纳米孔道单分子检测结直肠癌MicroRNAs

MicroRNA（miRNA）可用于癌症的早期诊断、预后判断,其分析检测具有重要临床意义.结直肠癌的发生、发展与miRNA 21、miRNA 92等的异常表达明显相关.本研究设计了以poly（dT）_n为引导链的DNA探针（probe）并尝试使用α-溶血素（α-HL）纳米孔道单分子检测方法检测结直肠癌miRNAs.miRNA·probe复合物分子穿过α-HL纳米孔道限域空间时,由于probe链长、序列不同导致probe-α-HL相互作用不同,miRNA 92·probe 92、miRNA 21·probe 21、miRNA 16·probe 16输出为形态、阻断时间不同的多台阶特征信号,实现了三种miRNAs的有效区分.实验证明,此方法可以用于检测血清实际样品.因此,未来有望使用α-HL构建miRNA超灵敏单分子生物传感器.     

基于电化学技术的microRNA生物传感器

MicroRNA(miRNA)是一种内源性的非编码单链RNA,通过与mRNA的3'端非翻译区(UTR)的不完全互补或完全互补结合抑制靶mRNA的翻译或促使靶mRNA的降解来调控基因的表达,参与细胞的增殖、凋亡、分化和代谢等重要过程。MiRNA表达的变化可以起到癌基因和抑癌基因的作用,是一种潜在的肿瘤标志物,因此,miRNA的检测技术引起了人们的关注。由于电化学检测方法具有灵敏、快速、低成本和低能耗等特点,研究者广泛开展了应用电化学技术来发展miRNA检测的研究。本文将对基于电化学技术的miRNA检测方法进行综述。     

MicroRNA分析方法进展*

MicroRNAs（miRNAs）是一类对基因表达具有调控作用的非编码小分子RNA (18–23 碱基)。MiRNA在动植物中普遍存在,并且在动植物的生长、发育、分化和生殖等过程中发挥着重要作用。人类约30%的基因受miRNA调控,并且miRNA的表达水平与人类重大疾病密切相关。MiRNA的定量检测和表达分析对深入理解其作用机制、疾病的诊断与治疗以及相关基因药物的开发等具有重要意义。MiRNA检测一般是基于核酸杂交和扩增原理,方法主要有Northern印迹、微阵列芯片、原位杂交、实时反转录聚合酶链式反应（PCR）、滚环扩增和基于共轭聚合物的检测等。随着miRNA在不同生物中的大量发现和对其功能的深入研究,miRNA的检测方法不断改进和完善,新的扩增、探针标记技术和检测技术不断被开发。本文综述了miRNA检测方法的研究进展,评述了各类方法的优缺点,并对miRNA检测的发展趋势进行了展望。     

基于DNA “Y”型结构的指数型核酸恒温扩增策略检测microRNA

该文基于“Y”型DNA模板和恒温指数扩增反应(EXPAR)技术的信号放大功能,建立了一种快速低序列依赖性的miRNA检测方法(Y-EXPAR)。以let-7b为检测目标,设计两条部分互补的DNA模板,与目标miRNA共同构成“Y”型结构,能够同时进行双向扩增,反应效率高,反应时间短,并降低了放大性能对模板序列的依赖。方法检出限低至0.3 amol/L(相当于10 μL溶液中有1.8拷贝的let-7b),并能区分单碱基差异的let-7家族同源序列。应用于肿瘤细胞裂解液let-7b的检测,POI值与细胞数目对数值呈线性关系,说明Y-EXPAR在实际样品miRNA的检测中具有优势。得益于扩增的高特异性、抗干扰性和低序列依赖性,该方法为miRNA的快速检测提供了新思路,在miRNA相关的疾病研究和临床诊断方面具有良好应用前景。     

双链特异性核酸切割酶等温循环扩增结合 高效液相色谱高灵敏检测双目标microRNA

通过双链特异性核酸切割酶(DSN)循环扩增策略结合高效液相色谱(HPLC)法,实现了对两种疾病标志物microRNA(miRNA)的选择性分离和高灵敏检测.采用酶循环等温扩增策略增强了目标miRNA的信号,提高了HPLC法检测核酸的灵敏度.利用固定在磁珠上的不同长度和碱基序列的DNA探针实现了不同miRNA信号的色谱分...     

