纳米荧光探针用于核酸分子的检测及成像研究

核酸,包括脱氧核糖核酸和核糖核酸,在生物的生长、发育、突变、炎症、癌症等正常或异常的生命活动中发挥着重要的作用,它们的异常表达与多种疾病的发生、发展也密切相关.因此,发展准确、有效的方法实现核酸分子的检测,对深入探究核酸的功能调控以及相关疾病的早期检测与治疗都具有重要的意义.荧光检测法与荧光成像技术具有灵敏度高、时空分辨率高等优点,为实时、准确的检测核酸分子提供了有力的工具.本文着重综述了近年来发展的纳米荧光探针用于疾病相关核酸分子的检测与细胞和活体成像工作的研究进展,最后提出了进一步构建新型纳米荧光探针用于核酸检测面临的挑战、未来发展方向与展望.     

锁核酸分子信标在分子识别与生物分析中的应用

分子信标是一种荧光探针,闭合时呈发夹结构。其5'末端修饰荧光基团,3'末端修饰猝灭基团。当目标存在时,环部与目标结合,发夹打开,发出荧光。锁核酸是一类双环状寡核苷酸衍生物,能够遵循碱基互补配对原则与核酸结合。锁核酸分子信标技术,结合了分子信标无需分离未结合探针而直接检测的优势和锁核酸亲合力强、热稳定性好、抗酶切以及体内无毒等特点,在核酸检测方面具有灵敏度高、特异性好的独特优势,近年来得到广泛关注。本文介绍了锁核酸修饰分子信标的结构、功能、设计要点,及其研究现状和一些重要进展,并讨论了目前锁核酸分子信标在分子识别及生物分析中的应用及存在的问题和发展前景。     

一种新型荧光探针--分子信标的研究及应用进展

分子信标是一种基于荧光能量转移原理而设计的发夹型寡聚核酸荧光探针。它通过与核酸等靶分子相互作用后发生构象的变化而产生荧光信号,对靶分子的检测具有灵敏度高、选择性强、适合于活体实时检测等优点。目前已广泛应用于生物化学分析、生物医学研究和环境监测等各领域。本文对分子信标的设计原理及其研究和应用进展进行了综述。     

基于核酸的纸基荧光生物传感器的设计及应用

纸基生物传感器由于其具有成本低、操作方便、生物可降解、识别元件用量低等优点,近年来受到了广泛的关注。其中,以功能核酸作为识别元件的纸基荧光生物传感器具有较高的灵敏度、瞬时响应以及实时检测等特性,在便携式传感设备方面展现出巨大的潜力。此外,将核酸作为识别元件的纸基无细胞蛋白合成平台,通过条件合成的报告荧光蛋白可实现对病毒、重金属等目标物的特异性检测,具有良好的应用前景。首先,本文介绍了基于核酸的纸基荧光生物传感器的设计,特别是基于核酸的识别元件与纸基材料的结合方式。其次,总结了基于核酸的纸基荧光生物传感器在临床诊断、食品安全检测、环境污染物检测等不同领域的最新研究进展,讨论了其优势与局限性。最后,探讨了基于核酸的纸基荧光生物传感器的发展方向与应用前景,以期为相关领域的研究提供参考。     

达安基因两产品获欧洲CE认证

达安基因公司近期通过了BSI（英国标准协会）的认证，荣获BSI颁发的医疗器械质量管理体系（ISO13485）认证证书、沙眼衣原体核酸扩增荧光检测试剂盒（CT）CE证书及人巨细胞病毒核酸扩增荧光检测试剂盒（CMV）CE证书。     

无标记型核酸适体荧光传感器检测氯霉素的含量

建立了DNA核酸适体检测氯霉素的荧光分析新方法。将前人筛选得到的80bp的氯霉素DNA核酸适体截短至40bp,实验发现截短并不影响核酸适体与氯霉素的结合能力。40bp的DNA核酸适体与另一条互补DNA链形成双链DNA,SYBR Green I荧光染料能插入双链并有强荧光发射,加入氯霉素后,氯霉素能和DNA核酸适体形成稳定的复合物,诱导DNA双链打开,SYBR Green I释放,荧光降低。实验结果显示:在10~200μmol/L范围内,荧光降低百分数和氯霉素之间有良好线性关系,检测限为6μmol/L。     

