A novel system for rapid screening of effective siRNA target sites by one step transfection with a single vector

siRNA technique has been widely used to study the gene functions and to develop disease therapeutics. One of the challenges of RNAi application is to obtain the most effective target specific siRNA sequences. Currently the process to select and validate optimal siRNA sites for a given gene, which is usually based on screening by using Western blot, Northern blot or Q-PCR, remains empirical and time consuming. Although few fluorescence-based siRNA sequence selection systems have proven useful, the rapid and efficient screening of siRNA target sites is still challenging. In the paper, we developed a quick and efficient method to screen siRNA target sites with a novel single vector system, which contains the following cassettes: (1) an eGFP reporter gene expression cassette followed by a multiple cloning site and SV40 pA for insertion of a target sequence; (2) siRNA expression cassette containing a dual PoI III promoter driving in opposite directions; and (3) an internal loading control, mCherry reporter gene. Based on this one-step transfection with single vector system, we could rapidly screen effective, target specific siRNA fragments in an unbiased manner by judging the fluorescence intensity ratio of eGFP to mCherry. The generation of this novel vector system will promote the application of siRNA in basic research and disease therapy.     

DSIR: Assessing the Design of Highly Potent siRNA by Testing a Set of Cancer-Relevant Target Genes

Chemically synthesized small interfering RNA (siRNA) is a widespread molecular tool used to knock down genes in mammalian cells. However, designing potent siRNA remains challenging. Among tools predicting siRNA efficacy, very few have been validated on endogenous targets in realistic experimental conditions. We previously described a tool to assist efficient siRNA design (DSIR, Designer of siRNA), which focuses on intrinsic features of the siRNA sequence. Here, we evaluated DSIR’s performance by systematically investigating the potency of the siRNA it designs to target ten cancer-related genes. mRNA knockdown was measured by quantitative RT-PCR in cell-based assays, revealing that over 60% of siRNA sequences designed by DSIR silenced their target genes by at least 70%. Silencing efficacy was sustained even when low siRNA concentrations were used. This systematic analysis revealed in particular that, for a subset of genes, the efficiency of siRNA constructs significantly increases when the sequence is located closer to the 5′-end of the target gene coding sequence, suggesting the distance to the 5′-end as a new feature for siRNA potency prediction. A new version of DSIR incorporating these new findings, as well as the list of validated siRNA against the tested cancer genes, has been made available on the web (http://biodev.extra.cea.fr/DSIR).     

Filtering of ineffective siRNAs and improved siRNA design tool

MOTIVATION: Short interfering RNAs (siRNAs) can be used to suppress gene expression and possess many potential applications in therapy, but how to design an effective siRNA is still not clear. Based on the MPI (Max-Planck-Institute) basic principles, a number of siRNA design tools have been developed recently. The set of candidates reported by these tools is usually large and often contains ineffective siRNAs. In view of this, we initiate the study of filtering ineffective siRNAs. RESULTS: The contribution of this paper is 2-fold. First, we propose a fair scheme to compare existing design tools based on real data in the literature. Second, we attempt to improve the MPI principles and existing tools by an algorithm that can filter ineffective siRNAs. The algorithm is based on some new observations on the secondary structure, which we have verified by AI techniques (decision trees and support vector machines). We have tested our algorithm together with the MPI principles and the existing tools. The results show that our filtering algorithm is effective. AVAILABILITY: The siRNA design software tool can be found in the website http://www.cs.hku.hk/~sirna/ CONTACT: smyiu@cs.hku.hk     

GMAP: a genomic mapping and alignment program for mRNA and EST sequences

MOTIVATION: We introduce GMAP, a standalone program for mapping and aligning cDNA sequences to a genome. The program maps and aligns a single sequence with minimal startup time and memory requirements, and provides fast batch processing of large sequence sets. The program generates accurate gene structures, even in the presence of substantial polymorphisms and sequence errors, without using probabilistic splice site models. Methodology underlying the program includes a minimal sampling strategy for genomic mapping, oligomer chaining for approximate alignment, sandwich DP for splice site detection, and microexon identification with statistical significance testing. RESULTS: On a set of human messenger RNAs with random mutations at a 1 and 3% rate, GMAP identified all splice sites accurately in over 99.3% of the sequences, which was one-tenth the error rate of existing programs. On a large set of human expressed sequence tags, GMAP provided higher-quality alignments more often than blat did. On a set of Arabidopsis cDNAs, GMAP performed comparably with GeneSeqer. In these experiments, GMAP demonstrated a several-fold increase in speed over existing programs. AVAILABILITY: Source code for gmap and associated programs is available at http://www.gene.com/share/gmap SUPPLEMENTARY INFORMATION: http://www.gene.com/share/gmap.     

