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siRNA design methods and algorithms
(siRNA design RNAi design tools)
1: Curr Protoc Bioinformatics. 2004 Sep;Chapter 12:Unit 12.3.

RNAi: design and analysis.

Sachidanandam R.

Sachidanandam Lab, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York,
USA.

RNAi is a novel genetic tool that is poised to revolutionize the study of
genetics and gene networks. Use of this tool requires knowledge of the underlying
mechanisms and application of this knowledge to the construction of a
gene-silencing library. This unit discusses approaches to rational design of this
library and its use in experiments. It focuses on the design algorithms developed
so far and briefly discuss several issues that could lead to improvements in the
current design algorithms.

2: Curr Protoc Bioinformatics. 2006 Mar;Chapter 12:Unit 12.1.

An overview of RNA structure prediction and applications to RNA gene prediction
and RNAi design.

Stormo GD.

Washington University School of Medicine, St. Louis, Missouri, USA.

This unit briefly describes the two fundamentally different methods for
predicting RNA structures. The first is to find that structure with the minimum
free energy of folding, as predicted by various thermodynamic parameters related
to base-pair stacking, loop lengths, and other features. If one has only a single
sequence, this thermodynamic approach is the best available method. The second
fundamental approach to RNA structure prediction is to use multiple, homologous
sequences for which one can infer a common structure, and then try and predict a
structure common to all of the sequences. Such an approach is referred to as a
comparative method or phylogenetic method of RNA structure prediction.

siRNA design tools:

siRNA Target Finder (Ambion)

siRNA target finder utilizes the basic siRNA design guidelines (Tuschl et.al) to find the siRNA target sequence. The efficiency for this tool is about 50%-50% (50% siRNA designed by the tool will reduce target gene expression by more than 50%).

pssRNAMiner

Ref: Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W114-8.

[ABSTRACT]: In plants, short RNAs including approximately 21-nt microRNA (miRNA) and 21-nt trans-acting siRNA (ta-siRNA) compose a 'miRNA --> ta-siRNA --> target gene' cascade pathway that regulates gene expression at the posttranscriptional level. In this cascade, biogenesis of ta-siRNA clusters requires 21-nt intervals (i.e. phasing) and miRNA (phase-initiator) cleavage sites on its TAS transcript. Here, we report a novel web server, pssRNAMiner, which is developed to identify both the clusters of phased small RNAs as well as the potential phase-initiator. To detect phased small RNA clusters, the pssRNAMiner maps input small RNAs against user-specified transcript/genomic sequences, and then identifies phased small RNA clusters by evaluating P-values of hypergeometric distribution. To identify potential phase-initiators, pssRNAMiner aligns input phase-initiators with transcripts of TAS candidates using the Smith-Waterman algorithm. Potential cleavage sites on TAS candidates are further identified from complementary regions by weighting the alignment expectation and its distance to detected phased small RNA clusters.

DEQOR

Ref: Biophys J. 2004 Jul;87(1):215-26.

[AbSTRACT]:RNA interference (RNAi) is a powerful tool for inhibiting the expression of a gene by mediating the degradation of the corresponding mRNA. The basis of this gene-specific inhibition is small, double-stranded RNAs (dsRNAs), also referred to as small interfering RNAs (siRNAs), that correspond in sequence to a part of the exon sequence of a silenced gene. The selection of siRNAs for a target gene is a crucial step in siRNA-mediated gene silencing. According to present knowledge, siRNAs must fulfill certain properties including sequence length, GC-content and nucleotide composition. Furthermore, the cross-silencing capability of dsRNAs for other genes must be evaluated. When designing siRNAs for chemical synthesis, most of these criteria are achievable by simple sequence analysis of target mRNAs, and the specificity can be evaluated by a single BLAST search against the transcriptome of the studied organism. A different method for raising siRNAs has, however, emerged which uses enzymatic digestion to hydrolyze long pieces of dsRNA into shorter molecules. These endoribonuclease-prepared siRNAs (esiRNAs or 'diced' RNAs) are less variable in their silencing capabilities and circumvent the laborious process of sequence selection for RNAi due to a broader range of products. Though powerful, this method might be more susceptible to cross-silencing genes other than the target itself. We have developed a web-based tool that facilitates the design and quality control of siRNAs for RNAi. The program, DEQOR, uses a scoring system based on state-of-the-art parameters for siRNA design to evaluate the inhibitory potency of siRNAs. DEQOR, therefore, can help to predict (i) regions in a gene that show high silencing capacity based on the base pair composition and (ii) siRNAs with high silencing potential for chemical synthesis. In addition, each siRNA arising from the input query is evaluated for possible cross-silencing activities by performing BLAST searches against the transcriptome or genome of a selected organism. DEQOR can therefore predict the probability that an mRNA fragment will cross-react with other genes in the cell and helps researchers to design experiments to test the specificity of esiRNAs or chemically designed siRNAs. DEQOR is freely available at http://cluster-1.mpi-cbg.de/Deqor/deqor.html.

