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What is RNAi and siRNA?

How does it work?
How are effective siRNA designed?
Why use fluorescent and modified siRNA’s?
What are my siRNA delivery options?
How do I quantify down regulation?
What are shRNA?
See Current Tuschl Lab’s siRNA User Guide

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How does it work?
Recent advances in molecular biology have shown that gene expression can be effectively silenced in a highly specific manner through the addition of double stranded RNA (dsRNA) (1-3). The term RNA interference (RNAi) was coined to describe this phenomenon and, while the mechanism was originally observed in plants and later in the worm Caenorhabditis elegans, subsequent studies have shown that RNAi is present in a wide variety of eukaryotic organisms including mammals (4-6). For the most part, it is believed that RNAi serves as an antiviral defense mechanism although there is preliminary evidence that it also plays a role in the formation and maintenance of heterochromatin during mitosis and meiosis (7,8).

Once dsRNA enters the cell, it is cleaved by an RNase III –like enzyme, Dicer, into double stranded small interfering RNAs (siRNA) 21-23 nucleotides in length that contain 2 nucleotide overhangs on the 3’ ends (9-11). In an ATP dependent step, the siRNAs become integrated into a multi-subunit protein complex, commonly known as the RNAi induced silencing complex (RISC), which guides the siRNAs to the target RNA sequence (12). At some point the siRNA duplex unwinds, and it appears that the antisense strand remains bound to RISC and directs degradation of the complementary mRNA sequence by a combination of endo and exonucleases (13).

Preliminary studies in mammalian systems using long dsRNAs to initate the RNAi response failed because they led to the induction of a non-specific Type I interferon response that produced extensive changes in protein expression and eventually resulted in cell death (14,15). Subsequent studies, however, using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer, sequence specific gene silencing could be achieved in mammalian cells without inducing the interferon response (6,16). siRNA technology is now extensively recognized as a powerful tool for the specific suppression of gene expression and is presently being used by researchers in a wide range of disciplines for the assessment of gene function.

How are effective siRNA designed?
siRNA Design Strategies:
Default parameters: AA(19mer) The default criteria selects target sequences of 21 nucleotides that begin with AA and are located within a region of the coding sequence that is within 50-100 nucleotides of the AUG start codon and within 50-100 nucleotides from the termination codon. The presence of AA at the start of the sequence allows for the use of dTdT at the 3’-end of the antisense sequence. The sense strand can also be synthesized with dTdT at the 3’ end, because only the antisense strand is involved in target recognition. The use of dTdT reduces the cost of synthesis and also makes the siRNA duplex more resistant to exonuclease activity. Because a number of reports have demonstrated that the presence of AA at the beginning of the target sequence is not an absolute requirement, the selection program includes the option to search for sequences that begin with other nucleotide pairs.

GC Content
The G-C content of the sequence is also used as a condition for selecting target sequences. Ideally the GC content will be less than 50%, although successful gene silencing has been reported with siRNAs that have G-C contents between 50 and 60%. The default parameter selects for a G-C content in the 40-50% range, however, options are available that allow for selection over wider ranges.

Stretches of Nucleotide Repeats
The default mode avoids sequences with repeats of three or more G’s or C’s, as their presence initiates intra-molecular secondary structures preventing effective siRNA silencing hybridization. As an option, repeat stretches of A’s and T’s can also be eliminated, as they tend to reduce the specificity of the target sequence. If possible, this option is highly recommended.

Blast Search
Once a target sequence has been chosen, a BLAST search is initiated to ensure that your target sequence is not homologous to other gene sequences. Target sequences that have more than 15 contiguous bases pairs of homology to other genes in the NCBI database are eliminated.

Why use fluorescent and modified siRNA’s?
It is not essential to monitor the subcellular localization of siRNA after transfection. When desired, the siRNA can be labeled with fluorescent dyes to track the delivery and uptake of siRNA. Usually after uptake the siRNA are present free in the cytoplasm and in complex formation with proteins in the endosomes.

The siRNA can be modified with various other modifications like 2’O methyl RNA, biotin or digoxigenin based on the researchers need. All these modifications are available from Gene Link.
Please click here to see the list of modifications.

What are my siRNA delivery options?
Delivery of siRNA directly in cells can be achieved by using microinjection or electroporation. Another popular option is the use of transfection reagent. Several companies offer specialized siRNA-delivery reagents. Please consult the transfection reagent vendor’s protocol for detailed information for the exact requirements and procedure. Careful optimization of variable factors should be ensured for all initial transfection experiment. It is based on this and further optimization that reproducible gene knock out results will be obtained. Usually RNAi effect is seen within 4 hours and the maximum down regulation observed in 24-48 hrs. The effect lasts several cell generations and from 4-10 days depending on cell culture type.

How does purity of synthetic siRNA’s affect RNAi?
RNAi is a sequence specific chain of events. Chemical synthesis of siRNA’s is based on coupling of bases to yield a particular sequence. The yield and purity depends on the coupling efficiency. Gene Link siRNA’s can be used without further purification, but Gene Link recommends purified siRNA’s for use in transfection.

