Algorithm——iRNA designer& Riboswitch designer

siRNA designer:

Tom Tuschl's rule (for cDNA)

1. Select targeted region from a given cDNA sequence beginning 50-100 nt downstream of start codon

2. Look for sequence motif AA+N19+TT first. If there is no suitable sequence look 23-nt sequence motif NA+N21 and convert the 3' end of the sense siRNA to TT

3. Or search for NAR+N17+YNN

PS: Target sequence should have a GC content of around 50%

R means A/G, and Y means T/C.

Rational siRNA design(for mRNA)

Evaluate potential candidates and assign scores to them, sequences with higher scores will have higher chance of success in RNAi.

The table below lists the 8 criteria and the methods of score assignment.

Criteria	Description	Score
		Yes	No
1	Moderate to low (30%-52%) GC Content	1 point
2	At least 3 A/Us at positions 15-19 (sense)	1 point /per A or U
3	Melting temperature-Tm*<20℃	1 point
4	A at position 19 (sense)	1 point
5	A at position 3 (sense)	1 point
6	U at position 10 (sense)	1 point
7	No G/C at position 19 (sense)		-1 point
8	No G at position 13 (sense)		-1 point

The "anti-sense" strand is the siRNA strand that is complementary to the target mRNA and that will be binding to the mRNA.

The melting temperature’ of a siRNA candidate can be calculated by the formula showed below:

wA, xT, yG, zC are separately the number of A, T, G, C in a siRNA candidate.

All siRNA candidates scored higher than 6 are acceptable.

 

Riboswitch designer:

The designer is based on a special promoter working in eukaryotes - internal ribosome entry site (IRES). We design the upstream sequence that can bind to IRES part so the second structure of IRES can be transformed by the ligand.

Up regulated:

Target sequence is assembled by five parts: MS-SL, aaIRES (anti-anti-IRES), aptamer, aIRES (anti-IRES), IRES.

The IRES, aIRES, aaIRES parts are settled, we get aptamer sequence according to customer’s quests from aptamer database and the MS-SL is paired to the combination part of aaIRES and aptamer. Then adjust the MS-SL part’s length to fit it’s free energy to -11.7 kcal/mol.

 

Down regulated:

The differences from down regulated to up regulated riboswitch are the order between aptamer and aaIRES, the sequence of MS part. The new structure is showed below:

We set MS to “CCUCU” under experimental data and the aptamer still comes from aptamer database.

 

Algorithm about free energy calculation comes from Turner’s team:

Hairpin loops:

In this equation, n (5) is the number of nucleotides in loop, the terminal mismatch parameter is the sequence-dependent term for the first mismatch stacking on the terminal base pair.

Watson-Crick Helices:

Specific data can be found on web site:

http://rna.urmc.rochester.edu/NNDB/turner04/index.html

 

References:

1.Elbashir SM et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411:494-498.

2.Elbahir SM et al. (2001). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877-6888.

3.Elbashir SM et al. (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods. 26:199-213.

4.Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 Mar;22(3):326-30.

5.Ogawa, A. (2011). Rational design of artificial riboswitches based on ligand-dependent modulation of internal ribosome entry in wheat germ extract and their applications as label-free biosensors. RNA (New York, N.Y.), 17(3), 478-88. doi:10.1261/rna.2433111

6.Ogawa, A. (2012). Rational construction of eukaryotic OFF-riboswitches that downregulate internal ribosome entry site-mediated translation in response to their ligands. Bioorganic & medicinal chemistry letters, 22(4), 1639-42. Elsevier Ltd. doi:10.1016/j.bmcl.2011.12.118

7.Turner, D. H. & Mathews, D. H.  (2009).  NNDB: The nearest neighbor parameter database for predicting stability of nucleic acid secondary structure.  Nucleic Acids Research.  38, D280-D282.