**2.1. Selection of target region**

262 Bioinformatics

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

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.

As discussed earlier, potency of siRNAs varies greatly with selection of target region. The parameters responsible for effectiveness of siRNAs being target accessibility, uniqueness of target region, absence of SNP sites etc. Other parameters worth considering while selecting the target site is the consideration of alternatively spliced isoforms of the target

A parameter which greatly influences siRNA potency is RISC loading of the intended antisense strand [6]. Only one strand of the siRNA duplex enters into RISC and guides the complex to the target mRNA for its silencing. So, the choice of strand that enters into RISC

siRNA selection algorithm and efficient parameter optimization.

**2. Guidelines for selecting potent siRNAs** 

gene [5].

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 alternatively spliced isoforms.

#### *2.1.1. Selection of target starting site*

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 codon.

## *2.1.2. Consideration for alternative splicing*

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 isoforms from a target gene.

## *2.1.3. Absence of homology with other mRNAs*

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 homology with any other mRNA should be avoided.

#### *2.1.4. Avoiding target sites having polymorphic locus*

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