基于阳离子荧光共轭聚合物和核酸适体探针的蛋白质检测新方法

以核酸适体为识别分子, 阳离子荧光共轭聚合物为报告分子, 建立了一种蛋白质检测新方法. 修饰有荧光熄灭基团的核酸适体探针通过静电作用与阳离子荧光共轭聚合物结合, 导致后者荧光熄灭. 当加入靶蛋白后, 核酸适体探针与其特异性结合, 荧光熄灭基团与阳离子荧光共轭聚合物远离, 聚合物荧光信号得以恢复. 实验结果表明, 荧光恢复程度与靶蛋白的浓度正相关. 采用该方法检测凝血酶的线性范围为17～40 nmol/L.     

核酸功能化金纳米探针在活细胞荧光成像方面的应用

荧光标记的核酸功能化金纳米探针结合了纳米材料与核酸技术的优势,具有增强的稳定性、良好的生物相容性、独特的光学性质及精确的可编程性,开辟了活细胞传感的新纪元.信号放大型的核酸功能化金纳米探针在原位检测含量较低但功能强大的RNA、蛋白质等生物标志物方面尤其表现出明显的优势.本文从活细胞成像分析的角度,重点介绍了荧光标记的核酸功能化金纳米探针的性质、设计原理及应用进展.     

基于金属有机骨架材料和酶切放大的适配体荧光法检测副溶血性弧菌

基于铜基金属有机骨架(Cu-MOF)的荧光猝灭性质、核酸适配体的选择识别能力及核酸外切酶的水解放大信号作用,建立了一种快速检测副溶血性弧菌(V.P)的适配体荧光法.该文在室温下合成Cu-MOF,对其形貌、结构、荧光猝灭机理进行了系统研究,考察了Cu-MOF的体积、猝灭时间、核酸外切酶用量、恢复时间及试剂加入顺序等分析条...     

核酸适配体荧光探针在生化分析和生物成像中的研究进展

核酸适配体是指通过体外筛选技术从核酸文库中筛选出来,能够高特异性、高亲和力识别靶标物的寡核苷酸序列,具有靶标类型广泛、合成简单、相对分子质量小、化学稳定性高、易于进行生物化学修饰等优点。 核酸适配体能够通过折叠成特定的二维或三维构型与靶标物特异性结合,加上合适的信号转导机制,为重要靶标物的研究提供理想的分子识别与分子检测探针。 荧光检测技术具有高灵敏、高分辨率、易于实现多元分析等优点。 将核酸适配体的分子识别特性与荧光优异的光学检测性能相结合,在生命科学研究领域有着广泛的应用空间。 本文主要综述了核酸适配体荧光探针常见的分子设计和信号响应方式,及其在细胞成像、亚细胞成像中的应用研究,并对核酸适配体探针目前面临的一些挑战进行了讨论,最后对其未来的发展方向进行了展望。     

Improving metal ion specificity during in vitro selection of catalytic DNA

Metal ions play important roles in both the structure and function of catalytic DNA and RNA. While most natural catalytic RNA molecules (ribozymes) are active in solutions containing Mg(2+), in vitro selection makes it possible to search for new catalytic DNA/RNA that are specific for other metal ions. However, previous studies have indicated that the in vitro selection protocols often resulted in catalytic DNA/RNA that were equally active or sometimes even more active with metal ions other than the metal ion of choice. To improve the metal ion specificity during the in vitro selection process, we implemented a negative selection strategy where the nucleic acid pool was subjected to a solution containing competing metal ions. As a result, those nucleic acids that were active with those metal ions are discarded. To demonstrate the effectiveness of the negative selection strategy, we carried out two parallel in vitro selections of Co(2+)-dependent catalytic DNA. When no negative selection was used in the selection process, the resulting catalytic DNA molecules were more active in solutions of Zn(2+) and Pb(2+) than in Co(2+). On the other hand, when the negative selection steps were inserted between the normal positive selection steps, the resulting catalytic DNA molecules were much more active with Co(2+) than in other metal ions including Zn(2+) and Pb(2+). These results suggest strongly that in vitro selection can be used to obtain highly active and specific transition metal ion-dependent catalytic DNA/RNA, which hold great promise as versatile and efficient endonucleases as well as sensitive and selective metal ion sensors.     

