三链DNA结构研究的新进展

本文详细总结了近年来三螺旋ＤＮＡ的结构和应用研究方面的进展，分析了各种因素（例如ｐＨ、抗衡离子、温度等）对三螺旋稳定性的影响，并提出了一些在三螺旋ＤＮＡ中富有挑战性的需进一步研究的问题     

三链DNA的形成抑制DNA结合蛋白与启动子的结合

电泳迁移分析方法及DNaseⅠ足迹实验表明21nt脱氧寡核苷酸G3TG2T GT2G5TG2TGT(CP1)与乙肝病毒(HBV)核心启动子(Cp)片段之间三链DNA的形成有较高的特异性及稳定性.凝胶滞留实验显示, 在大鼠肝细胞核提取物体外转录系统中, CP1可特异地抑制DNA结合蛋白与Cp片段的结合, 而不能与Cp结合形成三链DNA的脱氧寡核苷酸CP3(TGTG2TG5T2GTG2TG3)对蛋白与Cp的结合并无抑制作用.这些结果表明, 三链DNA的形成有可能抑制HBV DNA的转录.     

RecA蛋白介导的小粒野生稻STK类抗性基因富集文库的构建

利用RecA蛋白的同源重组特性,以生物素标记的STK类抗性基因PCR扩增产物为探针,对小粒野生稻DNA文库进行富集,构建了库容量为1 710个的STK类抗性基因富集文库,通过菌落原位杂交筛选获得21个阳性克隆.其中10个阳性克隆进行了两端测序,生物信息学分析表明:有3个阳性克隆可分别定位于粳稻抗性基因附近和抗性基因内,可能是新的候选抗性基因;有2个阳性克隆可以在粳稻上定位,但周围无抗性基因片段;其它5个阳性克隆由于与栽培稻有多处匹配,不能精确定位。     

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.     

三锭DNA结构研究的新进展

本文详细总结了近年来三螺旋ＤＮＡ的结构和应用研究方面的进展,研究了各种因素对三螺肇稳定性的影响,并提出了一些在三螺肇ＤＮＡ中富有挑战性的需进一步研究的问题。     

基于三链核酸的DNA计算

一种研究DNA计算的新模型——三链DNA计算模型在本文中提出。此模型是在近年三链核酸的研究成果的基础上建立的。并应用于求解可满足性问题（SAT）,这是一个困难的NP-完全问题。不同于以住的DNA计算方法,基于三链核酸的分子算法通过寡聚脱氧核苷酸（ODN）在RecA蛋白的介导下与同源的双链DNA匹配成三链DNA进行基本的运算,这样可以有效减少计算中的错误。依据分子生物学的实验方法,该算法切实可行并且有效。     

三链DNA的分离纯化

λ－ＤＮＡ Ｈｉｎｄ ＩＩＩ热变性可得到单、双、三链ＤＮＡ等混合物,本文利用ＤＮａｓｅＩ选择性地将其中的单、双链酶解后,经ＳｅｐｈａｄｅｘＧ－１５０分离,得到了纯化的三链ＤＮＡ,并对其进行了ＵＶ、荧光光谱、ＣＤ谱及Ｔｍ等性质测定。     

分子间三链DNA的应用

自Ｄｅｒｖａｎ等１９８７年首先证实三链ＤＮＡ的形成可介导对靶ＤＮＡ的特异切割以来,有关三链ＤＮＡ的研究进展和奶快。本综述了近几年分子间三链ＤＮＡ在基因表达调控、染色体作图、基因分离及定点诱变等方面的应用,并对三链ＤＮＡ应用中存在的几个问题及相应的解决方法作了简单介绍。     

乙肝病毒核衣壳启动子内三链DNA的形成

用电泳迁移分析方法研究了２１ｎｔ脱氧寡核苷酸Ｇ３ＴＧ２ＴＧＴ２Ｇ５ＴＧ２ＴＧＴ与１２９ｂｐ的乙肝病毒核衣壳启动子片段内一位点结合形成的三链ＤＮＡ的特异性及稳定性。在克隆有ＨＢＶ基因组的质粒ｐＣＲＰ１０的酶切产物中,ＣＰ１仅与含Ｃｐdisplay structure     

A novel means of asymmetric linker attachment to DNA molecule by RecA-mediated multistranded DNA formation

We have developed a novel asymmetric linker attachment technology that utilizes multistranded DNA formation mediated by the RecA protein and Exonuclease I. Multistranded DNA can readily be formed at the terminus of double-stranded DNA by a complementary oligonucleotide in the presence of RecA and Exonuclease I. We have explored the possibility of applying this finding to the asymmetric attachment of linkers to specific DNA termini. We show that these unique properties of the terminal triple-stranded structure can be applied to directly clone specific DNA sequences.     