双链特异性核酸酶介导的高灵敏度microRNA分析

基于氧化石墨烯(GO)对荧光标记单链DNA探针的荧光猝灭效应以及双链特异性核酸酶(DSN)选择性切割DNA/RNA杂合结构中DNA单链的特性,本文建立了一种新型恒温信号放大方法用于microRNA(miRNA)的高灵敏度检测.靶标miRNA首先与荧光DNA探针杂交,DSN能够特异性地将杂合双链中的DNA探针水解为碎片但不会降解miRNA,GO对酶切产生的寡核苷酸碎片吸附能力显著降低,使得荧光基团远离GO表面而不被猝灭.释放出的miRNA可再次发生与荧光DNA探针杂交、DSN酶切等反应,如此反复,可实现恒温条件下一个miRNA分子与多个探针杂交、酶切、释放荧光基团的循环过程,最终体系的荧光信号得到显著放大,通过记录体系的荧光信号即可实现对靶标miRNA的灵敏检测.     

基于illumina测序技术的IGROV-1/CP细胞中miRNA种类鉴定及相对丰度分析

从正常培养的IGROV-1/CP细胞中提取小RNA,构建小RNA的cDNA文库,然后用illumina通用测序平台对上述cDNA文库进行测序.从测序获得的序列数据中去除冗余数据后,与已知人类miRNA序列数据库进行比对,获得IGROV-1/CP细胞miRNA表达谱.以所获的序列长度与已知人类miRNA数据库中序列长度差异不大于2和无碱基错配为限制条件,共找到53种已知miRNA.按在cDNA文库测序中的出现频率计算,出现频率不大于10的低拷贝miRNA最多,占检测到所有miRNA种类的56.6%.说明illumina通用测序技术能够在检测细胞内的各种小RNA序列的同时反应相对丰度信息,该研究利用上述新技术获得了顺铂耐药性IGROV-1/CP细胞系miRNA表达种类和相对丰度的实验数据.     

MicroRNA体外检测的研究进展

MicroRNA (miRNA)是一类内源性、进化高度保守的小分子非编码RNA,通过识别同源序列及干扰转录、翻译或表观遗传以调节基因的表达。研究发现,某些miRNA的异常表达与疾病相关,可作为生物标志物或药物靶点为疾病诊断、治疗及预后提供新思路,而准确测定miRNA的表达是其应用于临床的关键。本文结合近年来研究成果对传统检测方法及其改进和等温核酸扩增的新技术进行概述,分析这些方法的优势与不足。     

RNA生物标志物体外检测方法研究进展

RNA生物标志物主要包括编码蛋白的mRNA和非编码蛋白的microRNA (miRNA)、环状RNA (circula rRNA, circRNA)及长链非编码RNA (lncRNA)等.RNA生物标志物种类多,生物信息量丰富,相比DNA更能动态反映细胞生物学功能和调控过程,因此,RNA生物标志物的定量检测和表达分析对于细胞功能研究、疾病的诊断和治疗及预后监测等具有重要意义.本文重点介绍了常见RNA生物标志物mRNA、miRNA、circRNA和lncRNA体外检测方法的原理及研究进展,并对RNA生物标志物检测面临的挑战及发展趋势进行了展望.     

