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.     

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.     

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.     

Insight into why pyrrolidinyl peptide nucleic acid binding to DNA is more stable than the DNA x DNA duplex

Molecular dynamics (MD) simulations and experimental measurements of the stability of a novel pyrrolidinyl PNA binding to DNA (PNA·DNA) in both parallel and antiparallel configurations were carried out. For comparison, simulations were also performed for the DNA·DNA duplex. The conformations of the three simulated systems were found to retain well-defined base pairing and base stacking as their starting B-like structure. A large gas-phase energy repulsion of the two negatively charged sugar-phosphate backbones of the DNA strands was found to reduce the stability of the DNA·DNA duplex significantly compared with that of the PNA·DNA complexes, especially in the antiparallel binding configuration. In addition, the antiparallel PNA·DNA was observed to be less solvated than that of the other two systems. The simulated binding free energies and the experimental melting temperatures for the three investigated systems are in good agreement, indicating that the antiparallel PNA·DNA is the most stable duplex.     

Preparation of radioiodinated peptide nucleic acids with high specific activity

Peptide nucleic acids (PNAs) have stronger affinity and greater specificity than do oligonucleotides for binding to DNA and RNA and, as such, have potential utility as probes in molecular biology applications. In this study, a novel approach for labeling the PNA with radioiodine that avoided solubility issues and poor labeling encountered when trying to radioiodinate PNAs directly in solution was developed. For this approach, a purpose-designed prosthetic group that incorporated both a radioiodinatable tyrosine and a triphenylphosphonium (TPP) moiety was synthesized. The latter is an organic cation that combines the properties of good solubility in both aqueous and organic solvents with a strong retention by reverse phase HPLC. Following radioiodination of the TPP-based prosthetic group in phosphate buffer, the prosthetic group was purified and coupled to the terminal amine of 15-mer PNA on the solid phase resin. After cleavage and deprotection of the PNA from the resin, the presence of the TPP group resulted in a clean separation of radioiodinated PNA from unlabeled PNA, yielding a high-specific activity probe in a single HPLC run. As an example of a potential molecular biology application of the resultant (125)I-labeled PNA probe, it was used to detect mRNA for the Lcn2 gene in Northern blotting.     

Light-directed synthesis of peptide nucleic acids (PNAs) chips

We report herein the light-directed synthesis of peptide nucleic acids (PNAs) microarray using PNA monomers protected by photolabile protecting groups and a maskless technique that uses a digital micromirror array system to form virtual masks. An ultraviolet image from the virtual mask was cast onto the active surface of a glass substrate, which was mounted in a flow cell reaction chamber connected to a peptide synthesizer. Light exposure was followed by automatic chemical coupling cycles and these steps were repeated with different virtual masks to grow the desired PNA probes in a selected pattern. In a preliminary experiment, an array of PNA probes with dimensions of 4.11 mm × 4.11 mm was generated on each slide. Each synthesis region in the final array measured 210 μm × 210 μm for a total of 256 sites. The center-to-center space was 260 μm. It was observed from the hybridization pattern of the fluorescently labeled oligonucleotide targets that the fluorescence intensities of the matched, and mismatched sequences showed substantial difference, demonstrating specificity in the identification of complementary sequences. This opens the way to exploit processes from the microelectronics industry for the fabrication of PNA microarrays with high densities.     

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.     

Synthesis of 1H-1,2,3-triazole derivatives as new α-glucosidase inhibitors and their molecular docking studies

Inhibition of α-glucosidase is an effective strategy for controlling the post-prandial hyperglycemia in diabetic patients. For the identification of new inhibitors of this enzyme, a series of new (R)-1-(2-(4-bromo-2-methoxyphenoxy) propyl)-4-(4-(trifluoromethyl) phenyl)-1H-1,2,3-triazole derivatives were synthesized (8a–d and 10a–e). The structures were confirmed by NMR, mass spectrometry and, in case of compound 8a, by single crystal X-ray crystallography. The α-glucosidase inhibitory activities were investigated in vitro. Most derivatives exhibited significant inhibitory activity against α-glucosidase enzyme. Their structure-activity relationship and molecular docking studies were performed to elucidate the active pharmacophore against this enzyme. Compound 10b was the most active analogue with IC₅₀ value of 14.2 µM, while compound 6 was found to be the least active having 218.1 µM. A preliminary structure-activity relationship suggested that the presence of 1H-1,2,3-triazole ring in 1H-1,2,3-triazole derivatives is responsible for this activity and can be used as anti-diabetic drugs. The molecular docking studies of all active compounds were performed, in order to understand the mode of binding interaction and the energy of this class of compounds.     

