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.     

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.     

Photo-cross-linking of psoralen-derivatized oligonucleoside methylphosphonates to single-stranded DNA.

The preparation of oligodeoxyribonucleoside methylphosphonates derivatized with 3-[(2-aminoethyl)carbamoyl]psoralen [(ae)CP] is described. These derivatized oligomers are capable of cross-linking with single-stranded DNA via formation of a photoadduct between the furan side of the psoralen ring and a thymidine of the target DNA when the oligomer-target duplex is irradiated with 365-nm light. The photoreactions of (ae)CP-derivatized methylphosphonate oligomers with single-stranded DNA targets in which the position of the psoralen-linking site is varied are characterized and compared to results obtained with oligomers derivatized with 4'-[[N-(aminoethyl)amino]methyl]-4,5',8-trimethylpsoralen [(ae)AMT]. It appears that the psoralen ring can stack on the terminal base pair formed between the oligomer and its target DNA or can intercalate between the last two base pairs of the oligomer-target duplex. Oligomers derivatized with (ae)CP cross-link efficiently to a thymidine located in the last base pair (n position) or 3' to the last base pair (n + 1 position) of the target, whereas the (ae)AMT-derivatized oligomers cross-link most efficiently to a thymidine located in the n + 1 position. The results show that both the extent and kinetics of cross-linking are influenced by the location of the psoralen-linking site in the oligomer-target duplex.     

Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

A new generation of PNAs, so-called pseudocomplementary PNAs (pcPNAs), which are able to target the designated sites on duplex DNA with mixed sequence of purines and pyrimidines via double-duplex invasion mode, has recently been introduced. It has been demonstrated that appropriate pairs of decameric pcPNAs block an access of RNA polymerase to the corresponding promoter. Here, we show that this type of PNAs protects selected DNA sites containing all four nucleobases from the action of restriction enzymes and DNA methyltransferases. We have found that pcPNAs as short as octamers form stable and sequence-specific complexes with duplex DNA in a very salt-dependent manner. In accord with a strand-invasion mode of complex formation, the pcPNA binding proceeds much faster with supercoiled than with linear plasmids. The double-duplex invasion complexes selectively shield specific DNA sites from BclI restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate DNA duplexes in a virtually sequence-unrestricted manner.     

Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis

Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets. We show that the tridecamer PNA (targeted to codon 149) containing a CTC(3)T pyrimidine stretch forms with the complementary oligoribonucleotide (ORN) a stable (PNA)(2)/ORN triplex at neutral pH (T(m) = 50 degrees C) and arrests Ha-ras mRNA translation elongation. Interestingly, the thermal stability of triplexes formed with PNAs designed to bind to the complementary ORN in a parallel orientation (the N-terminal of the PNA faces the 5'-end of target) was higher than that formed with antiparallel oriented PNAs (T(m) = 58 degrees C). Because parallel and antiparallel PNAs form stable triplexes with target sequence, they act as translation elongation blockers. These duplex-forming and partly triplex-forming PNAs targeted to Ha-ras mRNA also arrested translation elongation at specific polypurine sites contained in the mRNA coding for HIV-integrase protein. Furthermore, the tridecamer PNA containing the C(3)TC(4) motif was more active than a bis-PNA in which the Hoogsteen recognizing strand was linked to the Watson-Crick recognizing strand by a flexible linker. Pyrimidine-rich, short PNAs that form very stable duplexes with target Ha-ras mRNA inhibit translation by a mechanism that does not involve ribosome elongation arrest, whereas PNAs forming duplex and triplex structures arrest ribosome elongation. The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.     

RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligomer

Recently, we have shown that peptide nucleic acid (PNA) tridecamers targeted to the codon 74, 128 and 149 regions of Ha-ras mRNA arrested translation elongation in vitro. Our data demonstrated for the first time that PNAs with mixed base sequence targeted to the coding region of a messenger RNA could arrest the translation machinery and polypeptide chain elongation. The peculiarity of the complexes formed with PNA tridecamers and Ha-ras mRNA rests upon the stability of PNA-mRNA hybrids, which are not dissociated by cellular proteins or multiple denaturing conditions. In the present study, we show that shorter PNAs such as a dodecamer or an undecamer targeted to the codon 74 region arrest translation elongation in vitro. The 13, 12, and 11-mer PNAs contain eight and the 10-mer PNA seven contiguous pyrimidine residues. Upon binding with parallel Hoogsteen base-pairing to the PNA-RNA duplex, six of the cytosine bases and one thymine base of a second PNA can form C.G*C(+) and T.A*T triplets. Melting experiments show two well-resolved transitions corresponding to the dissociation of the third strand from the core duplex and to melting of duplex at higher temperature. The enzymatic structure mapping of a target 27-mer RNA revealed a hairpin structure that is disrupted upon binding of tri-, dodeca-, undeca- and decamer PNAs. We show that the non-bonded nucleobase overhangs on the RNA stabilize the PNA-RNA hybrids and probably assist the PNA in overcoming the stable secondary structure of the RNA target. The great stability of PNA-RNA duplex and triplex structures allowed us to identify both 1:1 and 2:1 PNA-RNA complexes using matrix-assisted laser desorption/ionization time-of -flight mass spectrometry. Therefore, it is possible to successfully target mixed sequences in structured regions of messenger RNA with short PNA oligonucleotides that form duplex and triplex structures that can arrest elongating ribosomes.     

