Nucleobase and ribose modifications control immunostimulation by a microRNA-122-mimetic RNA

Immune stimulation is a significant hurdle in the development of effective and safe RNA interference therapeutics. Here, we address this problem in the context of a mimic of microRNA-122 by employing novel nucleobase and known 2'-ribose modifications. The nucleobase modifications are analogues of adenosine and guanosine that contain cyclopentyl and propyl minor-groove projections. Via a site-by-site chemical modification analysis, we identify several immunostimulatory 'hot spots' within the miRNA guide strand at which single base modifications significantly reduce immune stimulation. A duplex containing one base modification on each strand proved to be most effective in preventing immune stimulation.     

Enzyme-free and multiplexed microRNA detection using microRNA-initiated DNA molecular motor

In this work, we have developed a sensitive, simple, and enzyme-free assay for detection of microRNAs (miRNAs) by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains. In the presence of miRNA target, it can hybridize with one of the stem-loop DNA to open the stem and to produce a miRNA/DNA hybrid and a single strand (ss) DNA, the ssDNA will in turn hybridize with another stem-loop DNA and finally form a double strand (ds) DNA to release the miRNA. One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence. The formation of dsDNA can produced specific fluorescence signal for miRNA detection. The released miRNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor, which results in great fluorescence amplification. With the efficient signal amplification, as low as 1 pmol/L miRNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained. Moreover, by designing different stem-loop DNAs specific to different miRNA targets and labeling them with different fluorophores, multiplexed miRNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum (SFS) technique.     

Functional characterization of plant small RNAs based on next-generation sequencing data

The currently developed next-generation sequencing (NGS) technology has significantly enhanced our capacity of small RNA (sRNA) exploration. Several ambitious sRNA projects have been established based on the technical support of NGS. Thanks to the high-throughput feature of NGS, huge amounts of sRNA sequencing data have been generated. However, much more research efforts are needed to further exploit these valuable data. In this study, we carried out functional analyses of sRNAs from 26 angiosperms by utilizing public sRNA NGS data. We proposed that the endogenous sRNAs largely represented by the 24-nt ones had a potential role in transposable element (TE) control in both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), based on the comparison of sRNA locus densities within the TEs to the non-TE genes. Functional analysis was performed for the predicted targets of the conserved sRNAs that were classified into eudicot-specific, monocot-specific, and angiosperm-conserved ones. Moreover, several miRNA families were found to be highly conserved, and miR396 was suggested to be the most conserved miRNA family between the eudicots and the monocots, indicating its essential role in angiosperm development. At last, we demonstrated that it was a great challenge for researchers to fully exploit these huge sRNA NGS datasets and numerous sRNA species remained to be uncovered and functionally characterized.     

Platinum interference with siRNA non-seed regions fine-tunes silencing capacity

Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ～20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to miRNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus indicating subtle differences between the abilities of cis- and oxaliplatin to interfere with si- and miRNA processing.     

Detection of MicroRNAs using target-guided formation of conducting polymer nanowires in nanogaps

A nanogapped microelectrode-based biosensor array is fabricated for ultrasensitive electrical detection of microRNAs (miRNAs). After peptide nucleic acid (PNA) capture probes were immobilized in nanogaps of a pair of interdigitated microelectrodes and hybridization was performed with their complementary target miRNA, the deposition of conducting polymer nanowires, polyaniline (PAn) nanowires, is carried out by an enzymatically catalyzed method, where the electrostatic interaction between anionic phosphate groups in miRNA and cationic aniline molecules is exploited to guide the formation of the PAn nanowires onto the hybridized target miRNA. The conductance of the deposited PAn nanowires correlates directly to the amount of the hybridized miRNA. Under optimized conditions, the target miRNA can be quantified in a range from 10 fM to 20 pM with a detection limit of 5.0 fM. The biosensor array is applied to the direct detection of miRNA in total RNA extracted from cancer cell lines.     

Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells.     

A cautionary tale of principal component analysis: an example from inductively-coupled plasma/atomic emission spectrometry

Principal component analysis (PCA) is a widely used chemometric technique, but there can be serious limitations on the validity of the conclusions. In the example given, PCA was applied to identify the causes of noise and drift in inductively-coupled plasma/atomic emission spectrometry (ICP/AES). The effects of ten possible instrumental variables on lines of 24 elements were measured independently to establish distinctive “fingerprints”. PCA showed that >90% of the variance in routine analysis was correlated between elements, rather than being random. However, the multi-element ‘fingerprints’ identified as the principal components did not correspond to any of those established as suspected causes. A model based on variation of two of the suspected causes of error was used to simulate analysis by ICP/AES. When subjected to PCA, the simulated data also gave erroneous fingerprints in the principal components that closely matched those found in the real data. The reason for the failure of PCA to identify the causes of the variation was found to be that the two apparently independent causes had correlated multi-element effects. Caution is therefore required in the identification of principal components in terms of recognisable features.     

Highly sensitive analysis strategies of microRNAs based on electrochemiluminescence

The ultrasensitive detection of microRNAs (miRNAs) is currently pursued for the diagnosis of diseases. Due to its outstanding sensitivity, electrochemiluminescence (ECL) is expected to be very effective toward the above goal. In this short review, bioanalytical strategies currently employed in ECL detections of miRNAs are summarized. ECL sensors based on electrochemiluminescent resonance energy transfer (ERET), hybridization chain reaction (HCR), strand displacement reaction (SDR), and other strategies, have an extremely low detection limit of 10^?18 M miRNA. In particular, the establishment of miniaturized ECL sensors has shown great potential for point-of-need testing of diseases.     

