Said to be the only commercial on-line design tool that allows users to view the secondary structure of the mRNA target sequence at the siRNA site before placing an order
The field of siRNA technology is still growing and discovering new concepts daily.
In response to this, MWG has recently improved its free on-line siRNA design tool.
MWG says it has the only commercial on-line design tool that allows users to view the secondary structure of the mRNA target sequence at the siRNA site before placing an order.
In the past, design algorithms focused mostly on the features of the siRNA duplex itself.
Attention was given to GC content, base composition, and length.
Aside from the GC content of the siRNA duplex, less attention was given to the secondary structure of the mRNA target site.
As siRNA research has progressed, scientists are debating the importance of secondary structure to the success of siRNA inhibition.
New papers published within the last year have further spurred the debate of this issue.
The focus of GC content within the siRNA duplex itself has naturally removed a large percentage of designs targeted to difficult areas of the mRNA sequence.
Recent articles are revealing that by reviewing the secondary structure of the mRNA target one can greatly increase the likelihood of knockdown success.
Yiu proposed an algorithm filtering ineffective siRNAs based on the secondary structure of the target site on the mRNA.
The filtering algorithm was applied to the design tools of all the major siRNA companies, and it was found that secondary structure analysis can filter up to 53% of ineffective siRNAs.
By reviewing secondary structure, the basic MPI Principles (which are used by MWG Biotech) would outperform the current algorithms of Ambion and Qiagen.
A more recent publication by Heale also showed the importance of secondary structure.
The team state that the siRNA and the mRNA must be able to form an A-helix for correct positioning of the scissile phosphate bond for cleavage in RNAi.
Their algorithm uses local structural features to calculate the secondary structure of a target site.
The higher the free energy of the helix in comparison to the stability of the target site secondary structure is, the better the effectiveness of the RNAi.
So, what is a siRNA researcher to do? How relevant is secondary structure and what rules of the surrounding area do you use? Many siRNA companies have built their reputation around their proprietary design tool, going as far as to provide entire libraries of siRNA duplexes - thus locking themselves into one design algorithm and siRNA length.
MWG's approach, however, is a more flexible one.
It believes users should know what algorithms are used, and have the ability to decide for themself, 1) if secondary structure analysis they wish to avoid.
The methodology of the research should be completely open in order to take it to its furthest level.
Why perform an experiment where the criteria used to design a siRNA are unknown? With MWG users have the flexibility to decide which rules they want to follow and have access to the latest bioinformatics trends, the company says.
How does MWG's siRNA Design Tool work? Step 1: enter your Fasta sequence or accession number.
Step 2: select the design criteria to use (such as GC content) or leave it at the default settings.
Step 3: The program will generate a list of possible siRNA sequences.
Select your sequence(s) of interest and click on 'View Secondary Structure'.
Step 4: review the mRNA structure(s).
The location of the siRNA will be displayed in red for each selected candidate.
Step 5: proceed to order the siRNA that meets your secondary structure requirements.
MWG says it has the only commercial on-line design tool that allows users to view the secondary structure of the mRNA target sequence at the siRNA site before making an order.
The standard default parameters have been intelligently designed to give the best chance of success for beginners and experts alike.
There is no hiding of the results, before ordering all candidate designs can be reviewed and re-designed if needed.
Each parameter including motif, GC content and sequence position can be customised to exactly fit the design needed.
A single or multiple target sequences can be entered in fasta format or as Genbank accession numbers.
Every design candidate is checked by Blast for specificity to distinguish between alternative splice forms and avoid off-target effects.
References.
1, Yiu, et al, Filtering of ineffective siRNAs and improved siRNA design tool - Bioinformatics 2005 Jan 15;21(2):144-51.
2, Heale, et al, siRNA target site secondary structure predictions using local stable substructures - Nucleic Acids Res 2005 Feb 18;33(3):e30.
