额外的错配碱基提高T4 DNA连接酶等位特异性连接（英文）

连接是一种主要的DNA处理过程。由于较低的商业成本以及核酸底物识别的灵活性,T4DNA连接酶被广泛应用于生物分子工程,特别是特定核酸序列的等位特异性连接检测。本文评估了在T4 DNA连接酶介导的连接反应中,引入额外的错配碱基对所产生的影响。设计了超过150组DNA/DNA或DNA/RNA带有的额外错配碱基对的组合。结果发现,引入额外的错配碱基对后,T4 DNA连接酶在DNA/DNA连接中特异性可提高60倍以上,而在DNA/RNA连接中特异性只能提高2倍。在等位特异性连接中,有的错配碱基对可使T4 DNA连接酶的特异性提高600多倍。     

基于多重置换扩增技术的RNA病毒基因组扩增方法研究

为了便于新发或罕见病毒性传染病的筛查检测,本研究利用多重置换扩增技术,以负链RNA病毒—发热伴血小板减少综合征病毒和正链RNA病毒—登革病毒为模拟样本探索临床样本中RNA病毒基因组非特异性扩增方法。研究中通过梯度稀释的RNA病毒模拟样本中可能存在的不同丰度的病原体,样本核酸依次加工成单链cDNA、双链cDNA、T4DNA连接酶处理后的双链cDNA以及添加外源辅助RNA后合成并连接的双链cDNA形式,然后进行Phi29DNA聚合酶等温扩增,使用荧光定量PCR方法比较各种方法对RNA病毒核酸扩增的影响。结果显示,对于不同类型的RNA病毒模拟标本,多重置换扩增对于单链及双链cDNA的扩增效果有限,而双链cDNA经DNA连接酶处理后的扩增能达到6×103倍;在cDNA合成过程中加入外源辅助RNA,模拟样本中病毒基因组的扩增可达2×105倍,尤其是对含有低丰度病原体的模拟样本扩增效果的改善更为明显。本研究摸索建立了基于多重置换扩增技术的RNA病毒基因组扩增方法,能够对样本中低丰度RNA病毒基因组实现有效扩增,可满足开展多种病原体筛查检测的需求。     

DNA末端硫修饰依赖的体外大片段DNA连接法

【背景】DNA组装技术是基因组合成中的一个关键技术。探索低成本、高效率的基因组合成技术一直是合成生物学的重要研究领域。在某些细菌如变铅青链霉菌中,DNA上有磷硫酰化修饰(简称硫修饰),而在另一些细菌如天蓝色链霉菌中存在一种含有硫修饰识别结构域(sulfur-binding domain, SBD)的识别蛋白,可以特异性识别DNA上的硫修饰,这启发了我们发展出一种新的DNA组装技术。【目的】探究在DNA末端硫修饰的连接中,T4 DNA连接酶与SBD相融合蛋白和单独的T4 DNA连接酶相比,是否有更高的连接效率。【方法】根据同源重组原理,设计硫修饰引物,扩增硫修饰的DNA片段。构建T4 DNA连接酶与SBD融合蛋白的3种表达载体T4-linker-SBD(Hga)、T4-linker-SBD(Spr)和T4-linker-SBD(Mmo),表达纯化以上3种融合蛋白。比较3组浓度梯度(2.4、0.24、0.024 mg/mL) T4 DNA连接酶与融合蛋白在2.5 kb和8.0 kb DNA片段连接上的差异。【结果】DNA末端硫修饰的2.5kb和8.0kb的两端片段均能扩增,而且3种融合蛋白...     

γPNA一种新型高效的肽核酸

肽核酸(peptide nucleic acid,PNA)是一种人工合成的具有类多肽骨架的DNA类似物,具有与核酸结合特异性强、组织和细胞内生物稳定性好、半衰期长等优点。通过靶向结合DNA/RNA而抑制其复制、转录和翻译过程,进行基因调控。在PNA骨架结构中γ位点引入带手性的官能团,能形成右手螺旋结构,显著提高其与靶DNA/RNA的杂交特性,这种PNA衍生物称之为γPNA。γPNA的溶解性、热稳定性和特异性等化学与生物学特性明显改善,在基因编辑和作为探针检测等方面具有良好的应用前景。通过对γPNA结构、性质及其研究进展进行总结,以期为γPNA反义应用提供理论依据和参考。     

