Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.     

Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA

Herein we describe the first application of direct linear analysis (DLA) to the mapping of a bacterial artificial chromosome (BAC), specifically the 185.1 kb-long BAC 12M9. DLA is a single molecule mapping technology, based on microfluidic elongation and interrogation of individual DNA molecules, sequence-specifically tagged with bisPNAs. A DNA map with S/N ratio sufficiently high to detect all major binding sites was obtained using only 200 molecule traces. A new method was developed to extract an oriented map from an averaged map that included a mixture of head-first and tail-first DNA traces. In addition, we applied DLA to study the conformation and tagging of highly stretched DNA. Optimal conditions for promoting sequence-specific binding of bisPNA to an 8 bp target site were elucidated using DLA, which proved superior to electromobility shift assays. DLA was highly reproducible with a hybridized tag position localized with an accuracy of ±0.7 µm or ±2.1 kb demonstrating its utility for rapid mapping of large DNA at the single molecule level. Within this accuracy, DNA molecules, stretched to at least 85% of their contour length, were stretched uniformly, so that the map expressed in relative coordinates, was the same regardless of the molecule extension.     

DNA detection using nanostructured SERS substrates with Rhodamine B as Raman label

A technique is demonstrated to detect DNA hybridization at low concentrations, based on Surface-Enhanced Raman Scattering (SERS) using silicon nanostructures coated with gold-silver as substrate. Standard silicon process technologies were employed to fabricate the SERS substrates featuring nanogaps with a characteristic distance of 15+/-10nm. Target DNA was hybridized with cysteine-modified Peptide Nucleic Acids (PNA), which was previously fixed into the nanogaps as the capture sites. After hybridization, the introduced phosphate groups from the backbone of the target DNA showed strong affinity to an inorganic linker, Zr(4+), so that resulting in the assembly substrate-PNA-DNA-Zr. Since PNA does not possess phosphate groups, the linker is avoided when there is no hybridization from the complimentary DNA. Subsequently, the assembly of substrate-PNA-DNA-Zr was incubated with a Raman label, Rhodamine B (RB). The carboxylic acid group in RB reacted with the linker Zr(4+) allowing this Raman Label to be attached to the assembly substrate-PNA-DNA-Zr. The Raman peaks corresponding to RB were selected to detect the target DNA, with a detection limit of 1x10(-12)M.     

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.     

Antisense PNA tridecamers targeted to the coding region of Ha-ras mRNA arrest polypeptide chain elongation

We have previously described the rational design of mutation-selective antisense oligonucleotides targeted to codon 12 of oncogenic Ha-ras mRNA. In order to further improve the biological efficacy of these unmodified oligonucleotides, we have studied three different classes of modifications: peptide nucleic acid backbone (PNA), sugar modification (2'-O-methyl) and phosphoramidate linkage (PN). We show that PNA is unique among the investigated steric blocking agents in its ability to specifically inhibit the translation of Ha-ras mRNA in vitro. The PNA-RNA hybrid (Tm=86 degrees C), which is not dissociated by cellular proteins and resists phenol extraction and urea denaturing conditions, specifically blocks the translation of mutated Ha-ras mRNA. A PNA tridecamer which forms with wild-type Ha-ras mRNA a duplex with a central mismatch had little effect on mRNA translation. Codon 12 is located close to the translation initiation site and hybridization of the PNA at this position may interfere with the assembly of the translation initiation complex. To test whether polypeptide chain elongation can also be blocked, we have targeted PNA tridecamers to codons in the 74, 128 and 149 regions. These PNAs form equally stable duplexes as that formed by the PNA targeted to the codon 12 region (ten G.C base-pairs out of 13). We show that PNA-RNA duplexes block the progression of the 80 S ribosome. Therefore, it is possible to arrest translation with concomitant production of a truncated protein by using duplex-forming PNA oligonucleotides targeted to a G+C-rich sequences. Our data demonstrate for the first time that a non-covalent duplex can arrest the translation machinery and polypeptide chain elongation.     

