The influence of helix morphology on co-operative polyamide backbone conformational flexibility in peptide nucleic acid complexes.

A systematic analysis of peptide nucleic acid (PNA) complexes deposited in the Protein Data Bank has been carried out using a set of contiguous atom torsion angle definitions. The analysis is complemented by molecular mechanics adiabatic potential energy calculations on hybrid PNA-nucleic acid model systems. Hitherto unobserved correlations in the values of the (alpha and epsilon) dihedral angles flanking the backbone secondary amide bond are found. This dihedral coupling forms the basis of a PNA backbone conformation classification scheme. Six conformations are thus characterised in experimental structures. Helix morphology is found to exert a significant influence on backbone conformation and flexibility: Watson-Crick PNA strands in complexes with DNA and RNA, that possess A-like base-pair stacking, adopt backbone conformations distinct from those in PNA.DNA-PNA triplex and PNA-PNA duplex P-helix forms. Solvation effects on Watson-Crick PNA backbone conformation in heterotriplexes are discussed and the possible involvement of inter-conformational transitions and dihedral angle uncoupling in asymmetric heteroduplex base-pair breathing is suggested.     

Synthesis and characterization of new chiral peptide nucleic acid (PNA) monomers.

PNAs are DNA analogues in which the nucleic acid's backbone is replaced by a chiral or achiral pseudopeptide backbone and nucleobases are attached to the backbone by methylene carbonyl linkers. The easy to modify PNA structure gives the possibility to obtain monomers, and subsequently oligomers, with improved properties. We have synthesised several new PNA monomers, starting from a series of 2'-substituted methyl N-(2-Boc-aminoethyl)glycinates. The pseudodipeptides were obtained using modified Kosynkina's method, based on the reductive amination of N-Boc-protected alpha-amino aldehydes [glycinal, isoleucinal, valinal, tryptophanal, serinal(Bzl), prolinal] with methyl glycinate. The compounds were then acylated with nucleic acid base derivatives by simplified procedure, and the purification was limited to the last step of the synthesis. The applied procedure is useful in synthesis of various chiral PNA monomers.     

Information transfer from peptide nucleic acids to RNA by template-directed syntheses.

Peptide nucleic acids (PNAs) are uncharged analogs of DNA and RNA in which the ribose-phosphate backbone is substituted by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. A PNA C10 oligomer has been shown to act as template for efficient formation of oligoguanylates from activated guanosine ribonucleotides. In a previous paper we used heterosequences of DNA as templates in sequence-dependent polymerization of PNA dimers. In this paper we show that information can be transferred from PNA to RNA. We describe the reactions of activated mononucleotides on heterosequences of PNA. Adenylic, cytidylic and guanylic acids were incorporated into the products opposite their complement on PNA, although less efficiently than on DNA templates.     

Information transfer from DNA to peptide nucleic acids by template-directed syntheses.

Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.     

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。     

Synthesis and evaluation of acylguanidine FXa inhibitors

A series of acylguanidine derivatives were prepared and investigated as inhibitors of Factor Xa (FXa). These compounds were made by guanidine acylation with carboxylic acids using carbonyl diimidazole (CDI) as the coupling reagent. Conditions for the rapid synthesis and purification of these compounds are described along with their ability to inhibit FXa. The best FXa inhibitor is 1 with a FXa IC(50) of 6 nM.     

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。     

肽核酸相关技术在杂交检测中的应用

肽核酸是一种寡核苷酸的类似物,它是由丹麦哥本哈根大学的Ｎｉｅｌｓｅｎ、Ｅｇｈｏｌｍ等人首先发明合成的。肽核酸与传统的寡核苷酸相比,骨架结构发生了根要变化。肽核酸的电中性骨架有许多ＤＮＡ所不具备的性质,例舅高灵敏度、高特异性、非盐依赖性等,从而使它成为一种优良的寡核苷酸的取代物,尤其是杂交检测领域。     

Evaluating the Effect of Ionic Strength on Duplex Stability for PNA Having Negatively or Positively Charged Side Chains

The enhanced thermodynamic stability of PNA:DNA and PNA:RNA duplexes compared with DNA:DNA and DNA:RNA duplexes has been attributed in part to the lack of electrostatic repulsion between the uncharged PNA backbone and negatively charged DNA or RNA backbone. However, there are no previously reported studies that systematically evaluate the effect of ionic strength on duplex stability for PNA having a charged backbone. Here we investigate the role of charge repulsion in PNA binding by synthesizing PNA strands having negatively or positively charged side chains, then measuring their duplex stability with DNA or RNA at varying salt concentrations. At low salt concentrations, positively charged PNA binds more strongly to DNA and RNA than does negatively charged PNA. However, at medium to high salt concentrations, this trend is reversed, and negatively charged PNA shows higher affinity for DNA and RNA than does positively charged PNA. These results show that charge screening by counterions in solution enables negatively charged side chains to be incorporated into the PNA backbone without reducing duplex stability with DNA and RNA. This research provides new insight into the role of electrostatics in PNA binding, and demonstrates that introduction of negatively charged side chains is not significantly detrimental to PNA binding affinity at physiological ionic strength. The ability to incorporate negative charge without sacrificing binding affinity is anticipated to enable the development of PNA therapeutics that take advantage of both the inherent benefits of PNA and the multitude of charge-based delivery technologies currently being developed for DNA and RNA.     