2′-氟取代对T7 DNA连接酶连接效率的阻滞

为更好地理解连接酶的接合机制,研究了2′-氟取代核苷酸（2′-fluorinated nucleotide, FN）对T7 DNA连接酶接合特性的影响。利用分子信标的荧光信号变化来检测连接酶接合活性。结果显示,在连接酶作用的分子信标缺口处的5′端和3′端分别进行F取代时,对T7 DNA连接酶的接合效果分别阻滞（48.7±6.7）%和（70.6±4.0）%。在取代同时发生在5′端和3′端时,接合效果被阻滞（76.6±1.3）%。动力学参数的回归显示,FN取代会导致连接反应最大速率（Vmax）降低,同时导致米氏常数Km增大,说明酶和底物之间的亲和力在取代后减小。缺口两端的碱基错配也对酶接合反应效果产生一定的阻滞效应。本研究的结果和结论使对氟取代后,T7 DNA连接酶的接合特性有了更进一步的认识,为更好地应用T7 DNA连接酶接合活性提供有力的实用依据。     

部分柔性的核酸双螺旋构象的模型化研究

基于核酸结构单元的基本思想,在分析给出任意碱基对片段的自由度集合和两碱基对片段的简化自由度集合的基础上,建立了DNA/m5cDNA/RNA双螺旋结构的理论模型和两碱基对部分柔性的构象计算方法.利用本方法获得的标准B-DNA双螺旋构象参数与实验基本一致.根据DNA嵌插受体的统计性实验约束,优化产生了适用于阿霉素类抗癌药的三碱基对DNA片段的理论嵌插受体模型.     

DNA连接酶、RNA连接酶、多核苷酸激酶和DNA聚合酶的同时分离纯化

本文报道从T4amN82噬菌体诱导的大肠杆菌E.coliB 同时分离和纯化四种酶的一般方法。先用硫酸链霉素沉淀把RNA连接酶,DNA连接酶与多核苷酸激酶和DNA聚合酶加以分离。然后用DEAE纤维素柱层析把DNA连接酶与RNA连接酶加以分离,用DEAE-Sephadex-A50柱层析把多核苷酸激酶与DNA聚合酶加以分离。本文着重介绍T4DNA聚合酶的分离纯化。     

噬菌体T4诱导的脱氧核糖核酸(DNA)连接酶的分离和纯化

本文介绍一个从噬菌体T4诱导的大肠杆菌中纯化DNA连接酶的简法。这是从同一起始原料(噬菌体T4感染的大肠杆菌菌体)同时提纯三种酶(多核苷酸激酶,DNA连接酶及RNA连接酶)的步骤的一部分。这个方法包括以下几步：超声破碎菌体,抽提粗酶,用硫酸链霉素沉淀去除多核苷酸激酶和DNA．甩I)EAE纤素素(DE-52)柱层折将DNA建接酶与RNA连接酶分离开来,用磷酸纤维素(P-II)分步冼脱DNA连接酶,对在Tris-HCI,pH7.6中含50％甘油的缓冲液透析加以浓缩。最终得到高浓度(5500单位／毫升)和高纯度酶制品。     

无缝克隆技术的研究进展

DNA克隆技术是分子生物学和基因工程研究必备的工具之一,但传统的以限制性内切酶酶切/连接为手段的克隆技术存在依赖限制性内切酶、连接效率低、耗时长和引入额外序列等缺点。近年来,无缝克隆技术发展迅猛,大量商业化克隆试剂盒也趋于成熟并在实验室被广泛应用,有力推动了蛋白质工程以及合成生物学的发展。本文从无缝克隆原理出发,对近年来发展出来的基于外切酶、PCR技术、同源重组、热稳定的连接酶等原理的无缝克隆技术进行了分类和讨论。特别是新酶资源的不断发现,如核酸外切酶XthA、FnCas12a突变体,与现有技术的相互融合,如Rec/ET重组和核酸外切酶共同使用,将有力推动无缝克隆技术的发展。     

