肽核酸的特性及作用

肽核酸（peptide nucleic acid,PNA)是一类DNA结构类似物,能与DNA和RNA特异性杂交。它通过与DNA结合而抑制基因转录,通过与mRNA结合而阻止蛋白质的合成;同时,PNA不易被核酸酶和蛋白酶降解,所有这些显示出其作为反基因药物和反义药物的良好应用前景。     

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。     

肽核酸及其应用

肽核酸（ＰＮＡ）是一咎人工合成的核苷酸类似物。它具有杂交特性,可选择性结合于双链ＤＮＡ,并可通过链置换形成（ＰＮＡ）２－ＤＮＡ三聚体。肽核酸在分子生物学和微生物学研究趴有广泛的用途,如作为杂交引物、ＤＮＡ绘图、ＰＣＲｃｌａｍｐｉｎｇ我杂交及反义药物等。     

肽核酸探针在微生物诊断领域的应用进展

肽核酸(Polyamide nucleic acid,PNA)是以中性酰胺键为骨架的脱氧核糖核酸(DNA)结构类似物,它可以特异性地与DNA杂交,且具有极高的生物稳定性.相比较传统的DNA探针技术,肽核酸探针以其特殊的结构和性质在食品、环境及临床等微生物快速诊断领域显示出独特的优势.就肽核酸探针在微生物诊断方面的应用进展做简要综述.     

肽核酸与核酸分子杂交的模式及其在基因诊断领域中的应用

在发明肽核酸(peptide nucleic acid,PNA)后的短短十年间,由于PNA分子独特的生物学性能,使之可以与DNA及RNA分子通过不同的机制形成稳定的复合物,导致PNA在基因诊断领域有得天独厚的优势。本文综述了PNA与核酸分子杂交的几种不同模式以及最近几年PNA在基因诊断领域的一些最新进展。     

肽核酸的分子生物学效应及应用

肽核酸(PNA)是一类以酰胺键连接骨架替代核酸中核糖磷酸二酯键骨架构成的核酸类似物,其中N-乙基甘氨酸骨架PNA与核酸链以Waston-Crick碱基配对形式稳定互补结合,具有广泛生物学效应,包括调节DNA识别蛋白质的功能以及调节转录和翻译.在分子生物学研究中PNA作为新的工具在多方面得到应用.除它的DNA(RNA)结合特性外PNA在生物稳定性、细胞摄取、结构修饰多方面的研究进展显示出作为基因调节药物具有良好前景.     

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。     

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。     

肽核酸—基因调控的利器

肽核酸(PNA)是具有类多肽骨架的DNA类似物,PNA的主链骨架是由N(2-氨基乙基)-甘氨酸与核酸碱基通过亚甲基羰基连接而成的。PNA可以特异性地与DNA或RNA杂交,形成稳定的复合体。PNA由于其自身的特点可以对DNA复制、基因转录、翻译等进行有针对的调控,同时作为杂交探针大大提高了遗传学检测和医疗诊断的效率和灵敏度。肽核酸（PNA）特异性地识别和结合互补核酸序列被引进用于医学和生物学的研究,展示了其独特的生化属性,成为了基因奥秘的探索者。     

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。     

Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules.     

Pseudocomplementary PNAs as selective modifiers of protein activity on duplex DNA: the case of type IIs restriction enzymes

This study evaluates the potential of pseudocomplementary peptide nucleic acids (pcPNAs) for sequence-specific modification of enzyme activity towards double-stranded DNA (dsDNA). To this end, we analyze the ability of pcPNA–dsDNA complexes to site-selectively interfere with the action of four type IIs restriction enzymes. We have found that pcPNA–dsDNA complexes exhibit a different degree of DNA protection against cleaving/nicking activity of various isoschizomeric endonucleases under investigation (PleI, MlyI and N.BstNBI) depending on their type and mutual arrangement of PNA-binding and enzyme recognition/cleavage sites. We have also found that the pcPNA targeting to closely located PleI or BbsI recognition sites on dsDNA generates in some cases the nicking activity of these DNA cutters. At the same time, MlyI endonuclease, a PleI isoschizomer, does not exhibit any DNA nicking/cleavage activity, being completely blocked by the nearby pcPNA binding. Our results have general implications for effective pcPNA interference with the performance of DNA-processing proteins, thus being important for prospective applications of pcPNAs.     

Targeting DNA with triplex-forming oligonucleotides to modify gene sequence

Molecules that interact with DNA in a sequence-specific manner are attractive tools for manipulating gene sequence and expression. For example, triplex-forming oligonucleotides (TFOs), which bind to oligopyrimidine.oligopurine sequences via Hoogsteen hydrogen bonds, have been used to inhibit gene expression at the DNA level as well as to induce targeted mutagenesis in model systems. Recent advances in using oligonucleotides and analogs to target DNA in a sequence-specific manner will be discussed. In particular, chemical modification of TFOs has been used to improve binding to chromosomal target sequences in living cells. Various oligonucleotide analogs have also been found to expand the range of sequences amenable to manipulation, including so-called "Zorro" locked nucleic acids (LNAs) and pseudo-complementary peptide nucleic acids (pcPNAs). Finally, we will examine the potential of TFOs for directing targeted gene sequence modification and propose that synthetic nucleases, based on conjugation of sequence-specific DNA ligands to DNA damaging molecules, are a promising alternative to protein-based endonucleases for targeted gene sequence modification.     

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson–Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37°C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.     

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.     

DNA mimics based on pyrrolidine and hydroxyproline

In recent years, a great number of analogues and mimics of nucleic acids have been developed with the aim of improving the physicochemical and biological properties of native oligonucleotides, in particular, to increase their affinity for nucleic acids, selectivity of action, and biological stability. This review summarizes the data on the synthesis and properties of DNA mimics, the analogues of peptide nucleic acids, which are the derivatives of pyrrolidine and hydroxyproline. Some physicochemical and biological properties of negatively charged mimics of this type are considered, which contain phosphonate residues in the back-bone and exhibit a high affinity for DNA and RNA, the selectivity of binding to nucleic acids, and stability in various biological systems. Examples of using these mimics as tools in molecular biology studies, in particular, functional genomics, are given. The prospects for their application in diagnosis and medicine are discussed.     

Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA

A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.     

Synthesis and application of negatively charged PNA analogues

Negatively charged DNA mimics containing phosphonate analogoues of peptide nucleic acids were designed, and their physicochemical and biological properties were evaluated in the comparison with natural oligonucleotides, classical peptide nucleic acids, and morpholino phosphorodiamidate oligonucleotide analogues. The results obtained revealed a high potential of phosphonate-containing PNA derivatives for a number of biological applications, such as diagnostic, nucleic acids analysis, and inhibition of gene expression.