**8. Discussion about selected siRNA designing tools**

Several siRNA sequence selection algorithms have been developed in the past decade that relies on intrinsic sequence, stability and target accessibility features of functional siRNAs. Different siRNA selection algorithms follow different set of rules derived from some wellknown siRNA design parameters as discussed above. In general, these algorithms rely on features like- low GC content, absence of siRNA self-alignment, absence of internal repeat, thermodynamic conditions favouring efficient RISC entry, absence of homology to other mRNAs and some position specific nucleotide compositions. Few of them also consider silencing of alternatively spliced isoforms of the given gene. Different algorithms use different techniques for combination of parameters and their weight distribution- ranging from empirical observation to sophisticated machine learning. In spite of a large number of online siRNA design solutions, few of them consider miRNA-like off-targeting potential of synthetic siRNAs. Consideration for minimization of such off-target effect involves imposing a threshold for number of off-target genes. Table 5 lists some of the online siRNA designing solutions with their designing parameters.

Computational Approaches for Designing Efficient and Specific siRNAs 273

**Off-target(near complementary)** 

Considered Considered Not

Considered Considered Considered

Considered Considered Considered

Considered Considered Not

Considered Considered Not

Considered Considered Considered

**miRNA-like off-target** 

Considered

Considered

Considered

**Alternative splicing** 

Considered Not Considered Considered Considered Considered

**siRNA selection tool** 

Promega siRNA Target Designer [http://www.promega. com/siRNADesigner/p

siDESIGN Center [http://www.dharmaco n.com/sidesign].

Imgenex siRNA tool [http://www.imgenex.c om/sirna\_tool.php].

[http://sonnhammer.cg b.ki.se/siSearch/siSearc

Whitehead WI siRNA Selection Program [http://jura.wi.mit.edu/ bioc/siRNAext/].

rogram/].

siDRM

siDirect

siSearch

h\_1.7.html].

**Thermodynamic deference between sense and antisense strand 5' end** 

[http://sidrm.biolead.org/] Considered Considered Not

[http://sidirect2.rnai.jp] Considered Considered Not

**9. Importance of functional off-target filtering** 

**Target accessibility** 

Considered Considered Not

Considered Considered Not

Considered Considered Not

Considered Considered Not

Considering only quantity of the off-targets and not the functions of individual off-targets can lead to inefficient handling of the miRNA-like off-target issue. Often in siRNA screening experiments, it has been reported that the desired output is affected because of silencing of unintended off-targets those sometimes are themselves member of the upstream pathway components of the direct target gene [26]. In such cases it can be useful to avoid specifically some off-targets that can cause more harmful or undesirable effects. It should be considered that during silencing process siRNA should not silence any mRNA from the same pathway the target mRNA is part of. In case of investigation of a gene function, if any gene from the same pathway is silenced rather than target gene then it will be difficult to investigate the actual phenotype of silencing the gene under investigation. For e.g. in a siRNA screening experiment designed for novel members of the transforming growth factor (TGF)-b pathway in a human keratinocyte cell line, dominant off-target effect was observed due to unintended silencing of two known upstream pathway components, the TGF-b receptors 1 and 2 (TGFBR1 and TGFBR2). Such off-target silencing activity poses threats of confusing and misleading results. Also the siRNAs suggested by the online siRNA selection tools often are predicted to have off-

**Table 5.** A comparison of siRNA designing parameters in different siRNA designing tools

targets that belong to the same pathway or somehow related to the direct target.



**Table 5.** A comparison of siRNA designing parameters in different siRNA designing tools