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.     

Peptide nucleic acids: deoxyribonucleic acid mimics with a peptide backbone

This tutorial briefly describes a new class of synthetic biopolymer, which is referred to as peptide nucleic acid (PNA). In PNA, individual nucleobases are linked to an achiral neutral peptide backbone. PNA exhibits the hybridization characteristic (e.g., Watson—Crick duplex formation) of DNA. The achiral peptide backbone provides similar interbase distances as natural DNA, and adequate flexibility to permit base pair interactions with complementary RNA or DNA strands. Several potential applications of PNA oligomers in biotechnology are suggested. These include the use of PNAs as a probe for specific recognition of a DNA or RNA sequence selective, purification of nucleic acids via designed high affinity binding to PNA, screening for DNA mutations, and as possible therapeutic agents.     

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.     

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.     

Structural diversity of target-specific homopyrimidine peptide nucleic acid-dsDNA complexes

Sequence-selective recognition of double-stranded (ds) DNA by homopyrimidine peptide nucleic acid (PNA) oligomers can occur by major groove triplex binding or by helix invasion via triplex P-loop formation. We have compared the binding of a decamer, a dodecamer and a pentadecamer thymine–cytosine homopyrimidine PNA oligomer to a sequence complementary homopurine target in duplex DNA using gel-shift and chemical probing analyses. We find that all three PNAs form stable triplex invasion complexes, and also conventional triplexes with the dsDNA target. Triplexes form with much faster kinetics than invasion complexes and prevail at lower PNA concentrations and at shorter incubation times. Furthermore, increasing the ionic strength strongly favour triplex formation over invasion as the latter is severely inhibited by cations. Whereas a single triplex invasion complex is formed with the decameric PNA, two structurally different target-specific invasion complexes were characterized for the dodecameric PNA and more than five for the pentadecameric PNA. Finally, it is shown that isolated triplex complexes can be converted to specific invasion complexes without dissociation of the Hoogsteen base-paired triplex PNA. These result demonstrate a clear example of a ‘triplex first’ mechanism for PNA helix invasion.     

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.     

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.     

Specific versus nonspecific binding of cationic PNAs to duplex DNA

Although peptide nucleic acids (PNAs) are neutral by themselves, they are usually appended with positively charged lysine residues to increase their solubility and binding affinity for nucleic acid targets. Thus obtained cationic PNAs very effectively interact with the designated duplex DNA targets in a sequence-specific manner forming strand-invasion complexes. We report on the study of the nonspecific effects in the kinetics of formation of sequence-specific PNA-DNA complexes. We find that in a typical range of salt concentrations used when working with strand-invading PNAs (10-20 mM NaCl) the PNA binding rates essentially do not depend on the presence of nontarget DNA in the reaction mixture. However, at lower salt concentrations (<10 mM NaCl), the rates of PNA binding to DNA targets are significantly slowed down by the excess of unrelated DNA. This effect of nontarget DNA arises from depleting the concentration of free PNA capable of interacting with DNA target due to adhesion of positively charged PNA molecules on the negatively charged DNA duplex. As expected, the nonspecific electrostatic effects are more pronounced for more charged PNAs. We propose a simple model quantitatively describing all major features of the observed phenomenon. This understanding is important for design of and manipulation with the DNA-binding polycationic ligands in general and PNA-based drugs in particular.     

Recognition of Double-Stranded Dna by Peptide Nucleic Acid

Abstract

Peptide nucleic acid (PNA) is an oligonucleotide mimic in which the backbone of DNA has been replaced by a pseudopeptide. We here show that there are distinct variations as to how PNA oligomers interact with double-stranded DNA depending on choice of nucleobases. Thymine-rich homopyrimidine PNA oligomers recognise double-stranded polynucleotides by forming PNA₂-DNA triplexes with the DNA purine strand. By contrast, cytosine-rich homopyrimidine PNAs add to double-stranded polynucleotides as Hoogsteen strands, forming PNA-DNA₂ triplexes, while homopurine, or alternating thymine-guanine, PNA oligomers invade DNA to form PNA-DNA duplexes.     

Peptide nucleic acid (PNA) amphiphiles: synthesis, self-assembly, and duplex stability

Peptide amphiphiles comprising a class of conjugates of peptide nucleic acid (PNA), natural amino acids, and n-alkanes were synthesized and studied. These PNA amphiphiles (PNAA) self-assemble at concentrations between 10 and 50 muM and exhibit water solubilities above 500 muM. The highly specific, stable DNA binding properties of PNAs are preserved by these modifications, with no significant differences between the thermodynamics of DNA binding of the PNA peptide and the PNA amphiphile. Proper solubilization of the PNAA required the attachment of (Lys)(2) and (Glu)(4) peptides to PNAs, which affected the PNAA-DNA duplex stability by electrostatic interactions between these charged amino acids and the negatively charged DNA backbone. These electrostatic effects did not affect the specificity of DNA binding, however. Electrostatic effects are screened with added salt, in a manner consistent with previous studies of PNA-DNA duplex stability and predictions from a charged-cylinder model for the duplex.