E-RNAi

Ref: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W582-8.

RNA interference (RNAi) has become a powerful genetic approach to systematically dissect gene function on a genome-wide scale. Owing to the penetrance and efficiency of RNAi in invertebrates, model organisms such as Drosophila melanogaster and Caenorhabditis elegans have contributed significantly to the identification of novel components of diverse biological pathways, ranging from early development to fat storage and aging. For the correct assessment of phenotypes, a key issue remains the stringent quality control of long double-stranded RNAs (dsRNA) to calculate potential off-target effects that may obscure the phenotypic data. We here describe a web-based tool to evaluate and design optimized dsRNA constructs. Moreover, the application also gives access to published predesigned dsRNAs. The E-RNAi web application is available at http://e-rnai.dkfz.de/.

OligoWalk

Ref: Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W104-8.

Given an mRNA sequence as input, the OligoWalk web server generates a list of small interfering RNA (siRNA) candidate sequences, ranked by the probability of being efficient siRNA (silencing efficacy greater than 70%). To accomplish this, the server predicts the free energy changes of the hybridization of an siRNA to a target mRNA, considering both siRNA and mRNA self-structure. The free energy changes of the structures are rigorously calculated using a partition function calculation. By changing advanced options, the free energy changes can also be calculated using less rigorous lowest free energy structure or suboptimal structure prediction methods for the purpose of comparison. Considering the predicted free energy changes and local siRNA sequence features, the server selects efficient siRNA with high accuracy using a support vector machine. On average, the fraction of efficient siRNAs selected by the server that will be efficient at silencing is 78.6%. The OligoWalk web server is freely accessible through internet at http://rna.urmc.rochester.edu/servers/oligowalk.

sFold

Ref:Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W135-41.

The Sfold web server provides user-friendly access to Sfold, a recently developed nucleic acid folding software package, via the World Wide Web (WWW). The software is based on a new statistical sampling paradigm for the prediction of RNA secondary structure. One of the main objectives of this software is to offer computational tools for the rational design of RNA-targeting nucleic acids, which include small interfering RNAs (siRNAs), antisense oligonucleotides and trans-cleaving ribozymes for gene knock-down studies. The methodology for siRNA design is based on a combination of RNA target accessibility prediction, siRNA duplex thermodynamic properties and empirical design rules. Our approach to target accessibility evaluation is an original extension of the underlying RNA folding algorithm to account for the likely existence of a population of structures for the target mRNA. In addition to the application modules Sirna, Soligo and Sribo for siRNAs, antisense oligos and ribozymes, respectively, the module Srna offers comprehensive features for statistical representation of sampled structures. Detailed output in both graphical and text formats is available for all modules. The Sfold server is available at http://sfold.wadsworth.org and http://www.bioinfo.rpi.edu/applications/sfold.

siDirect

Ref: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W124-9.

siDirect (http://design.RNAi.jp/) is a web-based online software system for computing highly effective small interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi). Highly effective siRNA sequences are selected using novel guidelines that were established through an extensive study of the relationship between siRNA sequences and RNAi activity. Our efficient software avoids off-target gene silencing to enumerate potential cross-hybridization candidates that the widely used BLAST search may overlook. The website accepts an arbitrary sequence as input and quickly returns siRNA candidates, providing a wide scope of applications in mammalian RNAi, including systematic functional genomics and therapeutic gene silencing.

siRNA Selection Server

Ref:Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W130-4.