An efficient and versatile synthesis of bisPNA-peptide conjugates based on chemoselective oxime formation

Oligomers with two identical peptide nucleic acid sequences joined by a flexible hairpin linker (bisPNA) can stably bind to specific DNA sequences without altering plasmid supercoiling, thus offering a unique opportunity to attach various functional entities to high molecular weight DNA. Current synthetic approaches, however, severely limit the possibility to link peptides or other chemical moieties (i.e., sugars, oligonucleotides, etc.) to bisPNA. Here we report a novel strategy for the synthesis of bisPNA-peptide conjugates in which chemoselective ligation of bisPNA to peptides was accomplished through oxime formation between an oxy-amine-containing peptide and a bisPNA-methyl ketone (complementary modifications can also be used). The described synthesis is highly efficient, does not require a protection strategy, and is carried out under mild aqueous conditions. Through this methodology long peptide sequences in either C to N or N to C polarity can be linked to bisPNA. In addition, this protocol makes the conjugation of cysteine-containing peptides feasible and allows disulfide bond formation to be controlled. This same approach can be exploited to link oligonucleotides, sugars, or other chemical entities to bisPNA.     

Measuring single-molecule DNA hybridization by active control of DNA in a nanopore

We present a novel application of active voltage control of DNA captured in a nanopore to regulate the amount of time the DNA is available to molecules in the bulk phase that bind to the DNA. In this work, the control method is used to measure hybridization between a single molecule of DNA captured in a nanopore and complementary oligonucleotides in the bulk phase. We examine the effect of oligonucleotide length on hybridization, and the effect of DNA length heterogeneity on the measurements. Using a mathematical model, we are able to deduce the binding rate of complementary oligonucleotides, even when DNA samples in experiments are affected by heterogeneity in length. We analyze the lifetime distribution of DNA duplexes that are formed in the bulk phase and then pulled against the pore by reversing the voltage. The lifetime distribution reveals several dissociation modes. It remains to be resolved whether these dissociation modes are due to DNA heterogeneity or correspond to different states of duplex DNA. The control method is unique in its ability to detect single-molecule complex assembly in the bulk phase, free from external force and with a broad (millisecond-to-second) temporal range.     

Conformational and chiral selection of oligonucleotides

In view of a better understanding of chiral selection of oligonucleotides, we have studied the hybridization of D- and L-CNA (cyclohexane nucleic acids) and D- and L-DNA, with chiral D-beta-homo-DNA and achiral PNA (peptide nucleic acids). PNA hybridizes as well with D-DNA, L-DNA as with D-beta-homo-DNA. The structure of the PNA x D-beta-homo-DNA complex is different from the PNA x DNA duplexes. D-CNA prefers D-DNA as hybridization partner, while L-CNA prefers D-beta-homo-DNA as hybridization partner. The conformation of the enantiomeric oligonucleotides D-CNA and L-CNA in the supramolecular complex with D-DNA and D-beta-homo-DNA, respectively, is different. These data may contribute to the confirmation of a hypothesis of the existence of achiral informative polymers as RNA predecessor, and to the understanding of homochirality of nucleic acids.     

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 NaPO(4), 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 NaPO(4), 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 > or =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.     

Taking control of gene expression with light-activated oligonucleotides

The recent development of caged oligonucletides that are efficiently activated by ultraviolet (UV) light creates opportunities for regulating gene expression with very high spatial and temporal resolution. By selectively modulating gene activity, these photochemical tools will facilitate efforts to elucidate gene function and may eventually serve therapeutic aims. We demonstrate how the incorporation of a photocleavable blocking group within a DNA duplex can transiently arrest DNA polymerase activity. Indeed, caged oligonucleotides make it possible to control many different protein-oligonucleotide interactions. In related experiments, hybridization of a reverse complementary (antisense) oligodeoxynucleotide to target mRNA can inhibit translation by recruiting endogenous RNases or sterically blocking the ribosome. Our laboratory recently synthesized caged antisense oligonucleotides composed of phosphorothioated DNA or peptide nucleic acid (PNA). The antisense oligonucleotide, which was attached to a complementary blocking oligonucleotide strand by a photocleavable linker, was blocked from binding target mRNA. This provided a useful method for photomodulating hybridization of the antisense strand to target mRNA. Caged DNA and PNA oligonucleotides have proven effective at photoregulating gene expression in cells and zebrafish embryos.     