NEBcutter: A program to cleave DNA with restriction enzymes

NEBcutter, version 1.0, is a program available via a web server (http://tools.neb.com/NEBcutter) that will accept an input DNA sequence and produce a comprehensive report of the restriction enzymes that will cleave the sequence. It produces a variety of outputs including restriction enzyme maps, theoretical digests and links into the restriction enzyme database, REBASE (http://www.neb.com/rebase). Importantly, its table of recognition sites is updated daily from REBASE and it marks all sites that are potentially affected by DNA methylation (Dam, Dcm, etc.). Many options exist to choose the enzymes used for digestion, including all known specificities, subsets of those that are commercially available or sets of enzymes that produce compatible termini.     

Efficient siRNA selection using hybridization thermodynamics

Small interfering RNA (siRNA) are widely used to infer gene function. Here, insights in the equilibrium of siRNA-target hybridization are used for selection of efficient siRNA. The accessibilities of siRNA and target mRNA for hybridization, as measured by folding free energy change, are shown to be significantly correlated with efficacy. For this study, a partition function calculation that considers all possible secondary structures is used to predict target site accessibility; a significant improvement over calculations that consider only the predicted lowest free energy structure or a set of low free energy structures. The predicted thermodynamic features, in addition to siRNA sequence features, are used as input for a support vector machine that selects functional siRNA. The method works well for predicting efficient siRNA (efficacy >70%) in a large siRNA data set from Novartis. The positive predictive value (percentage of sites predicted to be efficient for silencing that are) is as high as 87.6%. The sensitivity and specificity are 22.7 and 96.5%, respectively. When tested on data from different sources, the positive predictive value increased 8.1% by adding equilibrium terms to 25 local sequence features. Prediction of hybridization affinity using partition functions is now available in the RNAstructure software package.     

BIOFFORC: Tool development for biological file format conversion

The use of bioinformatics tools require different sequence formats at various instances. Every tool uses specific set of formats for processing. Sequence in one format is often required in another format. Thus, there is a need for sequence format conversion. A number of such tools are available in the public domain. Here, we describe BIOFFORC as a file format converter. The tool is developed with a graphical user interface in PERL.

Availability

http://www.winningpath.com/biofforc/     

OligoFaktory: a visual tool for interactive oligonucleotide design

SUMMARY: The OligoFaktory is a set of tools for the design, on an arbitrary number of target sequences, of high-quality long oligonucleotide for micro-array, of primer pair for PCR, of siRNA and more. The user-centered interface exists in two flavours: a web portal and a standalone software for Mac OS X Tiger. A unified presentation of results provides overviews with distribution charts and relative location bar graphs, as well as detailed features for each oligonucleotide. Input and output files conform to a common XML interchange file format to allow both automatic generation of input data, archiving, and post-processing of results. The design pipeline can use BLAST servers to evaluate specificity of selected oligonucleotides. AVAILABILITY: The web portal http://ueg.ulb.ac.be/oligofaktory/; the software for Macintosh: http://www.oligofaktory.org/     

29 Accelerating nucleic acid design using pre-selected sequences

Nanoscale nucleic acids could potentially be designed to be catalysts, pharmaceuticals, or probes for detecting pathogens. We hypothesize that designing nucleic acid molecules from pre-selected sequences, rather than from random sequences, would increase the speed of designing large molecules and also increase the accuracy of design. Helices should be formed in the optimal folding free energy change range, have maximal structure probability, and minimal ensemble defect. Loops should be composed of sequences with the lowest ensemble free energy change. All sequences should have low tendency to cross- and self-hybridize. These features are observed in RNA sequences with known structure.We demonstrate that preselected sequences and accelerate the design of structures that are mimics of biologically relevant structures. This is implemented as a new structure design component of RNAstructure (http://rna.urmc.rochester.edu/RNAstructure.html). This work is a collaboration with Celadon Laboratories, Inc. (http://www.celadonlabs.com/).     

miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs

miRDeepFinder is a software package developed to identify and functionally analyze plant microRNAs (miRNAs) and their targets from small RNA datasets obtained from deep sequencing. The functions available in miRDeepFinder include pre-processing of raw data, identifying conserved miRNAs, mining and classifying novel miRNAs, miRNA expression profiling, predicting miRNA targets, and gene pathway and gene network analysis involving miRNAs. The fundamental design of miRDeepFinder is based on miRNA biogenesis, miRNA-mediated gene regulation and target recognition, such as perfect or near perfect hairpin structures, different read abundances of miRNA and miRNA*, and targeting patterns of plant miRNAs. To test the accuracy and robustness of miRDeepFinder, we analyzed a small RNA deep sequencing dataset of Arabidopsis thaliana published in the GEO database of NCBI. Our test retrieved 128 of 131 (97.7%) known miRNAs that have a more than 3 read count in Arabidopsis. Because many known miRNAs are not associated with miRNA*s in small RNA datasets, miRDeepFinder was also designed to recover miRNA candidates without the presence of miRNA*. To mine as many miRNAs as possible, miRDeepFinder allows users to compare mature miRNAs and their miRNA*s with other small RNA datasets from the same species. Cleaveland software package was also incorporated into miRDeepFinder for miRNA target identification using degradome sequencing analysis. Using this new computational tool, we identified 13 novel miRNA candidates with miRNA*s from Arabidopsis and validated 12 of them experimentally. Interestingly, of the 12 verified novel miRNAs, a miRNA named AC1 spans the exons of two genes (UTG71C4 and UGT71C3). Both the mature AC1 miRNA and its miRNA* were also found in four other small RNA datasets. We also developed a tool, ??miRNA primer designer?? to design primers for any type of miRNAs. miRDeepFinder provides a powerful tool for analyzing small RNA datasets from all species, with or without the availability of genome information. miRDeepFinder and miRNA primer designer are freely available at http://www.leonxie.com/DeepFinder.php and at http://www.leonxie.com/miRNAprimerDesigner.php, respectively. A program (called RefFinder: http://www.leonxie.com/referencegene.php) was also developed for assessing the reliable reference genes for gene expression analysis, including miRNAs.     

siRNA specificity searching incorporating mismatch tolerance data

Artificially synthesized short interfering RNAs (siRNAs) are widely used in functional genomics to knock down specific target genes. One ongoing challenge is to guarantee that the siRNA does not elicit off-target effects. Initial reports suggested that siRNAs were highly sequence-specific; however, subsequent data indicates that this is not necessarily the case. It is still uncertain what level of similarity and other rules are required for an off-target effect to be observed, and scoring schemes have not been developed to look beyond simple measures such as the number of mismatches or the number of consecutive matching bases present. We created design rules for predicting the likelihood of a non-specific effect and present a web server that allows the user to check the specificity of a given siRNA in a flexible manner using a combination of methods. The server finds potential off-target matches in the corresponding RefSeq database and ranks them according to a scoring system based on experimental studies of specificity. AVAILABILITY: The server is available at http://informatics-eskitis.griffith.edu.au/SpecificityServer.     

Paternal care behaviors of Japanese giant salamander <Emphasis Type="Italic">Andrias japonicus</Emphasis> in natural populations

Parental care among salamanders is typically provided by females. A rare case of parental care by male salamanders appears to occur in Cryptobranchidae. Yet, paternal behaviors have rarely been reported from natural populations of any Cryptobranchid salamanders, and their adaptive significance is poorly understood. The present study aimed to examine paternal care behaviors in a fully aquatic Japanese giant salamander (Andrias japonicus) in situ. At the beginning of the summer breeding season, large males, called den-masters, occupy burrows along stream banks for breeding and nesting. We videotaped post-breeding behaviors of two den-masters that stayed with the eggs, one in a natural and the other in an artificial nest in natural streams. We identified three behaviors, tail fanning, agitating and egg eating, to be parental care. Tail fanning provides oxygenated water for the eggs. We found that the den-master in the artificial nest, where dissolved oxygen level was lower, displayed tail fanning more frequently. Agitating the eggs with its head and body likely prevents yolk adhesions. The den-masters selectively ate whiter eggs that appeared to be dead or infected with water mold. This behavior, which we termed hygienic filial cannibalism, likely prevents water mold from spreading over healthy eggs. Digital video images relating to this article are available at http://www.momo-p.com/showdetail-e.php?movieid=momo140906aj01a, http://www.momo-p.com/showdetail-e.php?movieid=momo140906aj02a, http://www.momo-p.com/showdetail-e.php?movieid=momo140906aj03a and http://www.momo-p.com/showdetail-e.php?movieid=momo140906aj04a.     