Coupling of iminodiacetic acid with amino acid derivatives in solution and solid phase

The IR studies for the preactivation step of N-protected iminodiacetic acid with different coupling reagents (TCFH,TFFH, HATU, HBTU, HSTU) were reported here and showed theformation of an anhydride as an active intermediate in caseof TCFH and TFFH. The formation of a mixture of an anhydrideand an active ester (–OBt, –OAt or –OSu) were observed forHBTU, HATU or HSTU coupling reagent. Dependent on the couplingconditions, acylation of N-protected iminodiacetic acid with amino acid ester or amide derivatives in solution phase gavemono- or di-substituted iminodiacetic acid derivatives. Couplingof N-protected iminodiacetic acid with an amino acid or peptideattached to a solid support (PAL-PEG-PS or Wang resin) gave onlythe monosubstituted iminodiacetic acid derivatives.     

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.     

A convenient route for the preparation of peptide nucleic acid monomers carrying acid-labile groups for the protection of the amino function

A convenient route for the preparation of peptide nucleic acid (PNA) monomers is described. Two different base-labile protecting groups (2-cyanoethyl and 4-nitrophenylethyl) are described for the protection of the carboxylic function of the N-(2-aminoethyl)glycine backbone during the assembly of the monomers. These groups are selectively removed yielding the desired PNA monomers in high yields, the 2-cyanoethyl group being faster and cleaner than the 4-nitrophenylethyl group. The use of PNA monomers for the preparation of DNA–PNA chimeric molecules is also discussed.     

Synthesis and properties of DNA-PNA chimeric oligomers.

Adenine, thymine and cytosine PNA monomers have been prepared using 3-amino-1,2-propanediol as a starting material. The benzoyl group was used to protect the exocyclic amines of the heterocyclic bases of A and C PNA monomers and the backbone primary amine was protected with the monomethoxytrityl group. The thymine and cytosine PNA monomers were used in conjunction with standard DNA synthesis monomers to produce chimeric PNA DNA (PDC) oligomers. Ultraviolet melting studies confirmed that these oligomers form stable hybrids with complementary DNA strands and that mismatches in the DNA but more so in the PNA sections lead to duplex destabilisation.     

Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions.     

Modulation of the pharmacokinetic properties of PNA: preparation of galactosyl, mannosyl, fucosyl, N-acetylgalactosaminyl, and N-acetylglucosaminyl derivatives of aminoethylglycine peptide nucleic acid monomers and their incorporation into PNA oligomers

A series of N-(2-aminoethyl)-alpha-amino acid thymine peptide nucleic acid (PNA) monomers bearing glycosylated side chains in the alpha-amino acid position have been synthesized. These include PNA monomers where glycine has been replaced by serine and threonine (O-glycosylated), derivatives of lysine and nor-alanine (C-glycosylated), and amide derivatives of aspartic acid (N-glycosylated). The Boc and Fmoc derivatives of these monomers were used for incorporation in PNA oligomers. Twelve PNA decamers containing the glycosylated units in one, two, or three positions were prepared, and the thermal stability (T(m)) of their complexes with a complementary RNA was determined. Incorporation of the glycosyl monomers reduced the duplex stability by 0-6 degrees C per substitution. A cysteine was attached to the amino terminus of eight of the PNA decamers (Cys-CTCATACTCT-NH(2)) for easy conjugation to a [(18)F]radiolabeled N-(4-fluorobenzyl)-2-bromoacetamide. The in vivo biodistribution of these PNA oligomers was determined in rat 2 h after intravenous administration. Most of the radioactivity was recovered in the kidneys and in the urine. However, N-acetylgalactosamine (and to a lesser extent galactose and mannose)-modified PNAs were effectively targeting the liver (40-fold over unmodified PNA). Thus, the pharmacodistribution in rats of PNA oligomers can be profoundly changed by glycosylation. These results could be of great significance for PNA drug development, as they should allow modulation and fine-tuning of the pharmacokinetic profile of a drug lead.     

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。     

Cell-dependent differential cellular uptake of PNA,peptides, and PNA-peptide conjugates

Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.