基于纳米金与纳米银簇间表面等离子增强能量转移效应特异性检测microRNA

microRNAs(miRNAs)的灵敏检测对临床诊断具有十分重要的意义.本研究采用偶联DNA聚合酶和核酸内切酶介导的恒温扩增反应实现靶标循环再生的策略,利用纳米金(AuNPs)与纳米银簇(AgNCs)间表面等离子增强能量转移效应,开发了一种miRNA定量检测方法.在AuNPs表面组装两种探针(Probe a和Probe b)制备响应元件Probe b-Probe a-AuNP,其中Probe a通过3′端巯基共价偶联到AuNPs表面,此外具有靶标miRNA互补序列、核酸内切酶酶切序列和Probe b互补序列,Probe b为荧光AgNCs合成模板.靶标miRNA存在时,启动酶级联恒温扩增反应,导致Probe b脱离AuNPs表面,抑制了Probe b为模板合成的AgNCs与AuNPs间表面等离子增强能量转移效应,使得反应体系荧光信号增强.本方法的检出限为2.5×10-11 mol/L,与miRNAs商业化检测试剂盒相比,避免了逆转录反应,而且操作简单,检测成本低,可应用于生物样本中miRNAs分析.     

Recent Applications of Carbon Nanomaterials for microRNA Electrochemical Sensing

MicroRNA (miRNA) is an important tumor marker in the human body, and its early detection has a great influence on the survival rate of patients. Although there are many detection methods for miRNA at present such as northern blotting, real-time quantitative polymerase chain reaction, microarrays, and others, electrochemical biosensors have the advantages of low detection cost, small instrument size, simple operation, non-invasive detection and low consumption of reagents and solvents, and thus they play an important role in the early detection of cancer. In addition, with the development of nanotechnology, nano-biosensors show great potential. The application of various nanomaterials in the development of electrochemical biosensor has greatly improved the detection sensitivity of electrochemical biosensor. Among them, carbon nanomaterials which have unique electrical, optical, physical and chemical properties have attracted increasing attention. In particular, they have a large surface area, good biocompatibility and conductivity. Therefore, carbon nanomaterials combined with electrochemical methods can be used to detect miRNA quickly, easily and sensitively. In this review, we systematically review recent applications of different carbon nanomaterials (carbon nanotubes, graphene and its derivatives, graphitic carbon nitride, carbon dots, graphene quantum dots and other carbon nanomaterials) for miRNA electrochemical detection. In addition, we demonstrate the future prospects of electrochemical biosensors modified by carbon nanomaterials for the detection of miRNAs, and some suggestions for their development in the near future.     

Feature importance analysis in guide strand identification of microRNAs

MicroRNA (miRNA) is the negative regulator of gene expression, also known as guide strand of transient miRNA:miRNA* duplex. It is critical in maintaining the normal physiological processes such as development, differentiation, and apoptosis in many organisms. With increasing miRNA data, it is desirable to design methods to identify guide strand based on machine learning algorithms. In this study, the random forest models based on local sequence-structure features were proposed to identify miRNA in four species. The accuracies achieved were 86.51% for Homo sapiens, 81.66% for Ornithorhynchus anatinus, 82.33% for Mus musculus and 85.71% for Schmidtea mediterranea, respectively. Furthermore, the important analysis of feature elements was carried out by using the conditional feature importance strategy. The analysis results revealed that most of the significant elements were related to guanine-cytosine (GC) base pair. We believed that our method could be beneficial to annotate the function of miRNA and help the further understanding of the RNA interference mechanism.     

Self-assembling protein platform for direct quantification of circulating microRNAs in serum with total internal reflection fluorescence microscopy

MicroRNA (miRNA) has recently emerged as a new and important class of cellular regulators. Strong evidences showed that aberrant expression of miRNA is associated with a broad spectrum of human diseases, such as cancer, diabetes, cardiovascular and psychological disorders. However, the short length and low abundance of miRNA place great challenges for conventional techniques in the miRNA quantification and expression profiling. Here, we report a direct, specific and highly sensitive yet simple detection assay for miRNA without sample amplification. A self-assembled protein nanofibril acted as an online pre-concentrating sensor to detect the target miRNA. Locked nucleic acid (LNA) of complimentary sequence was served as the probe to capture the target miRNA analyte. The quantification was achieved by the fluorescence intensity measured with total internal reflection fluorescence microscopy. A detection limit of 1 pM was achieved with trace amount of sample consumption. This assay showed efficient single-base mismatch discrimination. The applicability of quantifying circulating mir-196a in both normal and cancer patient’s serums was also demonstrated.     