不同价态金属离子对DNA构象的影响

用紫外分光光度法研究了不同价态金属离子对DNA大分子溶液构象的影响。研究结果显示: 金属离子与DNA的作用使DNA溶液的紫外吸收值下降, 即呈现减色效应, 同时减色效应的强弱与金属离子的价态有关。随着价态升高, 减色效应增强。由此说明, 金属离子与DNA的作用使DNA的构象趋于缩拢; 且随着金属离子价态升高, 缩拢程度增强, 甚至产生缩合。还用现代多聚电解质理论对以上现象进行了讨论, 结果是令人满意的。     

Influence of metal ions on the interaction between gatifloxacin and calf thymus DNA

To study the interaction between gatifloxacin (GT), metal ions (Cu²⁺, Cd²⁺, Co²⁺, Mg²⁺) and calf thymus DNA under condition of physiology pH, UV absorption and fluorescence methods were adopted. Result shows that metal ions and DNA are able to react with GT in ground state. In further research, by studying the influence of metal ions on binding of GT with DNA in metal ions–GT–DNA ternary system, we found that influential mechanism of Mg²⁺ on the binding of GT with DNA may be different from the other three. Mg²⁺ can act as a bridge in the binding of GT's carboxyl/carbonyl with DNA phosphate in certain concentration range; while Cu²⁺, Cd²⁺, Co²⁺ can combine directly with GT by reaction between GT carboxyl/carbonyl and DNA base, and enhance the binding ability of GT with DNA. The influence extent and type depend not only on the binding site of DNA with metal ions (phosphate or base), but also the binding ability of which. The stronger the binding ability of metal ions with DNA base is, the larger their promotion to binding of GT with DNA is. The order of metal ions’ influential ability on the binding of GT–DNA is identical to the binding ability order of metal ions with DNA base, that is: Cu²⁺ > Cd²⁺ > Co²⁺ > Mg²⁺.     

金属离子与DNA相互作用的研究进展

对金属离子与DNA相互作用的研究进展进行了综述,讨论了不同金属离子与DNA碱基的作用位点以及不同金属离子与DNA相互作用的特点.     

Evaluation of effects of bivalent cations on the formation of purine-rich triple-helix DNA by ESI-FT-MS

The GGA triplet repeats are widely dispersed throughout eukaryotic genomes. (GGA)n or (GGT)n oligonucleotides can interact with double-stranded DNA containing (GGA:CCT)n to form triple-stranded DNA. The effects of 8 divalent metal ions (3 alkaline-earth metals and 5 transition metals) on formation of these purine-rich triple-helix DNA were investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-MS). In the absence of metal ions, no triplex but single-strand, duplex, and purine homodimer ions were observed in mass spectra. The triple-helix DNA complexes were observed only in the presence of certain divalent ions. The effects of different divalent cations on the formation of purine-rich triplexes were compared. Transition-metal ions, especially Co²⁺ and Ni²⁺, significantly boost the formation of triple-helix DNA, whereas alkaline-earth metal ions have no positive effects on triplex formation. In addition, Ba²⁺ is notably beneficial to the formation of homodimer instead of triplex.     

二价金属离子对平阳霉素与DNA作用的影响

二价金属离子对平阳霉素与ＤＮＡ作用的影响王自春，黄登宇，袁静明（山西大学分子科学研究所，太原，０３０００６）关键词二价金属离子，平阳霉素，ＤＮＡ平阳霉素（简称ＢＬＭ－Ａ５）是抗肿瘤抗生素博莱霉素的成分之一，其化学结构、理化性质和药理作用虽基本相同［１...     

