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。     

LNA-modified oligonucleotides effectively drive intramolecular-stable hairpin to intermolecular-duplex state

Sequence-specific hybridization of antisense and antigene agent to the target nucleic acid is an important therapeutic strategy to modulate gene expression. However, efficiency of such agents falls due to inherent intramolecular-secondary-structures present in the target that pose competition to intermolecular hybridization by complementary antisense/antigene agent. Performance of these agents can be improved by employing structurally modified complementary oligonucleotides that efficiently hybridize to the target and force it to transit from an intramolecular-structured-state to an intermolecular-duplex state. In this study, the potential of variably substituted locked nucleic acid-modified oligonucleotides (8mer) to hybridize and disrupt highly stable, secondary structure of nucleic acid has been biophysically characterized and compared with the conventionally used unmodified DNA oligonucleotides. The target here is a stem-loop hairpin oligonucleotide-a structure commonly present in most structured-nucleic acids and known to exhibit an array of biological functions. Using fluorescence-based studies and EMSA we prove that LNA-modified oligonucleotides hybridize to the target hairpin with higher binding affinity even at lower concentration and subsequently, force it to assume a duplex conformation. LNA-modified oligonucleotides may thus, prove as potential therapeutic candidates to manipulate gene expression by disruption of biologically relevant nucleic acid secondary structure.     

锁核酸研究进展

锁核酸(locked nucleic acid,LNA)是一种新型的寡核酸衍生物,结构中β-D-呋喃核糖的2’-O,4’-C位通过缩水作用形成环形的氧亚甲基桥、硫亚甲基桥或胺亚甲基桥,呋喃糖的结构锁定在C3’内型的N构型,形成了刚性的缩合结构。LNA作为一种新的反义核酸,具有与DNA／RNA强大的杂交亲和力、反义活性、抗核酸酶能力、水溶性好及体内无毒性等优点。LNA在基因诊断和基因治疗上有很多优势,如：单链核酸的多态性基因分型、LNA寡聚体具有高效抑制端粒酶活性及LNA修饰的DNA核酶(LNAzymes)高效清除高级结构的RNA等,有良好的应用研究前景。     

A rapid coupling protocol for the synthesis of peptide nucleic acids

With the current interest in anti-sense and anti-gene technologies, an efficient, fast and less toxic synthesis protocol would be advantageous for the oligomerisation of Peptide Nucleic Acids (PNA). Most of the methods currently in use for the t-Boc synthesis of PNA's use TFA/m-cresol, pyridine, piperidine and capping reagents. In this work, a rapid synthesis protocol has been adapted from an earlier published peptide synthesis method allowing a reduction in cycle time from around 30 min down to 16 min. By utilising quantitative deprotection with 100% TFA, a coupling time of 10 min and a four-fold excess of monomer, this synthesis protocol has been used to synthesise a number of PNA's incorporating all four nucleotides of varying sequence, up to 17 residues in length.     

A rapid coupling protocol for the synthesis of peptide nucleic acids

Summary With the current interest in anti-sense and anti-gene technologies, an efficient, fast and less toxic synthesis protocol would be advantageous for the oligomerisation of Peptide Nucleic Acids (PNA). Most of the methods currently in use for thet-Boc synthesis of PNA's use TFA/m-cresol, pyridine, piperidine and capping reagents. In this work, a rapid synthesis protocol has been adapted from an earlier published peptide synthesis method allowing a reduction in cycle time from around 30 min down to 16 min. By utilising quantitative deprotection with 100% TFA, a coupling time of 10 min and a four-fold excess of monomer, this synthesis protocol has been used to synthesise a number of PNA's incorporating all four nucleotides of varying sequence, up to 17 residues in length.     

Specific oligonucleotide invasion into an end of a DNA duplex

A new phenomenon was described: a double-stranded DNA fragment interacted with a single-stranded oligonucleotide complementary to the terminal region of one strand of the duplex to yield a complex with oligonucleotide invasion. Generation of Holliday junctions by homologous linear DNA fragments was less efficient in the presence of single-stranded oligonucleotides complementary to duplex ends. The effect depended on the oligonucleotide concentration, size, and complementarity to a duplex strand. Sequence-specific complexes with single strand invasion were detected in mixtures containing radiolabeled oligonucleotides and duplexes. A single-stranded oligonucleotide invaded a duplex even when its concentration was far lower than the duplex concentration. Complexes with single strand invasion were analyzed by chemical cleavage of noncanonical base pairs. Analysis showed that an oligonucleotide interacts with the complementary region of one strand of the duplex, gradually displacing the other strand. The extent of oligonucleotide invasion into the duplex considerably varied. Oligonucleotide invasion into duplexes became more efficient with increasing oligonucleotide size.     

Very Stable End-Sealed Double Stranded DNA by Click Chemistry

An efficient and simple method has been established for the intermolecular click ligation of two complementary DNA strands to produce an end-sealed duplex with a triazole linkage at each end. The resultant end-sealed duplex is thermally very stable (ΔT_m ～ 30°C relative to a normal duplex) and a fluorescent version remained intact for up to 3 days in Fetal Bovine serum. In contrast a single strand was completely degraded in 2 hours. These favorable properties suggest that such cyclic DNA duplexes might have potential for in vivo applications and nanotechnology.     

Inhibition of the Human Immunodeficiency Virus Type 1 DNA Integration by Modified Oligonucleotides

Oligonucleotide inhibitors of the HIV-1 DNA integration identified to date are reviewed. Two basic strategies of blocking the integration are considered: shielding the integrase-binding sites on the viral DNA by triplex-forming oligonucleotides, and directly inhibiting the enzyme with oligonucleotide agents.     

