RNA切割型脱氧核酶功能化纳米材料荧光生物传感研究进展

脱氧核酶是通过体外筛选得到的一种具有酶活性的功能核酸。其中，具有RNA切割功能的脱氧核酶由于具有特异性强、可编程性高及易于合成修饰的特性，已在生物传感领域得到了广泛的发展，但易受核酸酶降解、胞内传输效率低等问题限制了其在体内的应用。纳米材料尺寸小、比表面积大及生物相容性好，在生物领域展现出了出色的应用前景，RNA切割型脱氧核酶与纳米材料的结合为解决上述问题提供了新思路。综述了基于RNA切割型脱氧核酶功能化纳米材料的荧光生物传感的研究进展，首先介绍了RNA切割型脱氧核酶功能化纳米材料的常见功能化策略，随后详细讨论了基于RNA切割型脱氧核酶功能化纳米材料的荧光传感器在体内和体外检测重要生物分子的最新应用，最后就此类传感器的开发所面临的挑战和未来发展方向进行了展望。     

核酶在病毒基因治疗中的研究进展

核酶是一类具有催化功能的核酸分子,能够与双链DNA或RNA分子结合,从而抑制其转录或翻译。由于核酶能够在相应位点切割核酸序列,具有酶的活性,使其在病毒的基因治疗中具有广阔的应用前景。随着有效的转运和表达方法的建立及核酸化学的进步,核酶有望成为基因治疗中最有效的工具之一。本文就核酶在病毒基因治疗中的研究进展作一综述。     

10-23脱氧核酶的研究进展

10-23脱氧核酶(Deoxyribozyme,DRz)是利用体外分子进化技术筛选得到的一种具有催化功能的单链DNA片段,具有高效的催化活性及特异的序列识别能力,能催化RNA特定部位的切割反应,抑制相应蛋白的表达.本文就10-23 DRz的结构、活性影响因素、反应机制、生物特性及应用的研究进展作一综述.     

基于脱氧核酶的生物传感器的实际应用

自近30年前发现脱氧核酶以来,脱氧核酶已广泛应用于生物传感领域。电化学法、荧光、比色法、电化学发光、化学发光、光电化学等检测方法在基于脱氧核酶的生物传感器中广泛应用于生物样品方面的检测。主要讨论了现有的基于脱氧核酶的生物传感器的几种常见检测方法在检测生物样品方面的实际应用,并且讨论了几种检测方法的优缺点,并指出了现有研究的不足之处。     

非标记脱氧核酶构象改变荧光法检测铀酰离子

在p H 5.5 MES缓冲溶液中,当体系中不存在铀酰离子时,SYBR GreenⅠ(SG)能通过嵌入和小沟结合方式与脱氧核酶作用,导致荧光增强;当铀酰离子存在时,脱氧核酶的r A碱基处断裂,游离出单链,此时SG与单链DNA作用减弱,荧光信号降低,且体系的荧光强度变化值与铀酰离子浓度在1.73×10-9⁴.40×10-8mol/L范围内呈良好线性关系,线性回归方程为ΔF=108.99C(×10-8mol/L)+79.22,相关系数r=0.990。根据空白管的标准偏差Sb和标准曲线的斜率k算出LOD为5.2×10-10mol/L。该方法简便、选择性好、灵敏度高。     

脱氧核酶电化学生物传感器高灵敏测定铅离子

基于铅离子脱氧核酶对铅离子的特异性识别作用,研制了用于测定铅离子的高灵敏电化学生物传感器。该传感器以固定于电极表面、末端标记了电信号基团的脱氧核酶作为识别元素和信号探针。当传感界面与含有铅离子的溶液接触时,脱氧核酶的酶链在铅离子的辅助作用下将修饰了电信号基团的底物链切断并使其脱离电极表面,从而使电极表面电信号分子的电流减小,产生用于铅离子定量检测的电信号。在0.5~12.5μg/L的浓度范围内,检测信号与Pb~(2+)浓度呈良好的线性关系,Pb~(2+)的检出限为0.3μg/L。同时,该电化学生物传感器对Pb~(2+)的检测表现出良好的特异性和选择性。     

脱氧核苷酸钠溶液电渗析法脱盐

<正> 核酸是生物体中传递遗传信息的一类非常重要的生物大分子。它们是由碱基、戊糖、磷酸所组成的核苷酸单体缩聚而成。根据所含戊糖的种类不同,核酸又分为核糖核酸(RNA)和脱氧核糖核酸(DNA)两大类。核酸在生物遗传变异、生长繁殖、分化发育等方面都起着决定性作用,核酸的研究对于肿瘤的发生和治疗也有重要意义。目前,核酸的研究已成为生物化学和分子生物学中一个十分活跃的领域。本文研究的脱氧核苷酸钠是由脱氧核糖核酸酶介而成的四种核苷酸单体钠盐的混合物(即脱氧腺嘌呤核苷酸钠、脱氧乌嘌呤核苷酸钠、脱氧胞嘧啶核苷酸     