A highly sensitive electrochemical assay for microRNA expression profiling

A simple and highly sensitive electrochemical assay for ligation-free and polymerase chain reaction (PCR)-free microRNA (miRNA) expression profiling is described in this work. The electrode used in the assay was made of a monolayer of stem-looped capture probes (CPs) comprising of a miRNA complementing region at one end and detection probes (DPs) receiving region at the other. It engaged an electrocatalytic reaction between electrochemically activated glucose oxidase (GOx) and glucose to enhance its sensitivity. Briefly, upon hybridizing to its target miRNA, the stem loop is unlocked exposing the DP receiving region. A subsequent hybridization with the DPs brought them together with an amplifier, the activated GOx, onto the electrode. The activated GOx exhibited excellent catalytic activity towards electrooxidation of glucose. MicroRNA detection could therefore be conducted in 60 mM glucose in phosphate-buffered saline. A detection limit of 4.0 fM and a linear calibration curve up to 10 pM were obtained under optimal conditions. The assay was applied to profile human let-7 miRNA expressions in cultured cancer cells.     

Os(VI)bipy-based electrochemical assay for detection of specific microRNAs as potential cancer biomarkers

Recently, altered expression levels of microRNAs (miRNAs) – short noncoding RNA molecules which bind to mRNAs and thus regulate gene expression – were observed in many cancer cells. miRNA expression profiling is therefore of great interest, but current standard methods are still considered relatively laborious and expensive. Electrochemistry has a potential to become quick and inexpensive alternative. Here, we describe modification of miRNA with an electroactive complex composed of six-valent osmium and 2,2′-bipyridine, Os(VI)bipy, specifically binding to the 3′-end of the ribose, which is detectable at hanging mercury drop electrode at femtomole level due to an electrocatalytic nature of a resulting signal. By combining miRNA labeling step with magnetic beads-based hybridization assay, detection of specific miRNA sequence from a mixture of other noncomplementary miRNAs was possible.     

Highly sensitive detection of microRNA by chemiluminescence based on enzymatic polymerization

We have developed a new methodology for miRNA assay using chemiluminescence imaging by poly(U) polymerase catalyzed miRNA polymerization. This method is very sensitive with a 50 fM limit of detection, which is comparable to or better than current assay methods. Multiplex detection for miRNA can be easily realized by introducing different capture probes onto the biosensor array, which will make it highly versatile for various research purposes.     

Dumbbell probe-mediated cascade isothermal amplification: A novel strategy for label-free detection of microRNAs and its application to real sample assay

Considering the great significance of microRNAs (miRNAs) in cancer detection and typing, the development of sensitive, specific, quantitative, and low-cost methods for the assay of expression levels of miRNAs is desirable. We describe a highly efficient amplification platform for ultrasensitive analysis of miRNA (taking let-7a miRNA as a model analyte) based on a dumbbell probe-mediated cascade isothermal amplification (DP-CIA) strategy. The method relies on the circularization of dumbbell probe by binding target miRNA, followed by rolling circle amplification (RCA) reaction and an autonomous DNA machine performed by nicking/polymerization/displacement cycles that continuously produces single-stranded G-quadruplex to assemble with hemin to generate a color signal. In terms of the high sensitivity (as low as 1 zmol), wide dynamic range (covering 9 orders of magnitude), good specificity (even single-base difference) and easy operation (one probe and three enzymes), the proposed label-free assay is successfully applied to direct detection of let-7a miRNA in real sample (total RNA extracted from human lung tissue), demonstrating an attractive alternative for miRNA analysis for gene expression profiling and molecular diagnostics, particularly for early cancer diagnosis.     

壳聚糖及其衍生物递送miRNA/siRNA的研究进展

微小RNA(microRNA,miRNA)和短链干扰RNA (small interfering RNA,siRNA)是两类具有调节基因表达功能的内源性非编码性小RNA分子.它们已成为多种疾病的潜在治疗药物,逐渐被应用于基因治疗中,而将小RNA应用于基因治疗亟需一种安全高效的递送载体.壳聚糖及其衍生物作为一种可降解、低...