dabPNA: design, synthesis, and DNA binding studies

In continuing our research efforts for developing new oligodeoxynucleotide (ODN)-like drugs and diagnostics, we designed diaminobutyric peptide nucleic acids (dabPNAs), nucleopeptides characterized by a diaminobutyric-based building block that is an isomer of the aminoethylglycyl PNA (aegPNA) unit and the acyclic modification of the aminoprolyl PNA (ampPNA) monomer. In this work we present the solid phase synthesis of a dabPNA oligomer and of two aegPNAs containing a single dabPNA unit. A study relative to their binding ability towards DNA is also reported even in comparison with the well known aegPNAs.     

Synthesis, characterization and hybridization studies of an alternate nucleo-ε/γ-peptide: complexes formation with natural nucleic acids

In order to develop new oligodeoxyribonucleotide (ODN) analogs to be used in biotechnological applications, we report here the synthesis, characterization and nucleic acid binding studies of novel nucleopeptides, that we called ε-lys/γ-dabPNAs, containing a backbone of alternated l-diaminobutyric acid and l-lysine moieties. Exploring the hybridization properties of the new ODN analog, we found, by circular dichroism and UV spectroscopies, that a homothymine ε-lys/γ-dabPNA hexamer binds both DNA and RNA of complementary sequence. Furthermore, human serum stability assays on the alternate nucleopeptide evidenced a noteworthy degradation resistance. These results encourage us to deepen the knowledge of this analog, in order to evaluate its possible use in antigene/antisense or diagnostic applications.     

Modulation of DNA and RNA by PNA

Peptide nucleic acid (PNA), a synthetic DNA mimic that is devoid of the (deoxy)ribose-phosphate backbone yet still perfectly retains the ability to recognize natural nucleic acids in a sequence-specific fashion, can be employed as a tool to modulate gene expressions via several different mechanisms. The unique strength of PNA compared to other oligonucleotide analogs is its ability to bind to nucleic acid targets with secondary structures such as double-stranded and quadruplex DNA as well as RNA. This digest aims to introduce general readers to the advancement in the area of modulation of DNA/RNA functions by PNA, its current status and future research opportunities, with emphasis on recent progress in new targeting modes of structured DNA/RNA by PNA and PNA-mediated gene editing.     

Evidences for complex formation between L-dabPNA and aegPNA

Continuing our research on the development of nucleopeptides as ODN analogs for biomedical and bioengineering applications, here we report the synthesis and the chemical–physical characterization of a homoadenine hexamer based on a l-diaminobutyric acid (l-DABA) backbone (dabPNA), and its binding studies with a complementary aegPNA. We demonstrated by CD and UV experiments that the l-dabPNA binds the aegPNA forming a complex with good thermal stability, that we identified as a left-handed triplex.     

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.     

Detection of the Hereditary Hemochromatosis Gene Mutation by Real-Time Fluorescence Polymerase Chain Reaction and Peptide Nucleic Acid Clamping

Hereditary hemochromatosis (HH), an iron overload disease, is the most common known inheritable disease. The most prevalent form of HH is believed to be the result of a single base-pair mutation. We describe a rapid homogeneous mutation analysis method that does not require post-polymerase chain reaction (PCR) manipulations. This method is a marriage of three emerging technologies: rapid cycling PCR thermal cyclers, peptide nucleic acid (PNA) probes, and a new double-stranded DNA-selective fluorescent dye, Sybr Green I. The LightCycler is a rapid thermal cycler that fluorometrically monitors real-time formation of amplicon with Sybr Green I. PNAs are DNA mimics that are more sensitive to mismatches than DNA probes, and will not serve as primers for DNA polymerases. PNA probes were designed to compete with PCR primers hybridizing to the HH mutation site. Fully complemented PNA probes at an 18:1 ratio over DNA primers with a mismatch result in suppression of amplicon formation. Conversely, PNA probes with a mismatch will not impair the binding of a complementary primer, culminating in amplicon formation. A LightCycler-based rapid genetic assay has been developed to distinguish HH patients from HH carriers and normal individuals using PNA clamping technology.     