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.     

Label-free fluorescence turn-on detection of microRNA based on duplex-specific nuclease and a perylene probe

A novel fluorescence turn-on microRNA (miRNA) detection method based on duplex-specific nuclease (DSN) and a perylene probe is presented in this study. A positively charged perylene derivative (compound 1) was used as the fluorescent probe. Compound 1 exhibits strong monomer fluorescence in an aqueous buffer solution. It is well known that single-stranded DNA is a polyanion in nature. Thus, it can induce the aggregation of compound 1 through strong electrostatic, hydrophobic and π−π stacking interactions. As a result, the fluorescence of compound 1 was efficiently quenched. When the target miRNA was added, the formation of DNA-RNA hybridized duplex initiated the cleavage of the DNA strand by DSN cycle reaction, which resulted in disaggregation of compound 1. A fluorescence turn-on signal was detected, and a novel miRNA sensing method was therefore established. The presented method is label-free, simple, cost effective, sensitive and selective.     

Toehold‐initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

The ability to quantitate and visualize microRNAs (miRNAs) in situ in single cells would greatly facilitate the elucidation of miRNA‐mediated regulatory circuits and their disease associations. A toehold‐initiated strand‐displacement process was used to initiate rolling circle amplification of specific miRNAs, an approach that achieves both stringent recognition and in situ amplification of the target miRNA. This assay, termed toehold‐initiated rolling circle amplification (TIRCA), can be utilized to identify miRNAs at physiological temperature with high specificity and to visualize individual miRNAs in situ in single cells within 3 h. TIRCA is a competitive candidate technique for in situ miRNA imaging and may help us to understand the role of miRNAs in cellular processes and human diseases in more detail.     

Synthesis, gene silencing, and molecular modeling studies of 4'-C-aminomethyl-2'-O-methyl modified small interfering RNAs

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (～1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.     

Hierarchical clustering analysis of flexible GBR 12909 dialkyl piperazine and piperidine analogs

Pharmacophore modeling of large, drug-like molecules, such as the dopamine reuptake inhibitor GBR 12909, is complicated by their flexibility. A comprehensive hierarchical clustering study of two GBR 12909 analogs was performed to identify representative conformers for input to three-dimensional quantitative structure–activity relationship studies of closely-related analogs. Two data sets of more than 700 conformers each produced by random search conformational analysis of a piperazine and a piperidine GBR 12909 analog were studied. Several clustering studies were carried out based on different feature sets that include the important pharmacophore elements. The distance maps, the plot of the effective number of clusters versus actual number of clusters, and the novel derived clustering statistic, percentage change in the effective number of clusters, were shown to be useful in determining the appropriate clustering level.Six clusters were chosen for each analog, each representing a different region of the torsional angle space that determines the relative orientation of the pharmacophore elements. Conformers of each cluster that are representative of these regions were identified and compared for each analog. This study illustrates the utility of using hierarchical clustering for the classification of conformers of highly flexible molecules in terms of the three-dimensional spatial orientation of key pharmacophore elements.     

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.     

Triangular silver nanoplates: Properties and ultrasensitive detection of miRNA

Triangular silver nanoplates (TSNPs) have been functionalised with probe strand miRNA that is complementary in part to the target nucleic acid, miR-132-3p, that is associated with neuroblastoma. These TSNPs were immobilised in gold microcavities via complementary miRNA hybridisation and can give plasmonic enhancement of the Raman response. Optimum enhancement can be achieved by using different excitation wavelengths and changing the distance between the nanoplate and the surface of the cavity. As silver is electrocatalytically active to the reduction of hydrogen peroxide, these probe-functionalised TSNPs can be used in a miRNA assay where a linear response was obtained for target concentrations from 100 fM to 1 μM target concentration, with a current generated of 100 μA.     

大鼠胰腺及癌组织红外光谱连续小波特征提取及径向基人工神经网络识别

采用水平衰减全反射(HATR)傅里叶变换红外光谱法(FTIR)测定了SD大鼠胰腺正常组织与非正常组织的谱图,提出了一种新的基于FTIR的连续小波特征提取与径向基人工神经网络分类方法以提高FTIR对早期SD大鼠胰腺癌的诊断准确率。利用连续小波多分辨率分析法提取FTIR特征量,对于提取的特征量采用径向基函数神经网络进行模式分类。对SD大鼠的胰腺正常组织、早期癌组织及进展期癌组织的FTIR,利用连续小波多分辨率分析法提取9个特征量,进行RBF神经网络分类判断。当目标误差为0.01,径向基函数的分布常数为5时,网络达到最优化,总的正确识别率为96.67%。并对影响分类结果的网络参数、目标误差和分布常数对分类样品的影响做了讨论。实验结果表明:此方法对早期胰腺癌具有较高的诊断率。     

Element profiles of onion producing districts in Japan, as determined using INAA and PGA

We carried out instrumental neutron activation analysis (INAA) as well as k ₀-based prompt gamma-ray analysis (k ₀-PGA) to measure the amount of the elements in onions and studied whether the onions collected from different sites can be categorized based on the elemental concentration profile. Six elements (Na, Mg, Cl, K, Ca, Mn) and 3 elements (B, S, Cl) were measured by INAA and PGA in the onions grown in two districts, Hokkaido and Saga, in Japan, respectively. After principal component analysis, it was found that Cl was an important element to feature the producing districts of onions.