黄地老虎颗粒体病毒DNA片段在枯草芽孢杆菌中的克隆和启动氯霉素抗性基因表达

黄地老虎颗粒体病毒(Agrotis segetum granulosis virus简称AsGv)DNA和pPL603质粒DNA,用限制性核酸内切酶EcoR I酶切后,再用T4连接酶连接,转化枯草芽孢杆菌BRl51感受态细胞。然后在loμg／m1氯霉素的sPBY选择平皿上筛选,得到了抗氯霉素的转化子。经过琼脂糖凝胶电泳检测,转化于中的重组质粒比原载体pPL603质粒分子量大。由于重组质粒上的抗性表达水平可达250μg／m1氯霉素,比pPL603质粒抗性表达水平(5μg／m1)提高50倍。所以重组质粒携带了有启动作用的DNA片段。启动功能片段的分子量,经琼脂糖凝胶电泳测定约为O．9kb。除进行DNA—DNA分子杂交确证外,并用重组质粒pAsGVPl5进行了第二次转化、抗性水平测定和分子量分析。     

Human DNA ligase IV and the ligase IV/XRCC4 complex: analysis of nick ligation fidelity

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.     

An error-prone viral DNA ligase

Our recent demonstration that DNA polymerase X (Pol X), the DNA repair polymerase encoded by the African swine fever virus (ASFV), is extremely error prone during single-nucleotide gap filling led us to hypothesize that it might contribute to genetic variability in ASFV. For the infidelity of Pol X to be relevant, however, the DNA ligase working downstream of it would need to be capable of sealing nicks containing 3'-OH mismatches. We therefore examined the nick ligation capabilities of the ASFV-encoded DNA ligase and here report the first complete 3' fidelity analysis, employing catalytic parameters, for any DNA ligase. The catalytic efficiency of nick sealing by both ASFV DNA ligase and bacteriophage T4 DNA ligase was determined in the steady state for substrates containing all 16 possible matched and mismatched base pair combinations at the 3' side of a nick. Our results indicate that ASFV DNA ligase is the lowest-fidelity DNA ligase ever reported, capable of ligating a 3' C:T mismatched nick (where C and T are the templating and nascent nucleotides, respectively) more efficiently than nicks containing Watson-Crick base pairs. Comparison of the mismatch specificity of Pol X with that of ASFV DNA ligase suggests that the latter may have evolved toward low fidelity for the purpose of generating the broadest possible spectrum of sealed mismatches. These findings are discussed in light of the genetic and antigenic variability observed among some ASFV isolates. Two novel assays for determining the concentration of active DNA ligase are also reported.     

Cognate base‐pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase

We present cognate base pair selectivity in template‐dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds‐Pa. T4 DNA ligase efficiently recognizes the Ds‐Pa pairing at the conjugation position, and Ds excludes the noncognate pairings with the natural bases. Our results indicate that the hydrophobic base pairing is allowed in enzymatic ligation with higher cognate base‐pair selectivity, relative to the hydrogen‐bond interactions between pairing bases. The efficient ligation using Ds‐Pa can be employed in recombinant DNA technology using genetic alphabet expansion, toward the creation of semi‐synthetic organisms containing UBPs.     

RNA-templated DNA ligation for transcript analysis

Ligase-mediated gene detection has proven valuable for detection and precise distinction of DNA sequence variants. We have recently shown that T4 DNA ligase can also be used to distinguish single nucleotide variants of RNA sequences. Here we describe parameters that influence RNA-templated DNA ligation by T4 DNA ligase. The reaction proceeds much more slowly, requiring more enzyme, compared to ligation of the same oligonucleotides hybridized to the corresponding DNA sequence. The reaction is inhibited at high concentrations of ATP and NaCl and both magnesium and manganese ions can support the reaction. We define reaction conditions where 80% of RNA target molecules can template a diagnostic ligation reaction. Ligase-mediated RNA detection should provide a useful mechanism for sensitive and accurate detection and distinction of RNA sequence variants.     

Enhanced mismatch selectivity of T4 DNA ligase far above the probe: Target duplex dissociation temperature