修饰性肽核酸的细胞转运

肽核酸是人工合成的寡核苷酸类似物,以N-(2-氨乙基)甘氨酸结构单元替代DNA分子中的戊糖-磷酸结构。与天然核酸相比,肽核酸可以更高效地与DNA或RNA特异性杂交,在分子生物学和基因药物领域具有良好的应用前景。但是,肽核酸骨架呈电中性,难以高效穿过细胞膜,这成为工程应用的最大障碍。为了改善肽核酸的细胞转运性能,对肽核酸进行化学修饰是近年来的研究热点。结合近十年来文献报道和本实验室的工作,对肽核酸的骨架修饰和配合物结合修饰两类增强细胞转运的修饰方法进行综述,并对修饰性肽核酸细胞转运研究中存在的问题以及未来的研究趋势及其应用提出了见解。     

Rapid detection, identification, and enumeration of Escherichia coli by fluorescence in situ hybridization using an array scanner

A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes and an array scanner for rapid detection, identification, and enumeration of Escherichia coli is described. The test utilizes Cy3-labeled peptide nucleic acid (PNA) probes complementary to a specific 16S rRNA sequence of E. coli. Samples were filtered and incubated for 5 h, the membrane filters were then analyzed by fluorescence in situ hybridization and results were visualized with an array scanner. Results were provided as fluorescent spots representing E. coli microcolonies on the membrane filter surface. The number of fluorescent spots correlated to standard colony counts up to 100 colony-forming units per membrane filter. Above this level, better accuracy was obtained with PNA FISH due to the ability of the scanner to resolve neighboring microcolonies, which were not distinguishable as individual colonies once they were visible by eye.     

肽核酸在基因诊断和治疗中的研究进展

肽核酸是一种以多肽为骨架,类似核苷酸的物质。它不带电荷,能抵抗核酸酶和蛋白酶的降解;它与DNA或RNA杂交特异性很强,可与靶基因形成稳定的三螺旋结构。肽核酸能够抑制基因的复制、转录、逆转录和翻译过程,在基因诊断及治疗方面有着广泛的用途。     

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。     

Antisense properties of peptide nucleic acid

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose phosphate backbone has been replaced by a pseudo-peptide polymer to which the nucleobases are linked. PNA-oligomers can be synthesized in relatively large amounts, are highly stable in biological environments, and bind complementary DNA and RNA targets with remarkably high affinity and specificity. Thus PNA possesses many of the properties desired for a good antisense agent. Until recently, limited uptake of PNA into cells has been the major obstacle for applying PNA as an antisense agent in cell cultures and in vivo. Here, the antisense properties of PNA in vitro and in vivo will be reviewed. In particular, we will focus on recent observations indicating that PNA equipped with or without various uptake moieties may function as an efficient and gene-specific inhibitor of translation in Escherichia coli and in certain mammalian cell types.     

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。     

Inhibition of multidrug resistance by SV40 pseudovirion delivery of an antigene peptide nucleic acid (PNA) in cultured cells

Peptide nucleic acid (PNA) is known to bind with extraordinarily high affinity and sequence-specificity to complementary nucleic acid sequences and can be used to suppress gene expression. However, effective delivery into cells is a major obstacle to the development of PNA for gene therapy applications. Here, we present a novel method for the in vitro delivery of antigene PNA to cells. By using a nucleocapsid protein derived from Simian virus 40, we have been able to package PNA into pseudovirions, facilitating the delivery of the packaged PNA into cells. We demonstrate that this system can be used effectively to suppress gene expression associated with multidrug resistance in cancer cells, as shown by RT-PCR, flow cytometry, Western blotting, and cell viability under chemotherapy. The combination of PNA with the SV40-based delivery system is a method for suppressing a gene of interest that could be broadly applied to numerous targets.     

