Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO₄, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO₄, 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of ≥100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10⁻²¹ M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.     

Affinity capture and recovery of DNA at femtomolar concentrations with peptide nucleic acid probes

The efficacy of PNA vs DNA oligomers for the recovery of femtomolar concentrations of 16S rDNA targets was determined with solution- and mixed-phase hybridization formats and limiting dilution quantitative PCR. Several results contradict existing perceptions of expected PNA behavior deduced from hybridization studies with oligonucleotide targets at high concentration. For example, DNA probes in the solution hybridization format performed as well as or better than PNA probes under high- or low-salt conditions, regardless of hybridization time or target size. In the mixed-phase hybridization format, however, PNA probes showed certain advantages, with more rapid and efficient binding/recovery of target nucleic acids regardless of target size. Recovery of target DNA with PNA probes was always more efficient in low-salt (20 mM in Na(+)) than high-salt (400 mM in Na(+-)) phosphate buffer. Recovery of target DNA by PNA probes was enhanced in the presence of excess, nontarget DNA, and differences in PNA efficacy under low- or high-salt conditions vanquished. In contrast, DNA probe performance was unaffected by the presence or absence of exogenous DNA in both solution- and mixed-phase hybridization formats. The absolute recovery and detection limit of the affinity purification method with either DNA or PNA probes was approximately 10(2) input target molecules at zeptamolar concentrations.     

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.     

PNA openers as a tool for direct quantification of specific targets in duplex DNA

We report a new approach for target quantification directly within DNA duplex. Our assay is based on the formation of a new biomolecular structure, the PD-loop. The approach takes advantage of a selective hybridization of a probe to double-stranded DNA (dsDNA), which is locally opened by a pair of bis-PNA oligomers. To optimize the technique, several experimental formats are tested with the use of PNA and oligonucleotide probes. The highest sensitivity is achieved when the hybridized probe is extended and multiply labeled with 125I-dCTP by DNA polymerase via strand displacement in the presence of single-strand binding (SSB) protein. In this case, the PNA-assisted probe hybridization combined with the method of multiphoton detection (MPD) allows to monitor sub-attomolar amounts of the HIV-1 target on the background of unrelated DNA at sub-nCi level of radioactivity. The developed robust methodology is highly discriminative to single mutations, thus being of practical use for DNA analysis.     

Snap-to-it probes: chelate-constrained nucleobase oligomers with enhanced binding specificity

We describe snap-to-it probes, a novel probe technology to enhance the hybridization specificity of natural and unnatural nucleic acid oligomers using a simple and readily introduced structural motif. Snap-to-it probes were prepared from peptide nucleic acid (PNA) oligomers by modifying each terminus with a coordinating ligand. The two coordinating ligands constrain the probe into a macrocyclic configuration through formation of an intramolecular chelate with a divalent transition metal ion. On hybridization with a DNA target, the intramolecular chelate in the snap-to-it probe dissociates, resulting in the probe ‘snapping-to’ and binding the target nucleic acid. Thermal transition analysis of snap-to-it probes with complementary and single-mismatch DNA targets revealed that the transition between free and target-bound probe conformations was a reversible equilibrium, and the intramolecular chelate provided a thermodynamic barrier to target binding that resulted in a significant increase in mismatch discrimination. A 4–6°C increase in specificity (ΔT_m) was observed from snap-to-it probes bearing either terminal iminodiacetic acid ligands coordinated with Ni²⁺, or terminal dihistidine and nitrilotriacetic acid ligands coordinated with Cu²⁺. The difference in specificity of the PNA oligomer relative to DNA was more than doubled in snap-to-it probes. Snap-to-it probes labeled with a fluorophore-quencher pair exhibited target-dependent fluorescence enhancement upon binding with target DNA.     

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.     

Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations

The detection of rare mutant DNA from a background of wild-type alleles usually requires laborious manipulations, such as restriction enzyme digestion and gel electrophoresis. Here, we describe a protocol for homogeneous detection of rare mutant DNA in a single tube. The protocol uses a peptide nucleic acid (PNA) as both PCR clamp and sensor probe. The PNA probe binds tightly to perfectly matched wild-type DNA template but not to mismatched mutant DNA sequences, which specifically inhibits the PCR amplification of wild-type alleles without interfering with the amplification of mutant DNA. A fluorescein tag (which undergoes fluorescence resonance energy transfer with the adjacent fluorophore of an anchor probe when both are annealed to the template DNA) also allows the PNA probe to generate unambiguous melting curves to detect mutant DNA during real-time fluorescent monitoring. The whole assay takes about only 1 h. This protocol has been used for detecting mutant K-ras DNA and could be applied to the detection of other rare mutant DNAs.     

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.     

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.     

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.     

Peptide nucleic acid (PNA)/DNA hybrid duplexes: intercalation by an internally linked anthraquinone.

Peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing. A set of reporter compounds that bind to DNA by intercalation are known, but these compounds do not intercalate in PNA/DNA hybrid duplexes. Analysis of the hybrid PNA duplexes requires development of reporter compounds that probe their chemical and physical properties. We prepared a series of anthraquinone (AQ) derivatives that are linked to internal positions of a PNA oligomer. These are the first non-nucleobase functional groups that have been incorporated into a PNA. The resulting PNA(AQ) conjugates form stable hybrids with complementary DNA oligomers. We find that when the AQ groups are covalently bound to PNA that they stabilize the hybrid duplex and are, at least partially, intercalated.     

