Differential DNA and RNA sequence discrimination by PNA having charged side chains

PNA sequences modified with charged side chains were evaluated for base-pairing sequence selectivity under physiological conditions. PNA having negatively charged aspartic acid side chains shows higher selectivity with RNA, while PNA having positively charged lysine side chains shows higher selectivity with DNA. These observations provide insight into the binding selectivity of modified PNA in antisense and antigene applications.     

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.     

PNA containing isocytidine nucleobase: synthesis and recognition of double helical RNA

Peptide nucleic acid (PNA1) containing a 5-methylisocytidine (iC) nucleobase has been synthesized. Triple helix formation between PNA1 and RNA hairpins having variable base pairs interacting with iC was studied using isothermal titration calorimetry. The iC nucleobase recognized the proposed target, C-G inversion in polypurine tract of RNA, with slightly higher affinity than the natural nucleobases, though the sequence selectivity of recognition was low. Compared to non-modified PNA, PNA1 had lower affinity for its RNA target.     

C3′-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60–80°, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated. A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived duplexes of modified PNA with complementary RNA were observed.     

Cationic nucleopeptides as novel non-covalent carriers for the delivery of peptide nucleic acid (PNA) and RNA oligomers

Cationic nucleopeptides belong to a family of synthetic oligomers composed by amino acids and nucleobases. Their capability to recognize nucleic acid targets and to cross cellular membranes provided the basis for considering them as novel non-covalent delivery agents for nucleic acid pharmaceuticals. Herein, starting from a 12-mer nucleopeptide model, the number of cationic residues was modulated in order to obtain new nucleopeptides endowed with high solubility in acqueous medium, acceptable bio-stability, low cytotoxicity and good capability to bind nucleic acid. Two candidates were selected to further investigate their potential as nucleic acid carriers, showing higher efficiency to deliver PNA in comparison with RNA. Noteworthy, this study encourages the development of nucleopeptides as new carriers to extend the known strategies for those nucleic acid analogues, especially PNA, that still remain difficult to drive into the cells.     

Effective delivery of antisense peptide nucleic acid oligomers into cells by anthrax protective antigen

Peptide nucleic acid (PNA) is highly stable and binds to complementary RNA and DNA with high affinity, but it resists cellular uptake, thereby limiting its bioavailability. We investigated whether protectiveantigen (PA, a non-toxic component of anthrax toxin) could transport antisense PNA oligomers into reporter cells that contain luciferase transgenes with mutant β-globin IVS2 intronic inserts, which permit aberrant pre-mRNA splicing and impair luciferase expression. PNA oligomers antisense to mutant splice sites in these IVS2 inserts induced luciferase expression when effectively delivered into the cells. PNA 18-mers with C-terminal poly-lysine tails [PNA(Lys)₈] demonstrated modest sequence-specific antisense activity by themselves at micromolar concentrations in luc-IVS2 reporter cell cultures. However, this activity was greatly amplified by PA. Antisense PNA(Lys)₈ with but not without PA also corrected the IVS2-654 β-globin splice defect in cultured erythroid precursor cells from a patient with β-thalassemia [genotype, IVS2-654(β⁰/β^E)], providing further evidence that anthrax PA can effectively transport antisense PNA oligomers into cells.     

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.     

Oligonucleotide-templated reactions based on Peptide Nucleic Acid (PNA) probes: Concept and biomedical applications

Sensing technologies based on Peptide Nucleic Acids (PNAs) and oligonucleotide-templated chemistry are perfectly suited for biomedical applications (e.g., diagnosis, prognosis and stratification of diseases) and could compete well with more traditional amplification technologies using expensive dual-labelled oligonucleotide probes. PNAs can be easily synthesised and functionalised, are more stable and are more responsive to point-mutations than their DNA counterpart. For these reasons, fluorogenic PNAs represent an interesting alternative to DNA-based molecular beacons for sensing applications in a cell-free environment, where cellular uptake is not required.     

Direct and nondestructive verification of PNA immobilization using click chemistry

The fluorogenic 1,3-Huisgen dipolar cycloaddition reaction was used as part of a novel immobilization strategy of PNA capture probes on a microarray. By using this click chemistry, azidocoumarin-anchored PNA probes were immobilized on phenyl acetylene-modified glass slides with the simultaneous generation of the fluorescent triazolylcoumarin moiety. Since the emitting moieties are generated in the immobilization reaction itself, fluorescent signals can be used to directly monitor the integrity of immobilization in a nondestructive manner. By using this strategy, PNA microarrays were prepared and successfully employed to perform microarray-based diagnosis of selected mutations in the breast cancer susceptibility gene BRCA1.     

Antisense effect of pyrrolidine-based oxy-peptide nucleic acids in Escherichia coli

To investigate the antisense effect of a pyrrolidine-based oxy-peptide nucleic acid (POPNA), we carried out the LacZ reporter assay using a 12-mer trans-l-POPNA conjugated with a cell-penetrating peptide (antisense reagent). The antisense effect of the conjugated POPNA (inhibition of LacZ activity) was comparable to that shown by a Nielsen-type peptide nucleic acid. Furthermore, the conjugated POPNA could switch the LacZ activity over a wide range of ambient temperatures.     

