sequence to bind by triplex formation, thereby placing the AQI closer to the 3'-end of the displaced strand, consistent with the observed photocleavage pattern. The ability of the PNA to directly report its binding site by photoinduced cleavage could have significant utility in mapping the secondary and tertiary structure of nucleic acids.     

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.     

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.     

Triplex formation by morpholino oligodeoxyribonucleotides in the HER-2/neu promoter requires the pyrimidine motif

Triplex-forming oligonucleotides (TFOs) are good candidates to be used as site-specific DNA-binding agents. Two obstacles encountered with TFOs are susceptibility to nuclease activity and a requirement for magnesium for triplex formation. Morpholino oligonucleotides were shown in one study to form triplexes in the absence of magnesium. In the current study, we have compared phosphodiester and morpholino oligonucleotides targeting a homopurine–homopyrimidine region in the human HER2/neu promoter. Using gel mobility shift analysis, our data demonstrate that triplex formation by phosphodiester oligonucleotides at the HER-2/neu promoter target is possible with pyrimidine-parallel, purine-antiparallel and mixed sequence (GT)-antiparallel motifs. Only the pyrimidine-parallel motif morpholino TFO was capable of efficient triple helix formation, which required low pH. Triplex formation with the morpholino TFO was efficient in low or no magnesium. The pyrimidine motif TFOs with either a phosphodiester or morpholino backbone were able to form triple helices in the presence of potassium ions, but required low pH. We have rationalized the experimental observations with detailed molecular modeling studies. These data demonstrate the potential for the development of TFOs based on the morpholino backbone modification and demonstrate that the pyrimidine motif is the preferred motif for triple helix formation by morpholino oligonucleotides.     

Synthesis and DNA binding properties of DNA-PNA chimeras

A systematic study to evaluate the ability of 5'-DNA-3'-p-(N)-PNA-(C) chimeras to form triple helix structures has been undertaken. Preliminary results carried out on a 16-mer chimera with three PNA monomers at the 3'-end showed the formation of a stable DNA-PNA/DNA/DNA triplex, having similar conformational behaviour to a canonical DNA/DNA/DNA triplex.     

Effect of temperature and ionic strength on the dissociation kinetics and lifetime of PNA-DNA triplexes

Dissociation kinetics of triplexes formed by molecules of peptide nucleic acid (PNA) and DNA have been studied. The complexes consisted of oligomeric PNA containing 10 thymine bases and the dA(10) target incorporated in single-stranded (ssDNA) or double-stranded DNA (dsDNA). Their dissociation was followed by means of the gel mobility shift assay at various temperatures and sodium ion concentrations. In all experiments, the dissociation kinetics of triplexes were exponential; the effective lifetime of a triplex, tau, depended on temperature in accordance with the Arrhenius law. The tau values for T(10) PNA complexes with ss- and dsDNA were equal within the accuracy of experiments. The activation energy, U, value for T(10) PNA-DNA complexes did not change when the NaCl concentration was increased from 50 to 200 or 600 mM. Conversely, the tau values decreased with the increase in NaCl concentration. The equal lifetimes of the T(10) PNA-DNA triplexes containing ss- and dsDNA suggest that the loop formed in dsDNA does not noticeably affect the triplex structure. The decrease in the triplex lifetime tau with an increase in ionic strength was accounted for by the fact that the PNA backbone is neutral. The lack of relationship between the activation energy of dissociation and salt concentration suggests that the dissociation enthalpy does not depend on the ionic strength. Thus, the effect of ionic strength on the lifetime is entropic by its nature. Contrary to this, for complexes of ssDNA with bis-PNA 1743, which also consists of 10 thymine bases but contains 2 additional positive charges inside the sequence in 1 of the PNA arms, an increase of the dissociation enthalpy at low salt concentration was observed. We suggest that this effect is a result of a direct electrostatic interaction of the positive charges of the PNA with the DNA backbone. Finally, our results allow an estimate of the lifetime of a 10-mer triplex invasion complex in dsDNA at 37 degrees C in excess of several hundred days.     

Relative specificities in binding of Watson-Crick base pairs by third strand residues in a DNA pyrimidine triplex motif.

The specificity of binding of Watson-Crick base pairs by third strand nucleic acid residues via triple helix formation was investigated in a DNA pyrimidine triplex motif by thermal melting experiments. The host duplex was of the type A10-X-A10: T10-Y-T10, and the third strand T10-Z-T10, giving rise to 16 possible triplexes with Z:XY inserts, 4 duplexes with the Watson-Crick base pairs (XY) and 12 duplexes with mismatch pairs (XZ), all of whose stabilities were compared. Two Z:XY combinations confirm the primary binding of AT and GC target pairs in homopurine.homopyrimidine sequences by T and C residues, respectively. All other Z:XY combinations in the T:AT environment result in triplex destabilization. While some related observations have been reported, the present experiments differ importantly in that they were performed in a T:AT nearest neighbor environment and at physiological ionic strength and pH, all of which were previously untested. The conclusions now drawn also differ substantially from those in previous studies. Thus, by evaluating the depression in Tm due to base triplet mismatches strictly in terms of third strand residue affinity and specificity for the target base pair, it is shown that none of the triplet combinations that destabilize qualify for inclusion in the third strand binding code for the pyrimidine triplex motif. Hence, none of the mismatch triplets afford a general way of circumventing the requirement for homopurine.homopyrimidine targets when third strands are predominated by pyrimidines, as others have suggested. At the same time, the applicability of third strand binding is emphasized by the finding that triplexes are equally or much more sensitive to base triplet mismatches than are Watson-Crick duplexes to base pair mismatches.     

Synthesis and DNA Binding Properties of DNA-PNA Chimeras

Abstract

A systematic study to evaluate the ability of 5′-DNA-3′-p-(N)-PNA-(C) chimeras to form triple helix structures has been undertaken. Preliminary results carried out on a 16-mer chimera with three PNA monomers at the 3′-end showed the formation of a stable DNA-PNA/DNA/DNA triplex, having similar conformational behaviour to a canonical DNA/DNA/DNA triplex.     

Relative stabilities of triple helices composed of combinations of DNA, RNA and 2'-O-methyl-RNA backbones: chimeric circular oligonucleotides as probes.

Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O-methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding.