三链 DNA 的形成、结构测定及可能的生物学作用

对三链核酸的历史发展进行了简单的回顾.对三链 DNA 的形成条件、制备方法、结构测定手段和量子生物学方法分别加以评述,指出了近期及今后的研究趋向.简单地讨论了三链核酸可能的生物学作用,指出它具有特异性裂解正常 DNA分子的功能并可阻断、诱导基因转录,在治疗遗传性、病毒性疾病方面可能具有较大应用价值.     

RecA蛋白介导的三链核酸结构形成及其序列提取

人工合成的单链DNA分子经PCR扩增形成双链DNA分子。将RecA蛋白与生物素标记的寡聚核酸探针序列在ATPγS存在的情况下共同哺育,使RecA蛋白包裹寡聚核酸探针,然后加入含同源序列的上述双链DNA分子经适当环境哺育形成了稳定的局部三链核酸结构。通过加入链亲和素包裹的磁珠吸附生物素化的探针,这样同源双链DNA分子与寡聚核酸探针形成的局部三链核酸结构也被吸附在磁珠上。使用磁分离装置提取这一结构,逐步降低盐离子浓度以洗脱双链DNA分子。将洗脱液中残留的蛋白质去除,经PCR扩增可获得目的DNA序列。同时使用同源探针和非同源探针在其它序列中提取目的DNA序列,结果显示目的DNA序列只被同源探针提取。实验结果显示了这一三链核酸结构形成的序列特异性,并且其稳定性随盐离子浓度降低而下降。提示在这一结构中同源的寡聚核酸单链与双链DNA分子形成了氢键结合,同时提示使用文中描述的方法可以提取特异的序列,用以克隆相应的基因。     

DNA芯片在0-1规划问题中的应用

生物芯片技术和DNA计算分别是近年来生命科学与信息科学的新兴研究领域,对信息高度并行的获取与处理是二者的本质特性.而0-1规划问题作为运筹学中一个重要的问题,到目前为止还没有好的算法.在DNA计算和DNA芯片基础上,提出了基于DNA芯片解决0-1规划问题的DNA计算新模型,与以往DNA计算模型相比,该模型具有高信息量和操作易自动化的优点.同时指出DNA芯片技术有望作为新型生物计算的芯片.     

基于分子信标的DNA计算

DNA计算是解决一类难以计算问题的一种新方法,这种计算随着问题的增大可以呈指数增长．迄今为止,许多研究成果已经成功地提高了它的性能和增加了它的可行性,本文在基于表面的DNA计算中采用了分子信标编码策略,并对分子信标在与对应的补链杂交形成双链时的受力进行分析,给出3-SAT问题的另一种解法．这种方法比现有的方法更有效,更具发展前景．因为它具有编码简单;耗材底;操作时间短;技术先进等优点．本文尝试了分子生物学,光学和力学的结合．这一工作为DNA计算能解决NP一完全问题提供了更有力的依据．     

分子信标芯片计算在0-1整数规划问题中的应用

生物芯片技术和DNA计算分别是近年来生命科学与信息科学的新兴研究领域,对信息高度并行的获取与处理是二者的本质特性．而0-1整数规划问题作为运筹学中一个重要的问题,到目前为止还没有好的算法．在DNA计算和DNA芯片基础上,提出了基于分子信标芯片解决0-1整数规划问题的DNA计算新模型．与以往DNA计算模型相比,该模型具有高信息量和操作易自动化的优点,同时指出分子信标芯片技术有望作为新型生物计算的芯片．     

全错位排列问题的基于表面的DNA计算模型

生物表面技术是DNA计算的一种实现方式,是近年来生命科学的新兴研究领域．而全错位排列问题作为组合数学中一个重要的问题,到目前为止还没有好的算法．在DNA表面技术的基础上,首次提出了全错位排列问题的基于表面的DNA计算模型,并对模型进行了简单的分析．     

心电逆问题的虚拟心脏模型参数解用于心室预激旁道定位的研究

提出了一种新的心电逆问题求解方法,即基于虚拟心脏模型的心电逆问题的模型参数解。详细研究了心室预激旁道位置与体表电位分布（ＢＳＰＭ：ＢｏｄｙＳｕｒｆａｃｅＰｏｔｅｎｔｉａｌＭａｐｐｉｎｇｓ）特征参数的关系,用ＢＳＰＭ的预激旁道位置敏感参数构造了优化系统的数学模型,给出了相应的优化算法及预激综合征旁道定位结果。研究结果表明：这种新的心电逆问题研究方法是可行、有效的,对心室内预激点的定位精度在三个心肌单元以内（即４．５ｍｍ）。     

不同G的四链体结构对DNAzyme活性的影响

富含鸟嘌呤的DNA序列在金属离子（通常是钠、钾离子）存在的条件下,可以形成稳定的G-四链体（G-quadruplex）。该G 四链体能够结合hemin（氯高铁血红素）形成具有过氧化物酶的活性的G四链体-hemin复合物DNAzyme。将这一原理联合滚环扩增技术可以对核酸进行可视化的检测。本研究旨在探索G-四链体-hemin复合物中,G-四链体结构以及两个G-四链体之间的链接长度与DNAzyme过氧化物酶活性之间的关系。实验分别选取了平行、反平行和混合结构的G-四链体,通过热差异光谱、紫外光谱、圆二色光谱对结构进行分析,不断加长链接序列并测定3种结构形成的DNAzyme活性,发现正平行结构的G-四链体具有更高的DNAzyme活性和更明显的可视化效果。综上所述,平行G-四链体结构可以用来满足裸眼可视化检测的需求,为无需复杂仪器的核酸检测奠定了方法基础。     

功能核酸DNA水凝胶的制备与组装

功能核酸DNA水凝胶是一种以DNA为构建单元通过化学反应或物理缠结自组装而成的新型柔性材料,其构建单元中包含1种或多种能够形成功能核酸的特定序列。功能核酸是通过碱基修饰和DNA分子之间的相互作用力组合的一类特定核酸结构,包括核酸适配体、DNA核酶、G-四联体(G-quadruplex,G4)和i-motif结构等。传统上,高浓度的长DNA链是制备DNA水凝胶的必要条件,而核酸扩增方法的引入为DNA水凝胶的组装方式提供了新的可能。因此,对常用于制备DNA水凝胶的多种功能核酸以及核酸的提取、合成和扩增手段进行了详细的介绍。在此基础上,综述了通过化学或物理交联方式组装功能核酸DNA水凝胶的制备方法。最后,提出了DNA纳米材料的组装所面临的挑战和潜在的发展方向,以期为开发高效组装的功能核酸DNA水凝胶提供参考。     

蛋白质- 核酸对接方法研究进展

分子对接技术作为预测蛋白质-核酸复合物结构的有效方法,为研究在生物学过程中蛋白质-核酸的相互作用提供了重要的工具。本文首先分析了当前蛋白质-核酸对接研究中的主要困难,例如构象变化和核糖磷酸骨架的带电性问题。然后从构象搜索、打分函数、柔性策略三个方面比较和总结了蛋白质-核酸对接中主要的计算方法。最后回顾了蛋白质-核酸对接计算模型的应用,并对未来的工作进行了展望。     

Structural alignment of RNA with triple helix structure

Structural alignment is useful in identifying members of ncRNAs. Existing tools are all based on the secondary structures of the molecules. There is evidence showing that tertiary interactions (the interaction between a single-stranded nucleotide and a base-pair) in triple helix structures are critical in some functions of ncRNAs. In this article, we address the problem of structural alignment of RNAs with the triple helix. We provide a formal definition to capture a simplified model of a triple helix structure, then develop an algorithm of O(mn(3)) time to align a query sequence (of length m) with known triple helix structure with a target sequence (of length n) with an unknown structure. The resulting algorithm is shown to be useful in identifying ncRNA members in a simulated genome.     

Pyrimidine morpholino oligonucleotides form a stable triple helix in the absence of magnesium ions

Oligonucleotides can be used as sequence-specific DNA ligands by forming a local triple helix. In order to form more stable triple-helical structures or prevent their degradation in cells, oligonucleotide analogues that are modified at either the backbone or base level are routinely used. Morpholino oligonucleotides appeared recently as a promising modification for antisense applications. We report here a study that indicates the possibility of a triple helix formation with a morpholino pyrimidine TFO and its comparison with a phosphodiester and a phosphoramidate oligonucleotide. At a neutral pH and in the presence of a high magnesium ion concentration (10 mM), the phosphoramidate oligomer forms the most stable triple helix, whereas in the absence of magnesium ion but at a physiological monovalent cation concentration (0.14 M) only morpholino oligonucleotides form a stable triplex. To our knowledge, this is the first report of a stable triple helix in the pyrimidine motif formed by a noncharged oligonucleotide third strand (the morpholino oligonucleotide) and a DNA duplex. We show here that the structure formed with the morpholino oligomer is a bona fide triple helix and it is destabilized by high concentrations of potassium ions or divalent cations (Mg(2+)).     

Triple helix stabilization by covalently linked DNA-bisbenzimidazole conjugate synthesized by maleimide-thiol coupling chemistry

Tethering of BBZPNH2, an analogue of the Hoechst 33258, with a 14 nucleotide long DNA sequence with the help of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a heterobifunctional crosslinking reagent, using DMF/ water as solvent yields a conjugate which effectively stabilizes the triple helix. The above conjugate was hybridized with 26 bp long double stranded (ds) DNA having 14 bp long polypurine-polypyrimidine stretch to form a pyrimidine motif triple helix. The above conjugate increases the thermal stability of both the transitions, that is, triple helix to double helix by 12 degrees C and double helix to single strand transition by 16 degrees C for the triple helix formed with conjugated TFO over the triple helix made from non-conjugated TFO. Fluorescence and circular dichroism spectra recorded at different temperatures confirm the presence of minor groove binding bisbenzimidazole in the AT-rich minor groove of dsDNA even after the major groove bound TFO separates out.     

