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  Technical Bulletin-Revision A
siRNA Oligonucleotides for RNAi Applications   Dharmacon siACE-RNAi^TM Options

Recent discoveries have revealed that double stranded RNA (dsRNA) effects silencing of the expression of gene(s) which are highly homologous to either of the RNA strands in the duplex. This phenomenon has been termed RNA interference or RNAi.¹ Gene silencing via RNAi results from degradation of mRNA sequences. Tuschl and co-workers now report this effect can be elicited very effectively by well-defined 21-base duplex RNAs, termed small interfering RNA, or siRNA.^{2, 3} When the siRNA duplex is added with a transfection agent to mammalian cell cultures, the 21-base RNA acts in concert with cellular components to silence the gene with sequence homology to one of the siRNA sequences. Of further note, siRNAs are effective at quantities much lower than alternative gene silencing methodologies, including antisense and ribozyme based strategies. RNA oligonucleotides are the key component of this technology.

As the world leader in RNA oligonucleotide synthesis, Dharmacon Research has the ability and the responsibility to assist with the application of siRNA methods. Dharmacon’s revolutionary 2’-ACE RNA synthesis technology offers the most advantageous avenue to produce the requisite siRNAs for RNAi gene silencing. With this Technical Bulletin, Dharmacon is initiating its siACE-RNAi line of products and options to ensure that siRNA technology is readily accessible and implemented with the highest quality RNA and services.

Dharmacon Research has worked closely with Dr. Tuschl and other leaders in the field to integrate 2’-ACE RNA synthesis technology with siRNA techniques. The following technical bulletin was prepared with valuable input from T. Tuschl, S. Elbashir, J. Harborth, and K. Weber. This bulletin is designed to provide guidance and direction to investigators interested in applying siRNA to their research and applications.⁴

 

Choosing Sequence and the Design of the siRNA Duplexes for the Target mRNA

Tuschl and co-workers have systematically analyzed the silencing efficiency of siRNA duplexes as a function of the length of the siRNAs, the length of the 3’-overhangs, and the sequence in the overhangs. The initial work was completed using Drosophila melanogaster lysates.⁵ Further research discovered that the most efficient silencing was obtained with siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 19-nucleotide duplex region and a 2-nucleotide overhang at each 3’-terminus.³ Symmetric 3'-overhangs ensure that the sequence-specific endonuclease complexes (siRNPs) are formed with approximately equal ratios of sense and antisense target RNA cleaving siRNPs.² The 3’-overhang in the sense strand provides no contribution to recognition as it is believed the antisense siRNA strand guides target recognition. Therefore, the UU or dTdT 3’-overhang of the antisense sequences is complementary to the target mRNA but the symmetrical UU or dTdT 3’-overhang of the sense siRNA oligo does not need to correspond to the mRNA. The use of deoxythymidines in both 3’-overhangs may increase nuclease resistance, although siRNA duplexes with either UU or dTdT overhangs work equally well. The recommended design of the siRNA duplex is as illustrated in Figure 1.

Figure 1. Recommended design of siRNA duplex comprised of 19-nucleotide duplex region and UU or dTdT overhangs at each 3’-terminus, where "X" equals the complement to "N." The 21-base complementary siRNA sequence corresponds to the target sequence of the mRNA.

The targeted region in the mRNA, and hence the sequence in the siRNA duplex, are chosen using the following guidelines. The open reading frame (ORF) region from the cDNA sequence is recommended for targeting, preferably at least 75 to 100 nucleotides downstream of the start codon. Both the 5’ and 3’ untranslated regions (UTRs) and regions near the start codon are not recommended for targeting as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex. As research into siRNA and RNAi continues, these parameters are expected to be further refined and optimized.

The sequence of the siRNA is selected as follows:

1. Start 75 bases downstream from the start codon.

2. Locate the first AA dimer.

3. Record the next 19 nucleotides following the AA dimer.

4. Calculate the percentage of guanosines and cytidines (G/C content) of the AA-N₁₉ 21-base sequence. Ideally the G/C content is ~50% but it must be less than 70% and greater than 30%. If the sequence does not meet this criteria, the search continues downstream to the next AA dimer until this condition is met.

5. The 21-base sequence is subjected to a BLAST-search (NCBI database) against EST libraries to ensure that only one gene is targeted. (The complement is automatically searched as well.)