SNUFER: A software for localization and presentation of single nucleotide polymorphisms using a Clustal multiple sequence alignment output file

SNUFER is a software for the automatic localization and generation of tables used for the presentation of single nucleotide polymorphisms (SNPs). After input of a fasta file containing the sequences to be analyzed, a multiple sequence alignment is generated using ClustalW ran inside SNUFER. The ClustalW output file is then used to generate a table which displays the SNPs detected in the aligned sequences and their degree of similarity. This table can be exported to Microsoft Word, Microsoft Excel or as a single text file, permitting further editing for publication. The software was written using Delphi 7 for programming and FireBird 2.0 for sequence database management. It is freely available for noncommercial use and can be downloaded from http://www.heranza.com.br/bioinformatica2.htm.     

Prediction of C-to-U RNA editing sites in plant mitochondria using both biochemical and evolutionary information

Although cytidine-to-uridine conversions in plant mitochondria were discovered 18 years ago, it was still an enigmatic process. Since the sequencing projects of plant mitochondrial genomes are providing more and more available sequences, the requirements of computationally identifying C-to-U RNA editing sites are also increasing. By incorporating both evolutionary and biochemical information, we developed a novel algorithm for predicting C-to-U RNA editing sites in plant mitochondria. The algorithm has been implemented as an online service called CURE (Cytidine-to-Uridine Recognizing Editor). CURE performs better than other methods that are based on only biochemical or only evolutionary information. CURE also provides the ability of predicting C-to-U RNA editing sites in non-coding regions and the synonymous C-to-U RNA editing sites in coding regions that are impossible for other methods. Furthermore, CURE can carry out prediction directly on the entire mitochondria genome sequence. The prediction results of CURE suggest the functional importance of synonymous RNA editing sites, which was neglected before. The CURE service can be accessed at http://bioinfo.au.tsinghua.edu.cn/cure.     

Accelerated off-target search algorithm for siRNA

MOTIVATION: Designing highly effective short interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi) is one of the hottest topics in molecular biology. The relationship between siRNA sequences and RNAi activity has been studied extensively to establish rules for selecting highly effective sequences. However, there is a pressing need to compute siRNA sequences that minimize off-target silencing effects efficiently and to match any non-targeted sequences with mismatches. RESULTS: The enumeration of potential cross-hybridization candidates is non-trivial, because siRNA sequences are short, ca. 19 nt in length, and at least three mismatches with non-targets are required. With at least three mismatches, there are typically four or five contiguous matches, so that a BLAST search frequently overlooks off-target candidates. By contrast, existing accurate approaches are expensive to execute; thus we need to develop an accurate, efficient algorithm that uses seed hashing, the pigeonhole principle, and combinatorics to identify mismatch patterns. Tests show that our method can list potential cross-hybridization candidates for any siRNA sequence of selected human gene rapidly, outperforming traditional methods by orders of magnitude in terms of computational performance. AVAILABILITY: http://design.RNAi.jp CONTACT: yamada@cb.k.u-tokyo.ac.jp.     

慢病毒GV115-AIF siRNA 重组表达系统的构建及鉴定

目的:构建高效的慢病毒GV115-AIF si RNA重组表达系统。方法:根据目的基因AIF以及RNA干扰序列设计原则,利用设计软件设计了3个可能的AIF si RNA序列。应用全基因合成技术和亚克隆技术构建GV115-AIF si RNA重组质粒,并采用聚合酶链反应技术(PCR技术)和基因测序技术对GV115-AIF si RNA重组质粒鉴定。利用GV115病毒包装辅助p Helper 1.0质粒和p Helper 2.0质粒进行病毒包装。病毒包装后转染人胚肾293T细胞,通过应用逆转录定量PCR技术(RT-PCR技术)检测转染后对人胚肾293T细胞中AIF基因的敲减效率,筛选最高效的AIF si RNA基因序列。结果:GV115-AIF si RNA质粒PCR鉴定显示位于341bp附近的条带。测序结果与设计的基因序列完全吻合。3个可能的AIF si RNA序列中基因敲减效率最高的可达到88.3%。结论:成功构建高效的慢病毒GV115-AIF si RNA重组表达系统。