In situ hybridisation in filamentous fungi using peptide nucleic acid probes

Fluorescent DNA and peptide nucleic acid (PNA) probes were used for in situ hybridisations in colonies of Schizophyllum commune and Aspergillus niger. DNA probes for 18S rRNA did not diffuse through the cell wall after mild chemical fixation. After permeabilising the cell wall with lysing enzymes or slow freezing and embedding, hybridisation was still poor and not reproducible. In contrast, PNA probes did diffuse through the cell wall after mild chemical fixation and reproducible fluorescent signals were obtained. The rRNA signal was most intense in the apical compartment of hyphae of S. commune. Within this compartment, the signal was lower at the extreme apex. Apparently, ribosomes are unevenly distributed in hyphae. In S. commune, the mRNA of the SC3 gene was also detected with a PNA probe. The ratio between 18S rRNA and SC3 mRNA signals were variable between hyphae and their compartments. This is the first report of using PNA probes for in situ hybridisation of mRNA in fungi. The method provides a powerful tool to study gene expression.     

Preparation, Tc-labeling and biodistribution studies of a PNA oligomer containing a new ligand derivative of 2,2′-dipicolylamine

A new azido derivative of 2,2′-dipicolylamine (Dpa), 2-azido-N,N-bis((pyridin-2-yl)methyl)ethanamine, (Dpa-N₃) was readily prepared from the known 2-(bis(pyridin-2-ylmethyl)amino)ethanol (Dpa-OH). It was demonstrated that Dpa-N₃ could be efficiently labeled with both [Re(CO)₃(H₂O)₃]Br and [^99mTc(H₂O)₃(CO)₃]⁺ to give [Re(CO)₃(Dpa-N₃)]Br and [^99mTc(CO)₃(Dpa-N₃)]⁺, respectively. Furthermore, Dpa-N₃ was successfully coupled, on the solid phase, to a Peptide Nucleic Acid (PNA) oligomer (H-4-pentynoic acid-spacer-spacer-tgca-tgca-tgca-Lys-NH₂; spacer = -NH-(CH₂)₂-O-(CH₂)₂-O-CH₂-CO-) using the Cu(I)-catalyzed [2 + 3] azide/alkyne cycloaddition (Cu-AAC, often referred to as the prototypical “click” reaction) to give the Dpa-PNA oligomer. Subsequent labeling of Dpa-PNA with [^99mTc(H₂O)₃(CO)₃]⁺ afforded [^99mTc(CO)₃(Dpa-PNA)] in radiochemical yields > 90%. Partitioning experiments in a 1-octanol/water system were carried out to get more insight on the lipophilicity of [^99mTc(CO)₃(Dpa-N₃)]⁺ and [^99mTc(CO)₃(Dpa-PNA)]. Both compounds were found rather hydrophilic (log D_o/w values at pH = 7.4 are −0.50: [^99mTc(CO)₃(Dpa-N₃)]⁺ and −0.85: [^99mTc(CO)₃(Dpa-PNA)]. Biodistribution studies of [^99mTc(CO)₃(Dpa-PNA)] in Wistar rats showed a very fast blood clearance (0.26 ± 0.1 SUV, 1 h p.i.) and modest accumulation in the kidneys (5.45 ± 0.45 SUV, 1 h p.i.). There was no significant activity in the thyroid and the stomach, demonstrating a high in vivo stability of the ^99mTc-labeled Dpa-PNA conjugate.     

肽核酸对基因调节作用的研究进展

肽核酸(PNA)是一类人工合成的核酸类似物,PNA与核酸链以Waston-Crick碱基配对方式稳定互补结合,具有高度的亲合性、稳定性、特异性特征,PNA能调节基因的复制、转录（或逆转录）和翻译过程,有着广泛的分子生物学效应,显示出其作为基因调节药物的应用潜力。     

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.     

反义基因治疗

除了反义核酸和核酶外,最近又发展了一种新型的反义药物———反义肽核酸(PNA)。反义治疗的经典策略是阻断异常基因的表达;随着研究的深入,又发现了以反义药物调整基因表达比例(即调控基因治疗)的反义治疗途径。本文对反义基因治疗的策略、反义药物的设计及稳定性等方面的新思路和该领域的发展与应用前景作了概括介绍。     

肽核酸在病毒检测与治疗中的应用 *

肽核酸(peptide nucleic acid, PNA)是以多肽骨架取代糖磷酸主链的寡核苷酸类似物,又称第三代反义核酸。PNA的电中性多肽骨架结构,使其保留类似糖磷酸链寡核苷酸高靶标亲和力的同时,比糖磷酸主链具有更强的酶稳定性和热稳定性,已成为当今寡核苷酸类似物研究的热点。一方面,PNA对病毒的复制与突变水平具有的快速、有效和准确的检测性能,对疾病的进一步治疗具有重要意义;另一方面,基于PNA的序列特异性和剂量依赖性,能在基因水平上对病毒的生命周期进行特异性的调控,从而能更有效地实现抑制病毒在宿主细胞中生存和复制的目的。结合近十年来的文献,综述了PNA应用于不同病毒的检测及病毒性疾病治疗的最新进展和作用机制,以期为PNA的临床产品研发提供新的思路。