Formation of the double helix: a mutational study

To investigate the mechanisms by which oligonucleotides hybridize to target molecules, the binding of two oligodeoxynucleotide probes to RNA targets was measured over a broad range of temperatures. Mutations were then scanned across each DNA/RNA hybrid to map, at single base resolution, sequences important for hybridization. Despite being unrelated in sequence, each hybrid formed by a similar mechanism. In the absence of secondary structure, two stretches of bases, termed nucleation regions, cooperated with one another by a looping mechanism to nucleate hybridization. Mutations inside each nucleation region strongly decreased hybridization rates, even at temperatures well below the melting temperature (T_m) of the hybridized duplex. Surprisingly, nucleation regions were detected in a RNA target but not a corresponding DNA target. When either nucleation region was sequestered in secondary structure, the hybridization rate fell and the mechanism of hybridization changed. Single-stranded bases within the nucleation region of the probe and target first collided to form a double helix. If sufficiently G + C rich, the double helix then propagated throughout the oligonucleotide by a strand invasion process. On the basis of these results, general mechanisms for the hybridization of oligonucleotides to complementary and mutant targets are proposed.     

Synthesis and Hybridization Properties of Modified Oligonucleotides with PNA-DNA Dimer Blocks

Abstract

Different modified PNA-DNA dimer-analogous synthons (I and II) were synthesized as phosphoramidites. These dimer units were assembled by a 5′-modified deoxythymidine and a modified PNA monomer. These synthons were used in the routine coupling procedure for oligonucleotides. Therefore no PNA coupling chemistry is necessary to synthesize PNA-DNA chimeric oligonucleotides. Various deoxyoligonucleotides were synthesized introducing the dimer blocks I and II at different positions in the sequences. Melting temperatures of the modified oligonucleotides with their complementary DNA analogues were determined.

Backbone modifications of oligonucleotides are required in the antisense strategy for protection against endonucleolytic cleavage in biological environment. Peptide nucleic acids (PNA fragments) are known to be nuclease resistant analogues, which show stable and discriminating hybridization. For this reason we prepared chimeric PNA-DNA oligomers by incorporation of two different modified PNA-DNA dimer blocks (Scheme A) into oligonucleotides. Melting temperatures of the modified oligonucleotides with their complementary DNA were determined.     

SNP genotyping using microsphere-linked PNA and flow cytometric detection.

BACKGROUND: Single nucleotide polymorphisms (SNPs) represent the most frequent form of genetic variations. Some of the most sensitive methods for SNP genotyping employ synthetic oligonucleotides, such as the peptide nucleic acid (PNA). We introduce a new method combining allele-specific hybridization, PNA technology, and flow cytometric detection. We tested the design by genotyping a Danish basal cell carcinoma cohort of 80 individuals for an A/C SNP in exon 6 of the XPD gene. METHODS: Genomic DNA was amplified by a two-step polymerase chain reaction (PCR) in the presence of fluorescein-dyed primers and fluorescein-12-dUTP. The allele-specific PNA molecules were covalently coupled to carboxylated microspheres with and without rhodamine. Allele-specific hybridization between PCR products and immobilized PNA was carried out at 60 degrees C followed by flow cytometric detection. RESULTS: We present a fully functional two-bead genotyping system based on PNA capture and flow cytometric detection used for the correct and fast regenotyping of a Danish basal cell carcinoma cohort. CONCLUSIONS: This new assay presents a simple, rapid, and robust method for SNP genotyping for laboratories equipped with a standard flow cytometer. Moreover, this system offers potential for multiplexing and will be operational for middle-scale genotyping.     

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.     

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.     

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.