DNA聚合与RNA转录联用分子机器的改进

建立了一个集成DNA聚合和RNA转录过程,能够重复产生RNA适体片段,并结合孔雀石绿产生荧光信号的分子机器,并探索了此分子机器在检测DNA方面的应用.转录产生的大量孔雀石绿适体序列被释放到溶液中,并可以与孔雀石绿结合产生荧光,实现信号的放大.85μL体系中的聚合转录的最适条件为：DNA聚合酶10 IU（0.118 IU·μL-1）,RNA聚合酶60 IU（0.706 IU·μL-1）,反应时间为3h.在上述条件下,荧光信号随着引发DNA用量的增加而增大.     

氧化石墨烯对DNA荧光分子探针FRET效应的研究

研究了氧化石墨烯的浓度、氧化石墨烯与DNA分子荧光探针的反应温度、DNA链长度对FRET效应的影响.研究表明,氧化石墨烯的浓度、氧化石墨烯与DNA分子荧光探针的反应温度、DNA链长度对FRET效应都有着重要的影响.氧化石墨烯浓度越大,其对DNA分子探针的荧光猝灭效率越高;氧化石墨烯与DNA分子荧光探针的反应温度越高,淬灭效率越高;DNA链越长,氧化石墨烯淬灭的效率越低.因此,氧化石墨烯的浓度、氧化石墨烯与DNA分子荧光探针的反应温度、DNA链长度对FRET效应都应该是设计生物传感器时考虑的因素.     

基于λ核酸外切酶辅助的级联型铜离子荧光检测体系的构建

通过将一种有效的级联信号放大策略引入铜离子荧光生物传感器中,构建了一种用于超灵敏检测铜离子的荧光检测体系。这种级联型荧光检测体系结合了λ核酸外切酶辅助的靶标回收和催化发夹自组装的循环信号扩增。在铜离子的存在下,用于识别铜离子的功能核酸的底物链被特异性切割和释放。释放出的单链DNA片段(tDNA)与5′端被磷酸标记的双链DNA(TQ-DNA)杂交形成具有平末端的双链DNA。随后,该DNA分子被λ核酸外切酶识别并切割,释放出游离的tDNA和TDNA。随着λ核酸外切酶的水解,越来越多的tDNA被回收,同时放出越来越多的TDNA。此时,TDNA作为催化发夹自组装反应的触发链,启动了新一轮的靶标循环。在TDNA的辅助下,作为分子信标的发夹DNA会与另外一个发夹DNA杂交形成新的双链DNA,同时,释放出高荧光强度。利用荧光光谱法对该体系不同反应阶段进行了表征。结果表明,该级联型铜离子荧光检测体系对痕量铜离子具有很好的响应。     

基于核酸序列依赖性扩增反应比色法检测沙门氏菌

本文以食源性致病菌中危害居于前列的沙门氏菌侵袭蛋白A基因作为检测靶标,通过改进传统的核酸序列依赖性扩增反应,开发了针对长链DNA的纳米金比色检测方法。本方法利用限制性内切酶和连接酶将T7启动子和终止子序列连接至靶标序列上,形成小型环状双链DNA,在T7 RNA聚合酶的作用下,转录出大量单链RNA序列进行NASBA反应,反应终产物能够促发纳米金探针发生交联聚集,溶液颜色从红色变成蓝色,从而实现对沙门氏菌的定量分析。     

基于金纳米粒子的DNA探针和Exo Ⅲ辅助信号放大平台用于HIV-DNA的检测

设计了一种金纳米粒子(AuNP)和核酸外切酶Ⅲ(ExoⅢ)辅助靶标循环信号放大的新型纳米探针用于HIV-DNA的检测.该纳米探针由AuNP和捕获探针p1和信号探针p2构成.在靶标HIV-DNA不存在的情况下,此时纳米探针抗ExoⅢ水解,信号探针p2与AuNP之间的距离较近产生荧光共振能量转移,体系的荧光基本被猝灭.但是,在靶标存在的情况下,靶标与信号探针p2杂交,将p1置换下来,此时与靶标杂交的信号探针p2双链体能在ExoⅢ的作用下发生水解,使荧光团释放,体系荧光增强.同时,靶标也被释放并与AuNP上的探针p2作用,以驱动下一个反应,达到靶标循环目的,因此这种方式检测时间较短且稳定性高.该方法的线性范围为100 pmol/L～50 nmol/L,检测限为38.68 pmol/L(信噪比S/N=3).该方法具有良好的抗干扰性,可用于复杂生物样品检测,回收率在98.72%～106.72%之间.     

基于DNA链置换反应的编码器逻辑运算模型研究

作为自组装DNA计算领域中一门新技术,DNA链置换反应在分子计算领域得到了广泛的应用.基于自组装DNA计算原理,设计了对应不同逻辑门的DNA分子电路.基于DNA链置换反应机理构建了编码器逻辑电路的分子计算模型.当输入DNA分子信号链时,将不同分子浓度比的DNA分子逻辑门电路混合,借助分子间的特异性杂交反应及分子间链置换反应,最终可输出信号链分子.Visual DSD仿真结果表明了本文设计的编码器逻辑计算模型的可行性与准确性.为拓展分子逻辑电路的应用做出有益的探索.     