Stabilization of duplex DNA and RNA by dangling ends studied by free energy simulations

Single unpaired nucleotides at the end of double‐stranded nucleic acids, termed dangling ends, can contribute to duplex stability. Umbrella sampling free energy simulations of dangling cytosine and guanine nucleotides at the end of duplex and single stranded RNA and DNA molecules have been used to investigate the molecular origin of dangling end effects. In unrestraint simulations, the dangling end nucleotides stayed close to placements observed in experimental structures. Calculated free energy contributions associated with the presence of dangling nucleotides were in reasonable agreement with experiment predicting the general trend of a more stabilizing effect of purine vs. pyrimidine dangling ends. In addition, the calculations indicate a more significant stabilizing effect of dangling ends at the 5′‐end vs. 3′‐end in case of DNA and the opposite trend in case of RNA. Both electrostatic and van der Waals interactions contribute to the duplex stabilizing effect of dangling end nucleotides. The free energy simulation scheme could also be used to design dangling end nucleotides that result in enhanced duplex stabilization. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 418–427, 2014.     

Nondisruptive, sequence-specific coupling of fluorochromes to plasmid DNA

A method to attach a fluorochrome sequence-specifically to supercoiled plasmid DNA (pDNA) without perturbing transgene expression would provide an invaluable aid in a variety of applications requiring probes for the intracellular tracking of transfected pDNA. Here we report a method to couple commercially available fluorochromes covalently and sequence-specifically to pDNA using a peptide nucleic acid (PNA) as a linker molecule. The terminal cysteine thiol group on the PNA peptide backbone is reacted with a maleimide moiety on the fluorochrome to produce a fluorescent conjugate which is in turn hybridized to a plasmid expression vector containing an 11-bp target sequence. Spectroscopic evaluation and an electrophoretic mobility shift assay showed that the pDNA hybridized to one PNA-fluorochrome conjugate molecule. The fluorescence signal comigrated with pDNA on acrylamide gels, confirming the stable attachment of the fluorescent conjugate to the pDNA. The utility of one of the conjugates, PNA-Oregon green 488/pCMVbeta-DTS, to probe pDNA transport across the nuclear envelope, a significant barrier to gene transfer, was undertaken using a digitonin-permeabilized HeLa cell assay. The PNA-Oregon green 488/pCMVbeta-DTS conjugate is able to efficiently traverse the nuclear membrane of the permeabilized cells, accumulating in the nuclei within 30 min and reaching maximal levels by 1h. When transfected into HeLa cells, the PNA-Oregon green 488/pCMVbeta-DTS conjugate retained 55% of the native plasmid's biological activity, as determined by a beta-galactosidase assay. Thus, this method allows for the sequence-specific coupling of commercially available fluorochromes to DNA expression vectors while retaining biological function.     

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.     

Solid-phase synthesis of peptide nucleic acid (PNA) monomers and their oligomerization using disulphide anchoring linkers

A new simple solid-phase method has been developed for synthesizing Boc-protected peptide nucleic acid (PNA) monomers. An immobilized backbone 3 was built on Expansin® resin using an ester disulphide handle: 2-hydroxypropyl-dithio-2′-isobutyric acid (HPDI). The base acetic acids of thymine 5 , Z-cytosine 9 , Z-adenine 12 , and 6-O-benzyl guanine 17 were prepared and coupled to the immoblized backbone. The HPDI handle was cleaved under mild conditions by cyanolysis or assisted hydrolysis with tris(2-carboxyethyl)phosphine (TCEP) to give undamaged PNA monomers. These monomers were coupled to form oligomers by solid-phase method with another disulphide linkage: aminoethyldithio-2-isobutyric acid (AEDI) grafted on an amino-functionalized TentaGel® resin, using in situ neutralization and TBTU as activating reagent. Final cleavage of the AEDI linker gave PNA bearing a cysteamide residue that could be useful for optimizing PNA properties. Oligomers of up to 16 residues long were assembled. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes

Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.     

The role of reactivity in DNA templated native chemical PNA ligation during PCR

DNA templated fluorogenic reactions have been used as a diagnostic tool for the sequence specific detection of nucleic acids; and it has been shown that the native chemical ligation between thioester- and 1,2-aminothiol-modified PNA probes is amongst the most selective DNA detection methods reported. We explored whether a DNA templated reaction can be interfaced with the polymerase chain reaction (PCR). This endeavor posed a significant challenge. The reactive groups involved must be sufficiently stable to tolerate the high temperature applied in the PCR process. Nevertheless, the ligation reaction must proceed very rapidly and sequence specifically within the short time available in the annealing and primer extension steps before denaturation is used after approx. 1 min to commence the next PCR cycle. This required a careful optimization of the ternary complex architecture as well as adjustments of probe length and probe reactivities. Our results point to the prime importance of the ligation architecture. We show that once suitable annealing sites have been identified less reactive probe sets may be preferable if sequence specificity is of major concern. The reactivity tuning enabled the development of an in-PCR ligation, which was used for the single nucleotide specific typing of the V600E (T1799A) point mutation in the human BRaf gene. Showcasing the efficiency and sequence specificity of native chemical PNA ligation, attomolar template proofed sufficient to trigger signal while a 1000-fold higher load of single mismatched template failed to induce appreciable signal.     