6. If the conditions in either step 4 or 5 are not met, repeat steps 2 - 5.⁶

The sequence selection process has no other constraints. It is important to note that structure within the targeted mRNA appears to have minimal effect on the availability of the mRNA target and efficacy of the siRNA silencing approach. To-date, successful silencing has been achieved using the above method to select the target sequence, although the method is essentially random with respect to accounting for mRNA structure.

Although siRNA silencing appears to be extremely effective by selecting a single target in the mRNA, it may be desirable to design and employ two independent siRNA duplexes to control for specificity of the silencing effect.⁷ This recommendation is only for specificity. It is as yet unknown if the targeting of a gene by two different siRNA duplexes is more effective than using a single siRNA duplex. It is believed that the rate-limiting component of the siRNA effect is the availability of cellular nuclease components and not mRNA target availability. Therefore, doubling the number of siRNA duplexes is not expected to double the rate or efficiency of silencing.

If the selected siRNA duplex(es) do not function for silencing, the following steps are recommended. First, a search is conducted for sequencing errors in the gene and possible polymorphisms. Initial studies on the specificity of target recognition by siRNA duplexes indicates that a single point mutation located in the paired region of an siRNA duplex is sufficient to abolish target mRNA degradation. Second, a re-examination is performed to confirm whether the cell line is from the expected species. Third, a second and/or third target are selected and the corresponding siRNA duplexes prepared.

 

Preparation of siRNA Duplexes

Preparation of the siRNA duplex comprises synthesizing the two RNA oligonucleotides, 2’-deprotection and desalting, and annealing of the two strands.

Dharmacon Research offers four options for ordering siRNA oligonucleotides and siRNA duplexes. The following explains these options in detail.

Custom siRNA synthesis: Custom order 2 RNA oligos of any sequence and length separately using any combination of scales and services available at Dharmacon. Orders can be conveniently placed by any one of three methods: via our website (www.dharmacon.com), fax order form, or Custom RNA email template. When choosing custom synthesis, the following choices need to be made:

Scale: Using 2’-ACE RNA synthesis chemistry, Dharmacon produces the highest yields of RNA per micromole.⁸ Dharmacon’s 0.2 μmole scale will yield ~1.0 mg (150 nanomoles) of full length material in the unpurified synthesis. These high yields lead to enough siRNA duplex sufficient for use with approximately one hundred 24-well plates. Larger 0.4 and 1.0 μmole scales are also available.

Purification: Tuschl and co-workers have demonstrated that Dharmacon RNA is consistently of suitable quality to form active siRNA duplexes without the need for isolating the full-length RNA oligo product. All Dharmacon RNA oligos are analyzed for purity and, for the average 21-base RNA oligonucleotide, must be >80% pure to pass. Further purification may be chosen to obtain the highest quality possible, >97%.

2’-Deprotection and Desalting: Dharmacon supplies RNA oligos with the 2’-hydroxyls protected in the 2’-ACE form⁷ to ensure the RNA is as stable as possible throughout handling and shipping. The ACE groups are readily removed with a pH 3.8 aqueous buffer. Both the buffer and 2’-deprotection protocol are provided with every RNA order. The 2’-ACE groups are readily removed to yield the fully-deprotected 2’-OH RNA product in 30 minutes. Following 2’-deprotection, the RNAs must be desalted prior to use in transfection. Desalting can be performed by any one of numerous standard techniques, e.g. ethanol precipitation or reversed-phase cartridge desalting. 2’-Deprotection and desalting must be performed under strictly RNase-free, sterile conditions. 2’-Deprotection and desalting can be readily completed by the end user. (Protocols are readily available at www.dharmacon.com.) Alternatively, the 2’-deprotection/desalting option may be selected to be completed at Dharmacon.

　

Annealing of siRNA Oligos to form siRNA Duplex

Annealing of the RNA oligos is necessary if Custom Synthesis, Option A, or Option B is selected. (Option C provides the siRNA duplex ready to be used as described later in the transfection section.) It is critical that all handling steps be conducted under sterile, RNase-free conditions. Please refer to suitable references for further instructions.⁹ To anneal the RNAs, the oligos must first be quantified by UV absorption at 260 nanometers (nm). This is included in Option B and quantification is only necessary with Custom Synthesis and Option A. Please refer to Appendix D for details on quantifying RNA oligos via UV absorption. After quantifying the RNA oligos, the following protocol is used for annealing:

1. Separately aliquot and dilute each RNA oligo to a concentration of 50 μM.

2. Combine 30 μl of each RNA oligo solution and 15 μl of 5X annealing buffer. Final buffer concentration is: 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate. Final volume is 75 μl.