Targeted gene delivery: the role of peptide nucleic acid

Receptor-mediated endocytosis can be exploited to achieve efficient cell-specific gene delivery. Our laboratory has used two approaches for targeted gene delivery. One uses polycation as a carrier for plasmid DNA and the other uses peptide nucleic acid (PNA) as a carrier. Targeted gene delivery using polycation carriers has been widely utilized with some success. This approach mainly suffers from large particle size and non-specific interaction with blood components. These drawbacks have limited use of this type of vector forin vivo applications. Using PNA as a carrier, on the other hand, allows for smaller particle size and less non-specific interactions. The stability of this vector in the circulation may be a limiting factor. In addition, both types of vector lack mechanisms for endosome escape and nuclear transport. In this chapter, current developments and uses for targeted gene delivery of each approach are reviewed.     

Membrane-based hybridization capture of intracellular peptide nucleic acid

Peptide nucleic acid (PNA) is a chimeric oligonucleotide with nucleotide-derived bases and a peptide backbone. Compared with natural nucleotides, PNA has several advantages, including improved stability and high sequence discrimination during duplex formation. Despite its potential for therapeutic application, analysis technologies have not been generalized, mainly due to ambiguous physiochemical properties resembling those of nucleic acids as well as protein. Here we present a PNA detection method: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrotransfer to a Western blotting membrane and then hybridization with a radiolabeled oligonucleotide probe. This method is useful for evaluating the quality of synthetic PNA and determining its intracellular localization.     

Targeted gene delivery: The role of peptide nucleic acid

Receptor-mediated endocytosis can be exploited to achieve efficient cell-specific gene delivery. Our laboratory has used two approaches for targeted gene delivery. One uses polycation as a carrier for plasmid DNA and the other uses peptide nucleic acid (PNA) as a carrier. Targeted gene delivery using polycation carriers has been widely utilized with some success. This approach mainly suffers from large particle size and non-specific interaction with blood components. These drawbacks have limited use of this type of vector for in vivo applications. Using PNA as a carrier, on the other hand, allows for smaller particle size and less non-specific interactions. The stability of this vector in the circulation may be a limiting factor. In addition, both types of vector lack mechanisms for endosome escape and nuclear transport. In this chapter, current developments and uses for targeted gene delivery of each approach are reviewed.     

Targeted gene delivery: the role of peptide nucleic acid

Receptor-mediated endocytosis can be exploited to achieve efficient cell-specific gene delivery. Our laboratory has used two approaches for targeted gene delivery. One uses polycation as a carrier for plasmid DNA and the other uses peptide nucleic acid (PNA) as a carrier. Targeted gene delivery using polycation carriers has been widely utilized with some success. This approach mainly suffers from large particle size and non-specific interaction with blood components. These drawbacks have limited use of this type of vector for in vivoapplications. Using PNA as a carrier, on the other hand, allows for smaller particle size and less non-specific interactions. The stability of this vector in the circulation may be a limiting factor. In addition, both types of vectorlack mechanisms for endosome escape and nuclear transport. In this chapter, current developments and uses for targeted gene delivery of each approach are reviewed.     

An introduction to peptide nucleic acid

Peptide Nucleic Acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes and biosensors.     

肽核酸(peptide nucleic acid,PNA)阵列

肽核酸(PNA)以N—(2—氨基乙基)甘氨酸替代DNA分子中的磷酸戊糖骨架。它能特异性地识别与DNA、RNA所形成的杂交体。PNA—DNA、PNA—RNA的热稳定性要比相应的DNA—DNA、DNA—RNA高,而且PNA识别单碱基的能力强于DNA和RNA,使之在微阵列,尤其是SNP检测领域有着广泛的应用前景。本文简述了PNA阵列从探针设计、阵列合成、杂交和检测的全过程。