3-Aminopyrrolidine-4-carboxylic acid as versatile handle for internal labeling of pyrrolidinyl PNA

Conformationally restricted pyrrolidinyl PNAs with an α/β-dipeptide backbone consisting of a nucleobase-modified proline and a cyclic five-membered amino acid spacer such as (1S,2S)-2-aminocyclopentanecarboxylic acid (ACPC) (acpcPNA) can form very stable hybrids with DNA with high Watson-Crick base pairing specificity. This work aims to explore the effect of incorporating 3-aminopyrrolidine-4-carboxylic acid (APC), which is isosteric to the ACPC spacer, into the acpcPNA. It is expected that the modification should not negatively affect the DNA binding properties, and that the additional nitrogen atom in the APC should provide a handle for internal modification. Orthogonally-protected (N(3)-Fmoc/N(1)-Boc and N(3)-Fmoc/N(1)-Tfa) APC monomers have been successfully synthesized and incorporated into the acpcPNA by Fmoc-solid-phase peptide synthesis. T(m), UV and CD spectroscopy confirmed that the (3R,4S)-APC could substitute the (1S,2S)-ACPC spacer in the acpcPNA with only slightly decreasing the stability of the hybrids formed between the modified acpc/apcPNA and DNA. In contrast, the (3S,4R) enantiomer of APC caused substantial destabilization of the hybrids. Furthermore, a successful on-solid-support internal labeling of the acpc/apcPNA via amide bond formation between pyrene-1-carboxylic acid or 4-(pyrene-1-yl) butyric acid and the pyrrolidine nitrogen atom of the APC spacer has been demonstrated. Fluorescence properties of the pyrene-labeled acpc/apcPNAs are sensitive to their hybridization states and can readily distinguish between complementary and single-mismatched DNA targets.     

肽核酸在分子生物学技术中的应用

肽核酸(PNA)作为一种人工合成的核酸类似物,以中性的肽链酰胺2-氨基乙基甘氨酸键取代了DNA中的戊糖磷酸二酯键骨架,其余部分与DNA相同。PNA可通过Watson-Crick碱基配对的形式识别并结合DNA或RNA序列,形成稳定的双螺旋结构。与传统的DNA或RNA相比,PNA具有生物学稳定性高、杂交特异性强、杂合体的稳定性高和杂交速度快等明显优点,使PNA具有良好的物理化学性质和生物学特性,在检测目的核酸序列中单碱基突变、PCR基因分子诊断与检测、荧光原位杂交定量分析、基因芯片和生物传感器技术等调控水平和临床应用上有自己的特点。简要综述了近年来肽核酸在上述分子生物学技术中的运用以及应用前景的展望。     

Investigating the kinetics of DNA–DNA and PNA–DNA interactions using surface plasmon resonance-enhanced fluorescence spectroscopy

Plasmon surface polaritons, resonantly excited in the Kretschmann format, are used to enhance the fluorescence emission of chromophore-labeled oligonucleotides (15mers) binding to surface-attached (via biotin–streptavidin linkages) complement catcher probes. A detailed analysis of the association and dissociation kinetics as well as the affinity constants is given for a mismatch 1 hybrid, emphasizing, in particular, the experimental conditions that are required to allow for an artifact-free determination of rate constants. A first comparison between DNA- and peptide nucleic acid (PNA-) probes shows similar affinities, however, significant deviations from single-exponential kinetics predicted by a simple Langmuir model for the PNA case are found.     

Electron microscopic studies of sequence-specific recognition of duplex DNA by different ligands

The following ligands were used to study sequence specific recognition of duplex DNA by electron microscopic techniques: methyltransferases BspR1 and EcoR124 (recognition sequences GGCC and GAAN7RTCG, respectively), a biotinylated deoxyoligonucleotide 5′-CTCTCTCTCTCTCT-3′ capable of forming triplex DNA, and PNA oligomer H-T₁₀-LysNH₂. For each ligand the best conditions for electron microscopic (EM)detection of stable specific complex formation were determined. It was demonstrated that EM allowed us to determine the position of the individual target site with an error of 15–20 bp, the relative affinities for individual target sites and kinetic parameters of the binding. These results open new possibilities for EM investigations of sequence-specific interactions with a wide range of other ligands of a similar nature. They also imply that a wide range of different sequences can be unambiguously and precisely mapped by EM and greatly extend the scope of EM applications for physical mapping of genomic DNA.