**6.3. miRNA-like off-target effect**

siRNAs down regulate a set of transcripts with 3' UTR complementarity to the 5' portion of the corresponding siRNA guide strand. These 5' ends of the guide strand resemble the seed region of endogenous miRNA which is responsible for target recognition. Such off-targets are regulated by translational repression like miRNA target regulation. That is why this

kind of off-target effect is called miRNA-like off-target effect [18, 19]. This kind of off-target cannot be fully avoided but can be reduced by computational design. Consideration for minimization of such off-target effect involves imposing a threshold for number of off-target genes. All of the present day online siRNA designing techniques consider only the quantitative approach for minimizing miRNA-like off-target effect by restricting the number of off-targets [20]. To go beyond mere quantitative approach and look for the functional correlation between the on-target and the genes off-targeted by the siRNA will certainly prove to be beneficial in minimizing miRNA-like off-target effect. A newly developed siRNA designing tool is aimed for such off-target minimization considering functional correlation of the off-target and the direct target (explained in section 9).

Computational Approaches for Designing Efficient and Specific siRNAs 271

siRNA seed region match in combination with 10

unintended target genes is used for pediction of off-targets. siRNAs predicted to have a large

prediction database, a threshold of seed match for a specific species is applied which is computed by a probability model which has general biological significance. siRNAs predicted to have a large

Chooses siRNAs that has a seed sequence with

siDirect selects siRNAs with lower melting temperature (Tm value) at the seed region, which contains 7 nucleotides at positions 2-8 from 5′ end of the guide strand as the capability of siRNA to induce this seed-dependent off-target effect is highly correlated with the thermodynamic stability of the duplex formed between the seed region of the siRNA guide strand and its target mRNA.

For each candidate siRNA, *siDRM* checks and reports if its seed region (position 2–8) has full homology to the 3' UTR region of another

Predicts off-targets based on the seed region complementarity as well as conservation of the target site among related species (human, mouse, rat, dog and chicken). siRNAs predicted to have a

large number of off-targets are rejected.

transcript, and this homology region is followed by four consecutive mismatches. siRNAs predicted to have a large number of off-targets are rejected.

or more bases of additional homology to

Depending on their proprietary off-target

siRNA off-target prediction problem. A study reported potential silencing of transcripts having consecutive 11 or more bases complementarity with miRNA or siRNAs including siRNA bases 9-12. These transcripts are more likely to be cleaved by the siRNA. Some other off-target prediction parameters include secondary structure analysis for target accessibility prediction and A/U base richness near target site [25]. For reliable off-target prediction, an

**miRNA-like off-target effects** 

number of off-targets are rejected.

number of off-targets are rejected.

lower seed compliment frequency.

optimized combination of all the above mentioned parameters is needed.

BIOPREDsi

[http://www.biopredsi.org/].

[https://www.genscript.com/ssl-

[http://www.dharmacon.com/sidesign].

siDirect [http://sidirect2.rnai.jp].

siDRM [http://sidrm.biolead.org/]

Whitehead WI siRNA Selection

[http://jura.wi.mit.edu/bioc/siRNAext/].

**Table 4.** Off-target minimization techniques of different siRNA designing tools.

Program

siRNA Target Finder

bin/app/rnai].

siDESIGN Center

**siRNA designing tool Approaches towards minimization of** 