What concentration of siRNA is most effective?
As low as 1 nM concentration of siRNA have been shown to be effective in exhibiting RNAi. Initial experiments should be done at varying concentrations from 1-10 nM. Some reports have used as high as 25nM concentrations. High quality siRNA’s should be used.

What is the optimal cell density for transfection?
This is another variable that has to be optimized and then maintained. A good starting point is 60-70% confluent cells. Time points should be taken after transfection to determine the maximum inhibition. Start at 4hrs and end at 72hrs initially.

How do I quantify down regulation?
RNAi down regulates a gene function without actually interacting with the gene. The subtle action is by mRNA degradation. Thus the degree of RNA interference achieved is directly proportional to the level of mature mRNA and the translated proteins. The options are:

1. Measurements of target protein(enzyme) activity. This option is suitable if a robust assay is available or has been in prior use. The assay would vary by the nature of the protein product.

2. Measurement of target mRNA level. This is the preferred method as it directly quantifies the level of mRNA. Quantitative PCR is very effective in measuring relative amount of target sequence. This can be achieved simply by SYBR green or by the use of TaqMan or Molecular Beacons. More information.

What are shRNA?
An alternate to individual chemical synthesis of siRNA is to construct a sequence for insertion in an expression vector. Several companies offer RNAi vectors for the transcription of inserts. Some use an RNA polymerase III (Pol III) promoter to drive expression of both the sense and antisense strands separately, which then hybridize in vivo to make the siRNA. Other vectors are based on the use of Pol III to drive expression of short "hairpin" RNAs (shRNA), individual transcripts that adopt stem-loop structures, which are processed into siRNAs by the RNAi machinery.

Designing Oligonucleotides for RNAi Expression Vectors
Select a target sequence that begins with AA and select the next 19-29 bases (depending on the vector supplier’s recommendations) as a template for the sense sequence. Do not include the initial AA in the sequence. The upper strand of the target sequence should start with a G or an A, as RNA polymerase III prefers to initiate transcription with a purine. If a G or A is not present, then it must be inserted immediately upstream of the target sequence. A terminator sequence consisting of 4-6 dTs should be added immediately downstream of the target sequence.

Appropriate cloning sequences can be added to the 5’ ends of the two complementary oligonucleotides.

Example for expression of siRNA (small interfering RNA):

Sense or Antisense

Oligo 1:5’-(cloning sequence)GN19Cttttt-3’
Oligo 2:3’-CN19Gaaaaa(cloning sequence) 5’

Example for expression of shRNA (short hairpin RNA):

Typical shRNA design consists of two inverted repeats containing the sense and antisense target sequences separated by a loop sequence. Commonly used loop sequences contain 8-9 bases. A terminator sequence consisting of 5-6 poly dTs is present at the 3’ end and cloning sequences can be added to the 5’ ends of the complementary oligonucleotides.

Example:

Oligo 1:5’-(cloning sequence)GN21GAAGCTTGN21TTTTTT-3’
 sense loopantisenseterminator
Oligo 2:3’-CN21CTTCGAACN21AAAAAA(cloning sequence)-5’
References
1. Fire,A., Xu,S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Nature, 391, 806 811.
2. Napoli, C., Lemieux, C. & Jorgensen, R. (1990) Plant Cell 2, 279 289.
3. Hannon, G.J. (2002) RNA interference. Nature, 418, 244 251
4. Billy,E., Brondani,V., Zhang,H., Muller,U. and Filipowicz,W. (2001) Proc. Natl. Acad. Sci. USA, 98, 14428 14433
5. Paddison, P.J., Caudy, A.A. and Hannon, G.J. (2002) Proc. Natl Acad. Sci. USA, 99, 1443 1448
6. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl,T. (2001) Nature, 411, 494 498
7. Volpe TA, Kidner C, Hall IM, Teng G, Grewal SI, Martienssen RA. (2002) Science.,297, 1833-1837.
8. Allshire R. (2002) Science, 297, 1818-1819.
9. Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001) Genes Dev., 15, 188 200.
10. Bernstein, E., Caudy, A.A., Hammond, S.M. and Hannon, G.J. (2001) Nature, 409, 363 366.
11. Hammond, S.M., Bernstein, E., Beach, L. and Hannon, G.J. (2000) Nature, 404, 293 296.
12. Nykanen, A., Haley, B. and Zamore, P.D. (2001) Cell, 107, 309 321.
13. Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R. and Tuschl, T. (2002) Cell, 110, 563 574.
14. Caplen, N.J., S. Parrish, F. Imani, A. Fire and R.A. Morgan. 2001. Proc Natl Acad Sci U S A 98:9742-9747.
15. Ullu E, Djikeng A, Shi H, Tschudi C., 2002, Philos Trans R Soc Lond B Biol Sci. 357, 65-70.
16. Ui-Tei, K., S. Zenno, Y. Miyata and K. Saigo, 2000, FEBS Lett 479:79-82.
Other Online References

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