基于链置换神经网络的印刷体汉字数字识别

链置换技术是一种体外恒温无酶的分子计算技术,近年来已成为DNA计算领域的常用技术,而人工神经网络是一种模仿生物神经网络结构和功能的计算模型。基于链置换技术可以用生物分子构建神经网络,并作为分类器用于执行各种模式识别任务。文章以链置换逻辑门为基础,构建了一个赢家通吃的分子神经网络计算系统,完成了印刷体汉字数字的模式识别任务。首先将代表数字模式的图片转化成用DNA序列编码的分子数据,再将人工合成的DNA数据链输入到分子神经网络计算系统中,该网络能够利用DNA链置换技术执行生物分子计算,从而实现对输入DNA数据模式的分类,最终的分类结果将会通过荧光分子修饰单链DNA输出,并通过光电信号转换自动识别。仿真实验和生物实验证明了基于链置换的分子神经网络可以出色地完成印刷体汉字数字识别的任务。     

连接酶反应介导的荧光策略用于高效检测尿嘧啶-DNA糖基化酶的活性

目的:灵敏检测尿嘧啶-DNA糖基化酶(UDG)活性有利于生物医学研究和疾病预后.这里,构建一种连接酶反应介导的荧光策略用于高效检测UDG活性.方法:在本策略中,两条短寡核苷酸链分别与发夹探针环部序列的一半杂交,形成含缺口的DNA复合物.在UDG作用下,发夹探针环部的单个U碱基被移除,产生无嘌呤/无嘧啶(AP)位点.AP位点能抑制缺口处的连接酶反应,使位于发夹探针末端的立足点和链迁移区域仍彼此邻近,从而引发杂交链式反应(HCR),产生大量G四倍体(G4)结构.最后,G4与N甲基卟啉IX(NMM)结合,产生增强的荧光信号.结果:本策略检测限低至0.000 20U/mL,并能区分UDG与其它DNA糖基化酶.结论:本策略为生物医学研究和疾病预后提供了一种有潜力的工具用于灵敏定量检测UDG活性.     

浅论基因芯片的研究

基因芯片是将大量核酸片段探针以预先设计的方式固定在载体上组成密集的探针集群,能1次检测大量的样品靶DNA(RNA)分子的存在和量,在研究基因表达、基因测序、发现新基因、疾病诊断和治疗等方面有广泛的应用.     

高灵敏度的精确DNA链置换逻辑门研究

随着生物化学技术的不断发展,以DNA分子作为存储数据和运算媒介的新型计算模型引起了研究者的广泛关注.采用编码DNA序列构建分子逻辑门是实现DNA可编程计算的基础,虽然传统的跷跷板门(seesaw gate)作为一种DNA逻辑门能够实现阶跃函数的效果,但是它在阈值附近的变化相对平缓,而且当输入超过阈值后,输出随输入增加难以稳定于期望数值,为此文章提出了基于DNA链置换的阶跃函数门.该逻辑门以四域信号链作为统一的DNA编码形式,通过湮灭反应设定阈值信号,通过支点互补程度控制反应顺序,使逻辑门的输出信号不但在阈值附近具有较高的突变灵敏度和准确度,而且当输入超过阈值后能稳定于期望值.随后基于阶跃函数门提出了能够区分"无信号输入信号"和"输入信号为低位"的与、或、非和异或门,解决了非门难以直接表示、需要通过双轨逻辑间接表示的问题.     

Complex shapes self-assembled from single-stranded DNA tiles

Programmed self-assembly of strands of nucleic acid has proved highly effective for creating a wide range of structures with desired shapes. A particularly successful implementation is DNA origami, in which a long scaffold strand is folded by hundreds of short auxiliary strands into a complex shape. Modular strategies are in principle simpler and more versatile and have been used to assemble DNA or RNA tiles into periodic and algorithmic two-dimensional lattices, extended ribbons and tubes, three-dimensional crystals, polyhedra and simple finite two-dimensional shapes. But creating finite yet complex shapes from a large number of uniquely addressable tiles remains challenging. Here we solve this problem with the simplest tile form, a 'single-stranded tile' (SST) that consists of a 42-base strand of DNA composed entirely of concatenated sticky ends and that binds to four local neighbours during self-assembly. Although ribbons and tubes with controlled circumferences have been created using the SST approach, we extend it to assemble complex two-dimensional shapes and tubes from hundreds (in some cases more than one thousand) distinct tiles. Our main design feature is a self-assembled rectangle that serves as a molecular canvas, with each of its constituent SST strands--folded into a 3 nm-by-7 nm tile and attached to four neighbouring tiles--acting as a pixel. A desired shape, drawn on the canvas, is then produced by one-pot annealing of all those strands that correspond to pixels covered by the target shape; the remaining strands are excluded. We implement the strategy with a master strand collection that corresponds to a 310-pixel canvas, and then use appropriate strand subsets to construct 107 distinct and complex two-dimensional shapes, thereby establishing SST assembly as a simple, modular and robust framework for constructing nanostructures with prescribed shapes from short synthetic DNA strands.     