T4 DNA ligase is a widely used ligase in many applications; yet in single nucleotide polymorphism analysis, it has been found generally lacking owing to its tendency to ligate mismatches quite efficiently. To address this lack of selectivity, we explored the effect of temperature on the selectivity of the ligase in discriminating single base pair mismatches at the 3′‐terminus of the ligating strand using short ligation probes (9‐mers). Remarkably, we observe outstanding selectivities when the assay temperature is increased to 7 °C to 13 °C above the dissociation temperature of the matched probe:target duplexes using commercially available enzyme at low concentration. Higher enzyme concentration shifts the temperature range to 13 °C to 19 °C above the probe:target dissociation temperatures. Finally, substituting the 5′‐phosphate terminus with an abasic nucleotide decreases the optimal temperature range to 7 °C to 10 °C above the matched probe:target duplex. We compare the temperature dependence of the T4 DNA ligase catalyzed ligation and a nonenzymatic ligation system to contrast the origin of their modes of selectivity. For the latter, temperatures above the probe:target duplex dissociation lead to lower ligation conversions even for the perfect matched system. This difference between the two ligation systems reveals the uniqueness of the T4 DNA ligase's ability to maintain excellent ligation yields for the matched system at elevated temperatures. Although our observations are consistent with previous mechanistic work on T4 DNA ligase, by mapping out the temperature dependence for different ligase concentrations and probe modifications, we identify simple strategies for introducing greater selectivity into SNP discrimination based on ligation yields.     

Chemical interrogation of mismatches in DNA-DNA and DNA-RNA duplexes under nonstringent conditions by selective 2'-amine acylation

2'-Amine-substituted nucleotides in hybridized duplexes can be chemically tagged in an acylation reaction that is faster for mismatched or flexible nucleotides than for residues constrained by base pairing. Here we explore mismatch and hybridization detection using probe oligodeoxynucleotides containing single 2'-aminocytidine or -uridine nucleotides annealed to DNA or RNA targets under nonstringent conditions, below T(m). Consistent with a mechanism in which 2'-amine acylation is gated by local nucleotide flexibility, we find that efficient acylation is correlated with formation of weaker or fewer hydrogen bonds in base pair mismatches. Using 2'-aminocytidine-containing probes annealed to both DNA and RNA targets, mismatches are reliably detected as rapid selective acylation of the 2'-amine group in two sequence contexts. For probe oligonucleotides containing 2'-aminouridine residues, good discrimination between U-A base pairs and U-G mismatches could be obtained for DNA-DNA but not for DNA-RNA duplexes upon the introduction of a single 2'-O-Me group 5' to the 2'-amino nucleotide. The 2'-O-Me group introduces a structural perturbation, presumably to a more A-form-like structure, that exaggerates local flexibility at mismatches in DNA strands. Thus, 2'-amine acylation can be used to interrogate all possible mismatches in DNA-DNA duplexes and mismatches involving 2'-amine-substituted cytidine nucleotides in DNA-RNA heteroduplexes. Applications of this chemistry include detecting and chemically proofreading single nucleotide polymorphisms in both DNA and RNA targets and quantifying absolute amounts of RNA.     

Site-directed modification of DNA duplexes by chemical ligation.

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.     

Crystal structure of human methyl-binding domain IV glycosylase bound to abasic DNA

The mammalian repair protein MBD4 (methyl-CpG-binding domain IV) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC), and downstream base excision repair proteins restore a G·C pair. MBD4 is also implicated in active DNA demethylation by initiating base excision repair of G·T mispairs generated by a deaminase enzyme. The question of how mismatch glycosylases attain specificity for excising thymine from G·T, but not A·T, pairs remains largely unresolved. Here, we report a crystal structure of the glycosylase domain of human MBD4 (residues 427-580) bound to DNA containing an abasic nucleotide paired with guanine, providing a glimpse of the enzyme-product complex. The mismatched guanine remains intrahelical, nestled into a recognition pocket. MBD4 provides selective interactions with the mismatched guanine (N1H, N2H(2)) that are not compatible with adenine, which likely confer mismatch specificity. The structure reveals no interactions that would be expected to provide the MBD4 glycosylase domain with specificity for acting at CpG sites. Accordingly, we find modest 1.5- to 2.7-fold reductions in G·T activity upon altering the CpG context. In contrast, 37- to 580-fold effects were observed previously for thymine DNA glycosylase. These findings suggest that specificity of MBD4 for acting at CpG sites depends largely on its methyl-CpG-binding domain, which binds preferably to G·T mispairs in a methylated CpG site. MBD4 glycosylase cannot excise 5-formylcytosine (fC) or 5-carboxylcytosine (caC), intermediates in a Tet (ten eleven translocation)-initiated DNA demethylation pathway. Our structure suggests that MBD4 does not provide the electrostatic interactions needed to excise these oxidized forms of mC.