**1. Introduction**

260 Bioinformatics

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Small RNA mediated RNA interference (RNAi) is a widely adopted mechanism towards immunity in plants and invertebrates. Two types of small RNAs- small interfering RNA (siRNA) and microRNA (miRNA), play key role in RNA interference either through cleaving or through translational repression of the target mRNA by guiding RNA induced silencing complex (RISC) to its target site. siRNA is a small RNA (19-23 nucleotides) which is complementary to part of their target mRNA [1]. siRNAs are very efficient in target gene knockdown that makes synthetic siRNA's perfect choice for use in experiments for silencing genes to examine their function. In addition, siRNA have good potential in drug development for therapeutic purpose [2]. Exogenous synthetic siRNAs are designed to target a part of the coding region in the target mRNA [3]. But, as evident from experiments, all siRNAs are not equally efficient in target gene silencing. The potency of siRNAs is largely dependent upon the selection of the region it targets. A displacement of 5-6 nucleotide position hugely alters the efficiency of siRNA. The reason behind this alteration of efficiency is the alteration in local sequence and structural features of the target region. These variations in sequence and structural features correlate with target accessibility, RISC loading or stimulation of immune response. A lot of study has been done in the field of rational siRNA designing to find appropriate parameters that facilitate designing of effective siRNAs [4]. Several commercial suppliers and non-profit educational institutes contribute to the research for searching appropriate siRNA selection parameters for improving potency of designed siRNAs. Progresses have been made in targeting success rate compared to the early days- from as low as 0-10% targeting success rate, today siRNAs have reached average 50% targeting success rate. Still there are many scopes to improve siRNA designing. For efficient designing, the siRNA selection parameters must be arranged and weighted in such a way that ensures optimal result while selecting the siRNA target site. There are some previously suggested guidelines about parameter weight assignment like rational or

© 2012 Ghosal et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Ghosal et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

weighted methods. These guidelines were made in the early days of siRNA selection research with small number of data, when experimentally knockdown validated siRNA dataset was scarce. But with increasing amount of knockdown validated siRNA datasets, new parameter optimization methods must come out to ensure selection of potent siRNAs in a bigger scenario. Today, high throughput siRNA screening experiments have become a common technique to investigate thousands of gene functions at a time to study some specific pathway. These experiments use siRNA libraries targeting transcripts in a genome wide range. But the success of these experiments relies on the knockdown success rate of the siRNAs in the library. After two decades of research, still many of these large scale screening experiments fail because of a large number of non-functional siRNAs present in the libraries used in these experiments. So, still there are needs for improvement in the siRNA selection algorithm and efficient parameter optimization.

Computational Approaches for Designing Efficient and Specific siRNAs 263

complex plays the vital role in the whole silencing process. Generally RISC complex chooses the siRNA strand which has weaker 5' end binding energy. So this thermodynamic property should be ensured while selecting siRNAs. Also there are a number of sequence compositions that are said to be important for recognition of the intended strand by RISC and some parameters for enhancing the stability of siRNA duplex are also worth considering while selecting siRNAs. These rules can be divided into three categories 1) Selection of target region, 2) Structural and Thermodynamic Consideration and 3) Sequence

Selection of the target region for siRNA should satisfy some constraints to ensure effective silencing. Target site should be analyzed for its position within the mRNA, presence of polymorphic site or other considerations like homology with other mRNAs or its

Generally siRNAs are targeted to a part of the coding region of an mRNA. The target site should be deep inside the open reading frame (ORF) of the target mRNA for efficient silencing. Generally it is advised to start from 50-100 nucleotides downstream of the AUG start codon. Besides targeting the coding region, some siRNAs may also be designed to target the 3' un-translated region (UTR) of an mRNA. This type of targeting is especially done in case of experiments conducted for restoration of original phenotype. In such cases it is advised to start targeting regions at least 15-20 nucleotides downstream of the stop

In eukaryotic organisms frequent alternative splicing results in diversification of mRNAs. To account for the alternative splicing, it is necessary to evaluate a common target region where siRNAs can be designed to knockdown a specific mRNA isoform or multiple mRNA

Cellular mRNAs with 15/16 or more consecutive base match with the siRNA, are likely to be silenced and degraded by the siRNA. So target sites with 15 or more consecutive base

Target regions having single nucleotide polymorphic (SNP) sites should be avoided.

Characteristics.

codon.

**2.1. Selection of target region** 

alternatively spliced isoforms.

*2.1.1. Selection of target starting site* 

*2.1.2. Consideration for alternative splicing* 

*2.1.3. Absence of homology with other mRNAs* 

homology with any other mRNA should be avoided.

*2.1.4. Avoiding target sites having polymorphic locus* 

isoforms from a target gene.

Another main challenge in computational siRNA designing lies in the specificity issue of siRNAs. Exogenous siRNAs often induce off-target effects that arise from near perfect or imperfect sequence complementarity with other mRNAs resulting in false positive phenotype during RNAi-based study of gene functions. The type of gene regulation resulting from imperfect sequence complementarity resembles target regulation by endogenous miRNAs- hence this type of off-targeting is called miRNA-like off-targeting. Minimization of miRNA-like off-targets involves choosing a siRNA with a seed region sequence that has fewer targets in the 3' UTR of other mRNAs. The siRNA seed region is a 6 or 7 nucleotide sequence (the 2-8th nucleotide position) from 5' end of siRNA guide strand and finding complementary sequence with this 6 or 7 nucleotide seed in the 3' UTRs of unintended transcripts results in a huge number of potential targets. A majority of these predicted targets are not practically relevant, as in practice, a large number of these predicted off-targets may not be silenced at all. Like miRNA targets, siRNA targets are also dependent on target accessibility and other sequence features around the target site. So, a more rational approach is needed when predicting the siRNA off-targets resulting from partial sequence complementarity. This chapter focuses on current siRNA designing parameters and the approaches towards minimizing the off-target effects as provided by the in-silico siRNA designing solutions and then discuss a bit about a customized off-target reducing algorithm that will be useful to avoid particular genes from being off-targeted.