Theoretical Study of Ethidium Intercalation in Triple-Stranded DNA and at Triplex-Duplex Junctions

Abstract

The contribution of different factors in the interaction of ethidium intercalated into various sequences of a triple helix, or in the region of the junction between the double- and triple-stranded DNA has been studied by energy minimization. It is found that in the total energy of the ethidium - triple helix complexes, a particular electrostatic contribution emerges due to the presence of protonated cytosines in the triple helix. This parameter is determinant in the sequence-specificity of ethidium binding to the triple helix. The preferred intercalation sites of ethidium in the triple helix are proposed. The interaction of ethidium at the triplex-duplex junction, and its effects are also discussed. This study is aimed at searching for new drugs specific for the triple helix, or for the triplex-duplex junctions.     

Molecular dynamics simulation of the DNA triplex d(TC)5.d(GA)5.d(C+T)5.

A molecular dynamics simulation of the DNA triple helix d(TC)5.d(GA)5.d(C+T)5 is described (C+ represents a protonated cytosine residue). The simulation has been performed using the program AMBER 3.1 and includes counterions and explicit solvent under periodic boundary conditions. Both the dynamic and time-averaged behaviour of the system has been analysed. Considerable deviations from the fibre-diffraction model for DNA triple helix structure are observed, including the repuckering of the purine strand sugars that has been identified in some nuclear magnetic resonance (n.m.r.) studies. The simulation suggests that this conformational change may be driven by the possibility of improved interactions between the phosphate groups of this strand and both the solvent and counterions. Several examples of a particular conformational transition are observed, involving correlated changes in the backbone angles alpha and gamma. These transitions provide a possible explanation for some unusual n.m.r. data that have been reported. The structure of the triple helix major groove also suggests an explanation for the observed stabilization of DNA triplexes by polyvalent cations, and their ability to interact with drugs that bind in the minor groove of DNA duplexes.     

A new solution for maximal clique problem based sticker model

In this paper, we use stickers to construct a solution space of DNA for the maximal clique problem (MCP). Simultaneously, we also apply the DNA operation in the sticker-based model to develop a DNA algorithm. The results of the proposed algorithm show that the MCP is resolved with biological operations in the sticker-based model for the solution space of the sticker. Moreover, this work presents clear evidence of the ability of DNA computing to solve the NP-complete problem. The potential of DNA computing for the MCP is promising given the operational time complexity of O(nxk).     

Internal dynamics in a DNA triple helix probed by (1)H-(15)N-NMR spectroscopy

The amino group of adenine plays a key role in maintaining DNA triple helical structures, being the only functional group in DNA that is involved in both Watson-Crick and Hoogsteen hydrogen bonds. In the present work we have probed the internal dynamics of the adenine amino group in the intramolecular YRY triple helix formed by the 31-mer DNA oligonucleotide d(AGAGAGAACCCCTTCTCTCTTTTTCTCTCTT). The DNA triple helix was specifically labeled with (15)N at the amino group of the adenine in the fifth position. The rotation rate of the labeled amino group was measured as a function of temperature using (1)H-(15)N heteronuclear NMR spectroscopy. The results indicate that, in the DNA triple helix, the rotation of the adenine amino group is greatly slowed relative to that in a DNA double helix. The temperature dependence of the rotation rate suggests a large entropic contribution to this effect, which may originate from different hydration patterns of the adenine amino group in the two structures.     

Complex between triple helix of collagen and double helix of DNA in aqueous solution

We demonstrate in this paper that one example of a biologically important and molecular self-assembling complex system is a collagen–DNA ordered aggregate which spontaneously forms in aqueous solutions. Interaction between the collagen and the DNA leads to destruction of the hydration shell of the triple helix and stabilization of the double helix structure. From a molecular biology point of view this nano-scale self-assembling superstructure could increase the stability of DNA against the nucleases during collagen diseases and the growth of collagen fibrills in the presence of DNA.     

The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-Pro-Gly units

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X-Y-Gly)(n) repeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)-hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)-hydroxyproline (hyp) at the X-position destabilizes the helix as demonstrated that the triple helix of (hyp-Pro-Gly)(15) is less stable than that of (Pro-Pro-Gly)(15) and that a shorter peptide (hyp-Pro-Gly)(10) does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro-Hyp-Gly)(4) -(hyp-Pro-Gly)(2) -(Pro-Hyp-Gly)(4) and analyzed its structure by X-ray crystallography and CD spectroscopy. In the crystal, the main-chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra-molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac-hyp-NMe(2) by quantum mechanical calculations.