AsiDesigner: exon-based siRNA design server considering alternative splicing
AsiDesigner is a highly performing, highly effective siRNA design program based on exon-based siRNA
design algorithm considering alternative splicing.
(* Refer to the User's manual for AsiDesigner).
It uses two effective algorithms for siRNA design: the algorithm for siRNA target
region selection considering alternative splicing and the optimization algorithm of
siRNA performance using relative binding energy profile.
Figure 1 The program structure and data flow for siRNA design in AsiDesigner.
The algorithm for siRNA target region selection considering alternative splicing
The purpose of this algorithm is to evaluate the target region to design
siRNAs targeting one or more mRNAs selectively from an isoform mRNA set.
After the target region evaluation, actual siRNA design is performed using
'the optimized siRNA design method using relative binding energy profile' within this target region.
The stepwise process for evaluating target region to
design siRNAs targeting one or more mRNAs from an isoform mRNA set is as follows:
(1) Retrieve all the mRNA isoforms from a target gene,
(2) Select a target mRNA isoform(s),
(3) Retrieve the physical coordinates of mRNA isoform from thegenome,
(4) Convert the physical position into that on the target mRNA,
(5) Evaluate a common (intersection) exon region among target mRNAs,
(6) Evaluate the union of the exons of non-target mRNAs,
(7) Inspect the target region,
(8) Convert the physical position of target mRNA into that on the genome,
(9) Extract the target region sequence.
Figure 2 Schematic diagram for evaluation of the target region to design siRNAs for specific
alternative spliced isoforms.
Figure 3 Schematic diagram for conversion process of the siRNA position from mRNA to genome.
The optimization algorithm using relative binding energy profile
The optimization algorithm of siRNA design using relative binding energy profile was adopted
in AsiDesigner utilizing the following selection rules and performance scoring rules:
1) siRNA selection rules
The candidate siRNA sequences are filtered or adopted based on the following selection rules.
2) siRNA scoring rules
- G/C content: users set up the range of GC content (default: 30 - 62%),
- Consecutive bases (default: maximum 5 for A or T, maximum 3 for G or C, maximum 7 for G/C mixed),
- Filtering siRNAs having SNP (single nucleotide polymor-phism) locus,
- Avoiding siRNA self-alignment (delta G < -4 kcal/mol),
- Maximum identical bases to the non-target mRNAs for off-target filtering (default: equal to or less than 15/19).
AsiDesigner uses a special performance scoring scheme to assess the performance of siRNAs designed.
AsiDesigner's performance scoring system assigns scores (Zi) for 5 main design parameters affecting the performance
of siRNAs and combines them linearly with the statistically evaluated weighting factors (Wi)
for each design parameters to get the final performance score (Zt).
where Mi is the maximum score for each design parameter and i is the index for each design parameter:
36 - 53 % GC content, 3 or more A/U existence at 15-19th bases from 5 sense position,
G/C existence at the 1st 5 sense position, A/U existence at the 19th base from 5 sense position,
and the relative binding energy profile. W1 = 0.11, W2 = 0.07, W3 = 0.5, W4 = 0.19, W5 = 0.90.
AsiDesigner was developed as a web application program based on the JAVA,
JSP/JSF technologies and MySQL relational database. Researchers can access and utilize AsiDesigner
under the user-friendly designed GUI environment interactively wherever the Internet is available.
Figure 4 Schematic diagram of the data processing flow for siRNA design in AsiDesigner.
Features of AsiDesigner
The main features of the AsiDesigner are as follows:
-Input: gene name, mRNA accession, target sequence.
-Organisms: Human, mouse, rat (3 organisms).
-siRNA type: AA (N19), NA (N19), NN (N19) overhang types.
- Target region selection (Full, CDS, and Range),
- G/C contents,
- SNP existence,
- Repeat existence,
- Melting temperature (Tm),
- Antisense/sense/middle 5' binding energy,
- Binding energy difference (3'-5'),
- Sequence similarity for specificity,
- Simple energy profile scoring,
- Secondary structure.
- Gene annotation and genome map (physical position) of mRNAs from
UCSC: hg19(Feb. 2009), mm10 (Dec. 2011), rn5 (Mar. 2012),
- mRNA sequence database from NCBI
RefSeq Release 61 (Sep. 2013),
- SNPs from NCBI
dbSNP Build 138 (Aug. 2013).
- NCBI BLAST for off-target filtering,
- FASTA for off-target filtering,
- mfold for analyzing siRNA secondary structure folding.
Figure 5 Performance of siRNAs designed by AsiDesigner. (A, B: Northern
blot for PP2A-a, PP2A-b gene; C, D: siRNA fraction of STE and Caspase gene
for mRNA knock-down rate.)
1) miRNA type off-target filtering
It is known that siRNA can induce an off-target effect through the miRNA-like mechanism
(Doench JG, Petersen CP, Sharp PA., 2003,
siRNAs can function as miRNAs. Genes Dev 17: 438-42,
At the present time, off-target filtering for this kind of effect is not considered in AsiDesigner
2) 25, 27mer siRNA design capacity