基于末端脱氧核苷酸转移酶协同G-四链体核酶的恩诺沙星检测新方法

恩诺沙星因抗菌活性强、抗菌谱广而广泛应用于动植物疾病治疗，但过度使用甚至滥用会导致其在动植物食品中的残留量超标，因此发展高效的恩诺沙星检测方法至关重要。本工作基于末端脱氧核苷酸转移酶（TdT）协同G-四链体核酶设计信号放大策略，建立了一种新型恩诺沙星（ENR）电化学检测方法。目标物恩诺沙星与特异性核酸适体的结合触发TdT在电极表面的扩增反应，产生G-四链体核酶纳米线结构，进而发挥辣根过氧化物酶活性催化信号放大，实现恩诺沙星的高灵敏和高特异性检测。本方法对恩诺沙星的线性检测范围为0.5 ~ 50 ng/mL，检测限低至0.043 ng/mL。此外，该无标记电化学生物传感器简单快速，成本低，并成功应用于对实际食品样本的分析检测，显示出较好的应用潜能。     

非标记脱氧核酶构象改变荧光法检测铀酰离子

在p H 5.5 MES缓冲溶液中,当体系中不存在铀酰离子时,SYBR GreenⅠ(SG)能通过嵌入和小沟结合方式与脱氧核酶作用,导致荧光增强;当铀酰离子存在时,脱氧核酶的r A碱基处断裂,游离出单链,此时SG与单链DNA作用减弱,荧光信号降低,且体系的荧光强度变化值与铀酰离子浓度在1.73×10-9~4.40×10-8mol/L范围内呈良好线性关系,线性回归方程为ΔF=108.99C(×10-8mol/L)+79.22,相关系数r=0.990。根据空白管的标准偏差Sb和标准曲线的斜率k算出LOD为5.2×10-10mol/L。该方法简便、选择性好、灵敏度高。     

2—甲基—1，4—萘醌对DNA光敏损伤的诱导作用

选择波长337nm的激光作为激励光源，借助凝胶电泳研究了2－甲基－1，4－萘醌诱导的DNA光敏损伤。结果表明：在氧气饱和、脱氧条件下光敏损伤显著，DNA损伤主要与光子剂量、核酸与萘醌浓度比及DNA存在形式有关。     

Nanopore-Based Analysis of Chemically Modified DNA and Nucleic Acid Drug Targets

Nucleic acids are central figures in many of life’s key molecular processes, e.g., enzymatic activity, epigenetics/gene regulation, viral replication, aging, cancer, and other diseases. Over the past two decades, nanopores have emerged as a new tool for studying the properties of nucleic acids at the single-molecule level. In this review, we summarize the use of nanopores as sensors of nucleic acid structure, particularly for studying chemically modified and damaged DNA, and for probing the interactions of small-molecule drugs with nucleic acid targets.     

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods

For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification‐free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra‐bright macrofluorophores of 9–84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target‐specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA.     

可控式citZ基因纳米盒的设计与研究

DNA分子具有自我识别的特殊能力，DNA折纸术就是利用这一特性进行核酸纳米材料精准设计和组装的一种新技术。研究者可以利用与DNA脚手架链互补的订书钉链，将长链核酸折叠成与预设模型一致的纳米结构。DNA折纸术最早是2006年由Rothemund提出，一直以来，人们利用M13mp18单链线性DNA进行各种纳米图形的自组装。为了寻找更多的核酸材料进行DNA折纸研究，本文以枯草芽孢杆菌（Bacillus. subtilis 168）citZ基因序列为研究对象，采用改进的DAEDALUS软件，引入“锁钥”结构设计，利用“从下向上”的方法使DNA分子进行自组装，设计出三维体积为50.71nm×50.71nm×50.71nm的citZ基因纳米盒，只有遇到可识别的基因和匹配的“钥匙”时，才可能打开盖子，释放盒中的内容物。这种核酸纳米材料还可以通过调节DNA序列长度调节盒子的内部空间，有望成为一种新型的靶向药物运送载体。     

Molecular dynamics applied to nucleic acids

In this Account, we focus on molecular dynamics (MD) simulations involving fully solvated nucleic acids. Historically, MD simulations were first applied to proteins and several years later to nucleic acids. The first MD simulations of DNA were carried out in vacuo, but nowadays fully solvated systems are common practice. Recently, technical improvements have made it possible to conduct accurate MD simulations of highly charged nucleic acids. The state-of-the-art of MD simulations and a number of applications on various nucleic acid systems are discussed.     