3. Incubate the solution for 1 minute at 90^o C, centrifuge the tube for 15 seconds, let sit for 1 hour at 37^o C, then use at ambient temperature. The solution can be stored frozen at -20^o C and freeze-thawed up to 5 times. The final concentration of siRNA duplex is 20 μMolar.

 

 

Transfection of Mammalian Cell Cultures with siRNA Duplexes

Tuschl and co-workers performed single transfections of siRNA duplexes using OLIGOFECTAMINE Reagent (product number: 12252011 from Life Technologies, now Invitrogen; www.invitrogen.com) and assayed for silencing 2 days after transfection. The guidelines for 24-well plate formats described in the 12252011.pdf file (see Invitrogen website) were followed. Transfection efficiencies of 90 - 95% were typically observed, although this may vary between cell lines. No silencing has been observed in the absence of transfection reagent.

The siRNA duplex is prepared for transfection as follows:

1. For each well of a 24-well plate, 60 pmole of siRNA duplex is used (3 μl of 20 μM annealed duplex).

2. Mix 3 μl of 20 μM siRNA duplex with 50 μl of Opti-MEM.

3. In a second tube, mix 3 μl of OLIGOFECTAMINE Reagent with 12 μl of Opti-MEM and incubate 7 to 10 minutes at room temperature.

4. Combine the two solutions from steps 2 and 3 and gently mix by inversion. Do not vortex.

5. After incubating another 20 to 25 minutes at room temperature; the solution turns turbid. Add 32 μl of fresh Opti-MEM to obtain a final volume of 100 μl.

6. Add the 100 μl of siRNA-OLIGOFECTAMINE to each well of cultured cells (40 to 50% confluent) in a 24-well plate. The cells were seeded the previous day in 24-well plates using 500 μl of DMEM tissue culture medium supplemented with 10% FBS and without antibiotics.

Tuschl and co-workers further report that transfection of 60 pmoles of single-stranded sense siRNA has no effect. Transfection with 60 pmoles of the complementary siRNA had a weak silencing effect when compared to 60 pmoles of the corresponding duplex siRNA. However, when the concentrations were reduced 100-fold, no silencing effect was apparent with the single-stranded sense or complementary RNA oligos, even though silencing was observed with the 100-fold diluted siRNA duplex. Therefore, future research may demonstrate it is possible to reduce the siRNA duplex concentration and still effect silencing.¹⁰

The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. Also, the time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) is critical. Please follow the instructions provided by the manufacturers.

Depending on the abundance and the life-time (turnover) of the targeted protein, a knock-down phenotype may become apparent after 1 to 3 days.¹⁰ In cases where no phenotype is observed, depletion of the protein may be assayed by immunofluorescence or Western blotting. If the protein is still abundant after 3 days, cells should be split and transferred for re-transfection. In the case of the lamin A/C knock-down (lamin A/C is not essential for cell viability), silencing was observed after 44 hours. The knock-down of lamin A/C persisted for more than 105 hours (6 generation times) but protein levels returned to normal levels after 170 hours incubation (10 generation times). This observation indicates that replication of siRNA duplexes may not occur in mammalian cells. It also appears that silencing does not spread to neighboring, non-transfected cells.¹⁰

 

Conclusion:

siRNA shows considerable promise in the study and application of mammalian gene expression. Dharmacon Research has produced this Technical Bulletin to facilitate the early use of this technology in advance of future experimental results and conclusions. The preceding protocols were prepared in collaboration with Dr. Tom Tuschl, Dr. Sayda Elbashir, Dr. Phillip Zamore, Dr. David Bartel and Dr. Phillip Sharp, and are based only on results reported through June 2001. As research and investigation continues into the study of siRNA and RNAi effects, these protocols are expected to be adjusted and changed as required. Pre-made kits of siRNA duplexes for common sequence targets and suitable controls will be available in the future as determined by feedback from Dharmacon’s customers and collaborators. Please feel free to contact Dharmacon with suggestions or requests. Finally, Dharmacon expresses its sincere gratitude to Dr. Tuschl and co-workers for their generous contributions to this Technical Bulletin.