A robust DNA mechanical device controlled by hybridization topology.

Controlled mechanical movement in molecular-scale devices has been realized in a variety of systems-catenanes and rotaxanes, chiroptical molecular switches, molecular ratchets and DNA-by exploiting conformational changes triggered by changes in redox potential or temperature, reversible binding of small molecules or ions, or irradiation. The incorporation of such devices into arrays could in principle lead to complex structural states suitable for nanorobotic applications, provided that individual devices can be addressed separately. But because the triggers commonly used tend to act equally on all the devices that are present, they will need to be localized very tightly. This could be readily achieved with devices that are controlled individually by separate and device-specific reagents. A trigger mechanism that allows such specific control is the reversible binding of DNA strands, thereby 'fuelling' conformational changes in a DNA machine. Here we improve upon the initial prototype system that uses this mechanism but generates by-products, by demonstrating a robust sequence-dependent rotary DNA device operating in a four-step cycle. We show that DNA strands control and fuel our device cycle by inducing the interconversion between two robust topological motifs, paranemic crossover (PX) DNA and its topoisomer JX2 DNA, in which one strand end is rotated relative to the other by 180 degrees. We expect that a wide range of analogous yet distinct rotary devices can be created by changing the control strands and the device sequences to which they bind.     

Molecular logic computing model based on DNA self-assembly strand branch migration

In this study,the DNA logic computing model is established based on the methods of DNA self-assembly and strand branch migration.By adding the signal strands,the preprogrammed signals are released with the disintegrating of initial assembly structures.Then,the computing results are able to be detected by gel electrophoresis.The whole process is controlled automatically and parallely,even triggered by the mixture of input signals.In addition,the conception of single polar and bipolar is introduced into system designing,which leads to synchronization and modularization.Recognizing the specific signal DNA strands,the computing model gives all correct results by gel experiment.     

DNA primase acts as a molecular brake in DNA replication

A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization. Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis. Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand.     

DNA计算的粘贴模型及在组合优化中的应用

讨论了分子计算的一种新的模型——粘贴模型。它使用DNA串作为底物来进行信息表达，杂交分离作为控制机制。粘贴模型有一个可随机访问的存储空间，而不需要DNA串的延伸，也无需用酶，并且它的材料是可重复使用的。     

Sequence-specific artificial photo-induced endonucleases based on triple helix-forming oligonucleotides

Homopyrimidine oligonucleotides bind to homopurine-homopyrimidine sequences of duplex DNA forming a local triple helix. This binding can be demonstrated either directly by a footprinting technique, gel assays, or indirectly by inducing irreversible reactions in the target sequence, such as photocrosslinking or cleavage. Binding occurs in the major groove with the homopyrimidine oligonucleotide orientated parallel to the homopurine strand. Thymine and protonated cytosine in the oligonucleotide form Hoogsteen-type hydrogen bonds with A.T and G.C Watson-Crick base pairs, respectively. Here we report that an 11-residue homopyrimidine oligonucleotide covalently attached to an ellipticine derivative by its 3' phosphate photo-induces cleavage of the two strands of a target homopurine--homopyrimidine sequence. To our knowledge, this is the first reported case of a sequence-specific artificial photoendonuclease. In addition we show that a strong binding site for a free ellipticine derivative is induced at the junction between the triplex and duplex structures on the 5' side of the bound oligonucleotide. On irradiation, cleavage is observed on both strands of DNA. This opens new possibilities for inducing irreversible reactions on DNA at specific sites by the synergistic action of a triple helix-forming oligonucleotide and an intercalating agent.     

Inequality in mutation rates of the two strands of DNA

As the mechanisms for replicating the two strands of duplex DNA differ it is, in principle, possible for the mutation rates to differ depending on which strand is being copied. In the absence of selection this would lead to a difference in the measured rate of a particular base substitution, such as T to C, depending on which DNA strand was analysed to determine the rate. Thus a change such as T to C on one DNA strand results from either a direct T-to-C mutation on that strand or an A-to-G mutation on the complementary strand; for the other strand the situation is reversed, and it can be seen that different processes are responsible for the two cases, allowing for asymmetry in substitution rate. We have tested whether such asymmetry indeed occurs by studying equivalent sequences from the beta-globin complexes of six species of primate. Our results reveal an asymmetry in substitution rates consistent with predictions based on strand-inequalities in mutation rates. Our sequence comparisons also allow us to make predictions about the positions of replication origins and the replication error rates of one strand relative to the other.