Synthesis,crystallization and preliminary crystallographic analysis of a 52-nucleotide DNA/2′-OMe-RNA oligomer mimicking 10–23 DNAzyme in the complex with a substrate

A 52-nucleotide DNA/2′-OMe-RNA oligomer mimicking 10–23 DNAzyme in the complex with its substrate was synthesized, purified and crystallized by the hanging-drop method using 0.8 M sodium potassium tartrate as a precipitant. A data set to 1.21 Å resolution was collected from a monocrystal at 100 K using synchrotron radiation on a beamline BL14.1 at BESSY. The crystal belonged to the P2₁ group with unit-cell a = 49.42, b = 24.69, c = 50.23, β = 118.48.     

三链核酸稳定性和生物学功能的研究进展

近10年来三链核酸的研究发展迅猛,已经成为分子生物学和基因工程的一个前沿领域.综述了最近在三链核酸稳定性和生物学功能方面的研究进展.较为详细地讨论了影响三链稳定性的各种内源和外源因素如：寡聚脱氧核苷酸(ODN)的长短 、序列、碱基修饰、骨架结构以及温度、酸度、离子强度 、配基等,同时对三链核酸的生物学功能进行了初步的总结,这些功能包括：控制基因转录,保护靶序列防止酶切,充当分子剪刀进行特定位点切割等.     

Dynamic states of the DNA repair enzyme AlkB regulate product release

The 2-oxoglutarate (2OG)- and Fe(2+)-dependent dioxygenase AlkB couples the demethylation of modified DNA to the decarboxylation of 2OG. Extensive crystallographic analyses have shown no evidence of significant structural differences between complexes binding either 2OG or succinate. By using nuclear magnetic resonance spectroscopy, we have shown that the AlkB-succinate and AlkB-2OG complexes have significantly different dynamic properties in solution. 2OG makes the necessary contacts between the metal site and the large beta-sheet to maintain a fully folded conformation. Oxidative decarboxylation of 2OG to succinate leads to weakening of a main contact with the large beta-sheet, resulting in an enhanced dynamic state. These conformational fluctuations allow for the replacement of succinate in the central core of the protein and probably contribute to the effective release of unmethylated DNA. We also propose that the inherent dynamics of the co-product complex and the subsequent increased molecular ordering of the co-substrate complex have a role in DNA damage recognition.     

Interaction of Keratin K1 with Nucleic Acids on the Cell Surface

The interaction of surface proteins from A431 cells and cellular extracts with nucleic acids was investigated using affinity modification with 32P-labeled reactive oligonucleotide derivatives. Proteins with molecular weights of 68, 46, 38, and 28 kD as well as several low molecular weight proteins capable of binding to nucleic acids were found on the surface of intact cells. It was demonstrated that a protein with molecular weight of 68 kD is exposed at the cell surface, since the treatment of cells with trypsin results in the cleavage of this protein. Disruption of the integrity of the cell membrane (scrapping, treatment with trypsin, or permeabilization of the cell membrane with streptolysin O or saponin) disrupts the interaction of the reactive oligonucleotides with the cell surface proteins. Affinity modification of the cytosolic and membrane-cytosolic cell fractions with labeled oligonucleotides results in the modification of a large number of proteins, where proteins with molecular weights of 68, 46, 38, and 28 kD can be found as minor components. Surface oligonucleotide-binding proteins with molecular weight of ~68 kD were isolated by affinity chromatography after the modification of intact A431 cells with a reactive oligonucleotide derivative. The isolated surface oligonucleotide-binding proteins from A431 cells were sequenced, and one of the proteins was identified as keratin K1.     

RNA—RNA原位杂交实验条件探讨

将Vigilin和qroal(Ⅰ)cDNA亚克隆到pGEM3Z和pGEM4Z载体,体外转录合成35s标记的cRNA探针。经RNA凝胶电泳,Southern Northern,杂交检查探针长度,杂交特性和特异性,通过系列实验探讨了RNA-RNA原位杂交实验中固定、杂交前处理、杂交温度,探针量、探针长度,洗脱严格性和RNA酶处理等对杂交结果的影响,建立了简化的RNA-RNA原位杂交方法。     

肽核酸——一种新型的反义物质

肽核酸是以肽为骨架的一种新型ＤＮＡ模拟物。已经证明肽核酸具有与ＤＮＡ和ＲＮＡ结合的高度亲合性、良好的稳定性及能方便地固相合成等特性。在反义技术和基因治疗中有着很好的前景。本文综述了肽核酸的生物化学特性及其在反义技术方面的应用。     

Experimental Evidence That GNA and TNA Were Not Sequential Polymers in the Prebiotic Evolution of RNA

Systematic investigation into the chemical etiology of ribose has led to the discovery of glycerol nucleic acid (GNA) and threose nucleic acid (TNA) as possible progenitor candidates of RNA in the origins of life. Coupled with their chemical simplicity, polymers for both systems are capable of forming stable Watson-Crick antiparallel duplex structures with themselves and RNA, thereby providing a mechanism for the transfer of genetic information between successive genetic systems. Investigation into whether both polymers arose independently or descended from a common evolutionary pathway would provide additional constraints on models that describe the emergence of a hypothetical RNA world. Here we show by thermal denaturation that complementary GNA and TNA mixed sequence polymers are unable, even after prolonged incubation times, to adopt stable helical structures by intersystem cross-pairing. This experimental observation suggests that GNA and TNA, whose structures derive from one another, were not consecutive polymers in the same evolutionary pathway to RNA. Reviewing Editor: Dr. Niles Lehman