Quenching of fluorophore-labeled DNA oligonucleotides by divalent metal ions: implications for selection, design, and applications of signaling aptamers and signaling deoxyribozymes

Recent years have seen a dramatic increase in the use of fluorescence-signaling DNA aptamers and deoxyribozymes as novel biosensing moieties. Many of these functional single-stranded DNA molecules are either engineered to function in the presence of divalent metal ion cofactors or designed as sensors for specific divalent metal ions. However, many divalent metal ions are potent fluorescence quenchers. In this study, we first set out to examine the factors that contribute to quenching of DNA-bound fluorophores by commonly used divalent metal ions, with the goal of establishing general principles that can guide future exploitation of fluorescence-signaling DNA aptamers and deoxyribozymes as biosensing probes. We then extended these studies to examine the effect of specific metals on the signaling performance of both a structure-switching signaling DNA aptamer and an RNA-cleaving and fluorescence-signaling deoxyribozyme. These studies showed extensive quenching was obtained when using divalent transition metal ions owing to direct DNA-metal ion interactions, leading to combined static and dynamic quenching. The extent of quenching was dependent on the type of metal ion and the concentration of supporting monovalent cations in the buffer, with quenching increasing with the number of unpaired electrons in the metal ion and decreasing with the concentration of monovalent ions. The extent of quenching was independent of the fluorophore, indicating that quenching cannot be alleviated simply by changing the nature of the fluorescent probe. Our results also show that the DNA sequence and the local secondary structure in the region of the fluorescent tag can dramatically influence the degree of quenching by divalent transition metal ions. In particular, the extent of quenching is predominantly determined by the fluorophore location with respect to guanine-rich and duplex regions within the strand sequence. Examination of the effect of both the type and concentration of metal ions on the performance of a fluorescence-signaling aptamer and a signaling deoxyribozyme confirms that judicious choice of divalent transition metal ions is important in maximizing signals obtained from such systems.     

New insights into the anti-hepatoma mechanism of Alisol G-metal ions complexes based on c-myc DNA

This study investigated the anti-hepatoma molecular mechanism of Alisol G, which is an effective component of the Chinese medicine Alisma orientalis, in the presence of metal ions Cu²⁺ and Fe³⁺ based on c-myc DNA. Here, a combination of Alisol G and metal ions (Cu²⁺, Fe³⁺) to augment anti-hepatoma efficiencies of Alisol G has been identified by methyl thiazolyl tetrazolium (MTT) assay. Network pharmacology revealed that c-myc DNA was the potential target of Alisol G with respect to its anti-hepatoma effects. By performing multi-spectroscopic analyses, we showed that the interaction of Alisol G with c-myc DNA was a process of static quenching. The binding constants and thermodynamic constants indicated that a 1:1 complex was formed between Alisol G and c-myc DNA. Moreover, metal ions strengthened the interaction between Alisol G and c-myc DNA. Molecular docking and molecular dynamics simulation further unveiled that the higher binding affinity between Alisol G-Fe³⁺ complex and c-myc DNA as compared to Alisol G-Cu²⁺ complex. This probably resulted from the polarization of metal ions and the structural flexion of Alisol G. The C22-O31-H76 and C18-O32-H77 of Alisol G were key groups in the interaction with c-myc DNA. Addition of metal ion, had greatly changed the c-myc DNA-binding domain of Alisol G while didn’t affect the kinetic stability of the interaction, thus facilitating the insertion of Alisol G into c-myc DNA A-T base pair. Importantly, the DG113 of c-myc DNA was important for its binding to metal ions. Together, our findings suggested that Alisol G in combination with metal ions may be an efficient and promising option for the treatment of liver cancer.     

Effect of divalent metal ions on DNA studied by capillary electrophoresis

Divalent metal ions, such as Zn(2+), Co(2+), and Ni(2+), are capable of incorporating into DNA under certain conditions to form complexes termed M-DNA. To better understand the effects of these cations on DNA we used capillary electrophoresis (CE). The presence of these metal ions in a typical genotyping buffer led to broad peaks with low fluorescence intensities. In addition, some of the metal-complexed DNA molecules had different electrophoretic mobilities than their normal DNA counterparts. It is likely that the mobility shifts observed in the electropherograms of these affected fragments are due to the divalent cations causing structural changes in the single-stranded DNA. However, as can be seen from the resulting peak shapes, the structure, charge, and/or mass changes due to metal binding are not conserved among all of the DNA fragments. The extent of both peak-broadening and mobility shifts were found to be dependent on the metal cation and its concentration, the length of time that the DNA sample existed in formamide prior to injection into the capillary, and also the fragment size and sequence. These results suggest that the presence of metal ions might be responsible for the poor CE performance that occurs when genotyping certain kinds of DNA samples.