 

References and Footnotes:

1. (a) S. M. Hammond, A. A. Caudy and G J. Hannon, "Post-Transcriptional Gene-Silencing by Double-Stranded RNA," Nature, p110-119 (2001). (b) P. A. Sharp, "RNA interference - 2001" Genes Dev., 15, p485-490 (2001).

2. S. M. Elbashir, W. Lendeckel, T. Tuschl, "RNA interference is mediated by 21- and 22-nucleotide RNAs." Genes Dev., 15, p188-200 (2001).

3. S. M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells." Nature, 411, p494-498 (2001).

4. Additional detail can be found at the following website set-up by Dr. Tuschl: www.mpibpc.gwdg.de/abteilungen/100/105/.

5. (a) T. Tuschl, P. D. Zamore, R. Lehmann, D. P. Bartel, P. A. Sharp, "Targeted mRNA degradation by double-stranded RNA in vitro." Genes Dev., 13, p3191-3197 (1999). (b) P. D. Zamore, T. Tuschl, P. A. Sharp, D. P. Bartel, "RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals," Cell, 101, p25-33 (2000).

6. If no suitable AA-N19^TM target is identified, search for a suitable CA-N19 target and contact Dharmacon Research for further instructions.

7. T. Tuschl, personal communication.

8. S. A. Scaringe, F. E. Wincott and M. H. Caruthers, J. Am. Chem. Soc., 129, p11820-11821 (1998).

9. (a) J. Sambrook, E.F. Fritsch, T. Maniatis, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor, NY: Cold Spring Harbor Press (1989). (b) F.M. Ausubel, et al, "Current Protocols in Molecular Biology," New York: Greene Pub. Associates and Wiley-Interscience (1987).

10. S. M. Elbashir and T. Tuschl: unpublished results, manuscript in preparation.

 

Appendix A

パッケ?ジA：

一般的なAA-N19?20?21のmRNAをタ?ゲットとした配列の供給
個?のsiRNAをご希望の合成スケ?ルで受注
合成されたオリゴは1本鎖の?態で配送
オリゴは2’の?保護????アニ?リング?理が必要です。
必要に?じて精製?理も行なってください。

パッケ?ジB：

一般的なAA-N19?20?21mのRNAをタ?ゲットとした配列の供給
個?のsiRNAをご希望の合成スケ?ルで受注
2’の?保護???は逆相クロマトグラフィ?によって?理?み
アニ?リング?理?み
必要に?じて精製?理も行なってください。

パッケ?ジC：

一般的なAA-N19?20?21のmRNAをタ?ゲットとした配列の供給
個?のsiRNAをご希望の合成スケ?ルで受注
オリゴは精製し、アニ?リング?理し、すぐにトランスフェクションできる2本鎖の?態で配送(精製とアニ?リング?理は最適?件で行なわれます。)
オリゴは97%以上に精製後、最終濃度が20-80uM溶液となるように凍結乾燥してお?けいたします。

　※各種修飾基、????保護等のオプションとの組み合わせも可能です。
　　(RNAカスタム合成の?格表をご?照ください。)

　

Appendix B - Pricing for siRNA Options

Please see siRNA Pricing Guide for current prices.

Appendix C - Delivery Times for siRNA Options

Q&A.

　

Appendix D - Protocol for the Quantification of RNA Oligonucleotides

Materials for each oligonucleotide sample:

Supplies:

1. Variable wavelength UV spectrophotometer

2. 0.5 mL Quartz cuvettes

3. 1.5 mL microfuge tubes

Solutions:

1. 7 M urea, buffered with 1M TrisCl, pH 7.0

2. Molecular biology grade water.

Procedure:

1. Pipet ultra-pure water into the sample so that the final volume is 1.00 ml to provide the Stock Solution.
2. Dilute 10 mL sample into 990 ml of buffered 7 M urea at room temperature to provide the 100X Dilution Sample.
3. Blank the UV spec at 260 nm with 450 mL of buffered 7M urea at room temperature.
4. Rinse the cell out with 450 ml of 100X dilution sample, and discard.
5. Again fill the cell with 450 m l of 100X dilution sample.
6. Record the absorbance reading at 260 nm. To safely remain within the linear range of most photomultiplier tubes, assure that the value is between 0.2 and 1.0 absorbance units.
7. Calculations:
8.