The Whitehead siRNA (short interfering RNA) Selection Web Server (http://jura.wi.mit.edu/bioc/siRNA) automates the design of short oligonucleotides that can specifically 'knock down' expression of target genes. These short sequences are about 21 nt in length, and when synthesized as double stranded RNA and introduced into cell culture, can reduce or eliminate the function of the target gene. Depending on the length of a gene, there are potentially numerous combinations of possible 21mers. Some experimental evidence has already shown that not all 21mers in a gene have the same effectiveness at silencing gene function. Our tool incorporates published design rules and presents the scientist with information about uniqueness of the 21mers within the genome, thermodynamic stability of the double stranded RNA duplex, GC content, presence of SNPs and other features that may contribute to the effectiveness of a siRNA.

siRNAdb

# siRNA efficacy prediction

A Support vector regressor (SVR) for predicting efficacy for siRNAs


# siRNA specificity prediction

Specificity search for siRNAs to avoid off-target effects


#siRNAdb

A database of known siRNA molecules


# siSearch (under reconstruction)

Rule-based siRNA design

siVirus

Ref: Nucleic Acids Res. 2006 Jul 1;34(Web Server issue):W448-50.

siVirus (http://siVirus.RNAi.jp/) is a web-based online software system that provides efficient short interfering RNA (siRNA) design for antiviral RNA interference (RNAi). siVirus searches for functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences. These siRNAs are expected to resist viral mutational escape, since their highly conserved targets likely contain structurally/functionally constrained elements. siVirus will be a useful tool for designing optimal siRNAs targeting highly divergent pathogens, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus and SARS coronavirus, all of which pose enormous threats to global human health.

TROD

Ref: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W121-3.

We have developed T7 RNAi Oligo Designer (TROD), a web application for RNA interference studies. TROD greatly facilitates the design of oligodeoxynucleotide sequences for the in vitro production of siRNA duplexes with T7 RNA polymerase. Given a query cDNA sequence, the program scans for appropriate target sequences based on the constraints of the T7 RNA polymerase method and published criteria for RNA interference with siRNAs. The output is an ordered and prioritized list of ready-to-order DNA oligonucleotide sequences, with links to perform a BLAST search to ascertain target sequence specificity. The TROD web service is available at http://www.cellbio.unige.ch/RNAi.html.

(Selected siRNA design papers)
1: Gong W, Ren Y, Xu Q, Wang Y, Lin D, Zhou H, Li T.
Integrated siRNA design based on surveying of features associated with high RNAi effectiveness.
BMC Bioinformatics. 2006 Nov 27;7:516.

2: Naito Y, Ui-Tei K, Nishikawa T, Takebe Y, Saigo K.
siVirus: web-based antiviral siRNA design software for highly divergent viral
sequences.
Nucleic Acids Res. 2006 Jul 1;34(Web Server issue):W448-50.

3: Shabalina SA, Spiridonov AN, Ogurtsov AY.
Computational models with thermodynamic and composition features improve siRNA
design.
BMC Bioinformatics. 2006 Feb 12;7:65.

4: Scherer L, Rossi JJ.
Therapeutic applications of RNA interference: recent advances in siRNA design.
Adv Genet. 2004;52:1-21. Review. No abstract available.

5: Yiu SM, Wong PW, Lam TW, Mui YC, Kung HF, Lin M, Cheung YT.
Filtering of ineffective siRNAs and improved siRNA design tool.
Bioinformatics. 2005 Jan 15;21(2):144-51. Epub 2004 Aug 27.

6: Naito Y, Yamada T, Ui-Tei K, Morishita S, Saigo K.
siDirect: highly effective, target-specific siRNA design software for mammalian
RNA interference.
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W124-9.

7: Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A.
Rational siRNA design for RNA interference.
Nat Biotechnol. 2004 Mar;22(3):326-30. Epub 2004 Feb 1.