Charge-reversal lipids, peptide-based lipids, and nucleoside-based lipids for gene delivery

Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy's appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to myriad diseases through treatments tailored to a specific individual's genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when non-viral vectors are used have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: (1) The release of the nucleic acid from the carrier will increase gene transfection. (2) The use of biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery. (3) Mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will produce unique supramolecular assembles with high transfection efficiencies. The results presented in this Account demonstrate that engineering the chemical components of the lipid vectors to enhance nucleic acid binding and release kinetics can improve the cellular uptake and transfection efficacy of nucleic acids. Specifically, our research has shown that the incorporation of a charge-reversal moiety to initiate a shift of the lipid from positive to negative net charge improves transfection. In addition, by varying the composition of the spacer (rigid, flexible, short, long, or aromatic) between the cationic headgroup and the hydrophobic chains, we can tailor lipids to interact with different nucleic acids (DNA, RNA, siRNA) and accordingly affect delivery, uptake outcomes, and transfection efficiency. The introduction of a peptide headgroup into the lipid provides a mechanism to affect the binding of the lipid to the nucleic acid, to influence the supramolecular lipoplex structure, and to enhance gene transfection activity. Lastly, we discuss the in vitro successes that we have had when using lipids possessing a nucleoside headgroup to create unique self-assembled structures and to deliver DNA to cells. In this Account, we state our hypotheses and design elements as well as describe the techniques that we have used in our research to provide readers with the tools to characterize and engineer new vectors.     

Circular Nucleic Acids: Discovery,Functions and Applications

Circular nucleic acids (CNAs) are nucleic acid molecules with a closed-loop structure. This feature comes with a number of advantages including complete resistance to exonuclease degradation, much better thermodynamic stability, and the capability of being replicated by a DNA polymerase in a rolling circle manner. Circular functional nucleic acids, CNAs containing at least a ribozyme/DNAzyme or a DNA/RNA aptamer, not only inherit the advantages of CNAs but also offer some unique application opportunities, such as the design of topology-controlled or enabled molecular devices. This article will begin by summarizing the discovery, biogenesis, and applications of naturally occurring CNAs, followed by discussing the methods for constructing artificial CNAs. The exploitation of circular functional nucleic acids for applications in nanodevice engineering, biosensing, and drug delivery will be reviewed next. Finally, the efforts to couple functional nucleic acids with rolling circle amplification for ultra-sensitive biosensing and for synthesizing multivalent molecular scaffolds for unique applications in biosensing and drug delivery will be recapitulated.     

Simulations of nucleic acids and their complexes

Recent years have seen considerable progress in simulations of nucleic acids. Improvements in force fields, simulation techniques and protocols, and increasing computer power have all contributed to making nanosecond-scale simulations of both DNA and RNA commonplace. The results are already helping to explain how nucleic acids respond to their environment and to their base sequence and to reveal the factors underlying recognition processes by probing biologically important nucleic acid-protein interactions and medically important nucleic acid-drug complexation. This Account summarizes methodological progress and applications of molecular dynamics to nucleic acids over the past few years and tries to identify remaining challenges.     

Unlocked Nucleic Acids with a Pyrene‐Modified Uracil: Synthesis,Hybridization Studies,Fluorescent Properties and i‐Motif Stability

The synthesis of two new phosphoramidite building blocks for the incorporation of 5‐(pyren‐1‐yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene‐modified nucleobase component were found to destabilize an i‐motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene‐modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene‐modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady‐state fluorescence emission studies of oligonucleotides containing pyrene‐modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene‐modified UNA monomers was observed after formation of i‐motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene‐modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.     

Fluorescence-based assay formats and signal amplification strategies for DNA microarray analysis

DNA microarrays are powerful tools for the high throughput analysis of nucleic acids due to their parallel detection capabilities. To realize the apparent power of DNA microarray, an efficient assay format is essential and a variety of assay formats have been developed for nucleic acid detection on microarrays. Many of them employ fluorescence-based methods because fluorescence detection is straightforward and easy to implement. Herein we broadly review fluorescence-based assay formats with a focus on PCR-associated target preparation. For the heterogeneous assay of nucleic acid on DNA microarrays, sensitivity is one of the most important factors and special emphasis has been given to recently developed signal amplification strategies aimed at achieving high sensitivity.