Preparation and evaluation of a peptide nucleic acid gene chip system associated with surface plasmon resonance

The aim of this study was to construct a gene chip system based on a surface plasmon resonance technique, where peptide nucleic acid (PNA) oligomers are used as probes. Since the self-assembled monolayer (SAM) technology offers good control at the molecular level, we prepared 2D surface chemistry via SAM for probe attachments. PNA, which was designed according to the bioinformatics, was immobilized on the SAM-modified chip, and subsequently, relevant parameters of the experiment were ensured and optimized. Our results suggest that the ion strength and pH value of the buffer solution do not play significant roles in PNA or its complementary strand hybridization. The PNA probe binds to its complementary nucleic acid strand with a higher sensitivity and specificity compared to those of a traditional DNA probe. The PNA probe combined with surface plasmon resonance (SPR) technology has the benefits of being a label-free and in-real time monitor, as well as having improved hybridization and stability efficiency, which highlight the PNA gene chip detection system as a promising biosensor for clinical applications.     

Rapid detection of human papilloma virus using a novel leaky surface acoustic wave peptide nucleic acid biosensor

A novel leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor with double two-port resonators has been constructed successfully for the quantitative detection of human papilloma virus (HPV). The bis-PNA probe can directly detect HPV genomic DNA without polymerase chain reaction (PCR) amplification, and it can bind to the target DNA sequences more effectively and specifically than a DNA probe. When the concentrations varied from 1 pg/L to 1000 μg/L, with 100 μg/L being the optimal, a typical linearity was found between the quantity of target and the phase shifts. The detection limit was 1.21 pg/L and the clinical specificity was 97.22% of that of real-time PCR. The bis-PNA probe was able to distinguish sequences that differ only in one base. Both the intraassay and interassay coefficients of variance (CVs) were <10%, and the biosensor can be regenerated for ten times without appreciable loss of activity. Therefore, this technical platform of LSAW biosensor can be applied to clinical samples for direct HPV detection.     

Synthesis of polyacrylamides N-substituted with PNA-like oligonucleotide mimics for molecular diagnostic applications.

Two types of oligonucleotide mimics relative to peptide nucleic acids (PNAs) were tested as probes in nucleic acid hybridisation assays based on polyacrylamide technology. One type of mimic oligomers represented a chimera constructed of PNA and phosphono-PNA (pPNA) monomers, and the other one contained pPNA residues alternating with PNA-like monomers on the base of trans -4-hydroxy-L-proline (HypNA). A chemistry providing efficient and specific covalent attachment of these DNA mimics to acrylamide polymers using a convenient approach based on the co-polymerisation of acrylamide and some reactive acrylic acid derivatives with oligomers bearing 5'- or 3'-terminal acrylamide groups has been developed. A comparative study of polyacrylamide conjugates with oligonucleotides and mimic oligomers demonstrated the suitability and high potential of PNA-pPNA and HypNA-pPNA chimeras as sequence-specific probes in capture and detection of target nucleic acid fragments to serve current forms of DNA arrays.     

Synthesis of labelled PNA oligomers by a post-synthetic modification approach

The preparation of t-butoxycarbonyl (Boc)-protected O(4)-(o-nitrophenyl) thymine peptide nucleic acid (PNA) monomer is described. This PNA monomer was incorporated into PNA oligomer sequences. The post-synthetic modification of the oligomers to yield fluorescently-labelled PNA oligomers was studied before and after the removal of the protecting groups. In both cases, the desired fluorescently-labelled PNA oligomer was obtained in good yields.     

Affinity capture of specific DNA fragments with short synthetic sequences

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine, and 5-propynyl-2′-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments in mixtures.     

Detection of peptide nucleic acids in tissue extracts of treated animals by gel mobility shift assay

We have developed a sensitive and reproducible gel mobility shift assay to detect PNA oligomers in tissue of treated animals. PNA present in purified tissue extracts of treated animals is hybridized to a 33P-labelled DNA oligomer probe, and analyzed by polyacrylamide gel electrophoresis. The PNA-DNA hybrid migrates more slowly than the DNA probe alone and can be quantified relative to a standard curve. This detection method is useful for detecting PNAs in many different tissues, including brain, heart, kidney, liver, spleen, and serum, as well as cells in culture.     

Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs

The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of L-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.     

Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells

In the search of facile and efficient methods for cellular delivery of peptide nucleic acids (PNA), we have synthesized PNAs conjugated to oligophosphonates via phosphonate glutamine and bis-phosphonate lysine amino acid derivatives thereby introducing up to twelve phosphonate moieties into a PNA oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range as inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. Antisense activity depended on the number of phosphonate moieties and the most potent hexa-bis-phosphonate-PNA showed at least 20-fold higher activity than that of an optimized PNA/DNA hetero-duplex. These results indicate that conjugation of phosphonate moieties to the PNA can dramatically improve cellular delivery mediated by cationic lipids without affecting on the binding affinity and sequence discrimination ability, exhibiting EC(50) values down to one nanomolar. Thus the intracellular efficacy of PNA oligomers rival that of siRNA and the results therefore emphasize that provided sufficient in vivo bioavailability of PNA can be achieved these molecules may be developed into potent gene therapeutic drugs.     

Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.