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology

The RecA protein ofEscherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.     

RecA-mediated joint molecule formation between O-methylated RNA/DNA hairpins and single-stranded targets

Chimeric oligonucleotides consisting of one DNA strand paired with an O-methylated RNA strand interrupted by six DNA residues have been used in gene targeting experiments. Here we demonstrate that these hairpins can form a heteroduplex (or joint molecule) with single-stranded DNA targets in a reaction mediated by the E. coli RecA protein. One end of the double hairpin may unwind to form a 14-base-RecA filament which initiates the reaction. Chimeric oligonucleotides containing only O-methylated RNA residues on one strand or truncated hairpins lacking this 14-base segment did not participate in RecA-driven heteroduplex formation under these reaction conditions. The results presented here represent a first step in studying one facet of a strategy which uses O-methylated RNA residues as participants in homologous pairing events. Received: 2 June 1997 / Accepted: 29 September 1997     

Sequence Specific DNA Purification by Triplex Affinity Capture: Using Solid Support Linked Oligodeoxynucleotide

Abstract

Sequence-specific DNA isolation by triplex affinity capture using solid support linked oligonucleotide has been described. It has been demonstrated that it is possible to separate oligonucletides differing in single point mutation by this method.     

一个能与水稻未成熟种子核蛋白特异结合的31bp的DNA片段

水稻蜡质基因5'上游序列中有5个能与水稻胚乳核蛋白结合的片段,其中HinfId和RsaIe片段有部分重叠,而重叠部分含AACGT顺序。按重叠区上下游顺序合成了其中含有3次重复的AACGT顺序的31bp寡核苷酸,并以此为探针进行凝胶滞后实验,表明它不仅能与未成熟水稻种子的胚乳核蛋白特异结合,而且也与HinfId和RsaIe片段有强烈的竞争作用。因此31bp顺序中含有与水稻胚乳核蛋白专一结合位点,从而有可能应用此31bp的寡核苷酸作探针来分离编码蜡质基因的调控蛋白基因。     

RecA-mediated strand invasion of DNA by oligonucleotides substituted with 2-aminoadenine and 2-thiothymine

Sequence-specific recognition of DNA is a critical step in gene targeting. Here we describe unique oligonucleotide (ON) hybrids that can stably pair to both strands of a linear DNA target in a RecA-dependent reaction with ATP or ATPγS. One strand of the hybrids is a 30-mer DNA ON that contains a 15-nt-long A/T-rich central core. The core sequence, which is substituted with 2-aminoadenine and 2-thiothymine, is weakly hybridized to complementary locked nucleic acid or 2′-OMe RNA ONs that are also substituted with the same base analogs. Robust targeting reactions took place in the presence of ATPγS and generated metastable double D-loop joints. Since the hybrids had pseudocomplementary character, the component ONs hybridized less strongly to each other than to complementary target DNA sequences composed of regular bases. This difference in pairing strength promoted the formation of joints capable of accommodating a single mismatch. If similar joints can form in vivo, virtually any A/T-rich site in genomic DNA could be selectively targeted. By designing the constructs so that the DNA ON is mismatched to its complementary sequence in DNA, joint formation might allow the ON to function as a template for targeted point mutation and gene correction.     

Population Studies of Intact Vitamin D Binding Protein by Affinity Capture ESI-TOF-MS

Blood plasma proteins with molecular weights greater than approximately 30 kDa are refractory to comprehensive, high-throughput qualitative characterization of microheterogeneity across human populations. Analytical techniques for obtaining high mass resolution for targeted, intact protein characterization and, separately, high sample throughput exist, but efficient means of coupling these assay characteristics remain rather limited. This article discusses the impetus for analyzing intact proteins in a targeted manner across populations and describes the methodology required to couple mass spectrometric immunoassay with electrospray ionization mass spectrometry for the purpose of qualitatively characterizing a prototypical large plasma protein, vitamin D binding protein, across populations.     

A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp

Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.     

Structure and interactions of RecA: plasticity revealed by molecular dynamics simulations

Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from Mycobacterium tuberculosis, Mycobacterium smegmatis and Escherichia coli and their Adenosine triphosphate (ATP) complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural framework within which RecA molecules from different eubacteria function. Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins.