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Make siRNA by T7 Transcription

The first T7 transcription method was reported by Olivier Donzã© and Didier Picard in Nucleic Acids Research, 30:e46, 2002 and the following figure was used by them for illustrating the method:

Briefly, the strategy they used to generate siRNAs in vitro is: an 18mer oligonucleotide encompassing the T7 promoter is annealed to a 38mer (39mer) oligonucleotide with the complementary sequence of the T7 promoter downstream of the target sequence preceded by two additional nucleotides (reading the sequence 5'3').

The transcribed sequence is 19 nt (20 nt) plus 2 nt, which can be any nucleotides in the case of the sense RNA but must be complementary nucleotides in the antisense RNA, since it has been shown that the antisense RNA of the siRNA guides target recognition.

It is noteworthy that T7 RNA polymerase can transcribe a template where only the promoter is double stranded. The last guanosine of the T7 promoter is the first ribonucleotide that is incorporated into the RNA by the T7 RNA polymerase during transcription and, therefore, all siRNAs designed by this method will start with a G. Thus, the design of T7 siRNA requires that the sequence starts with a G and has a C at position 19 (position 20) to allow annealing with the complementary RNA, which also starts with a G. This G-N17 (N18)-C rule does not restrict the T7 siRNA design since this sequence is frequently found in any gene (on average about five times in a random sequence of 100 bp).

To obtain dsRNA for RNAi, only two DNA oligonucleotides corresponding to the sense and antisense sequences of the target gene have to be ordered. The T7 promoter oligonucleotide is invariant and common to any target gene. Following transcription reactions, sense and antisense transcripts are annealed and ethanol precipitated, yielding what they refer to as T7 siRNAs.

Detailed procedure for T7 siRNAs

1. Numbering sense coding sequence of target gene (N1, N2, N22, N23, are numbered positions)
2. Find 5'-N1 N2 G/A G/A NNNNNNNNNNNNNNN C C N22 N23-3'
3. Drop N22 and N23 first
   add 5'-TATAGTGAGTCGTATTA-3' (T7 Promoter) to 3' END
   to get 5'-N1 N2 G/A G/A NNNNNNNNNNNNNNN C C TATAGTGAGTCGTATTA-3' = oligo A
4. From (2) 5'-N1 N2 G/A G/A NNNNNNNNNNNNNNN C C N22 N23-3' drop N1 N2 convert G/A G/A to G G to get 5'-G G NNNNNNNNNNNNNNN C C N22 N23-3'
5. Add 5'-TAATACGACTCACTATA-3' to 5'END to get 5'-TAATACGACTCACTATA G G NNNNNNNNNNNNNNN C C N22 N23-3'
6. Get reverse complement of (5) 5'-N23' N22' G G NNNNNNNNNNNNNNN' C C TATAGTGAGTCGTATTA-3' = oligo B
7. Rank the initial target sequences with GG preferable to G G/A preferable to G/A G with A A not even considered unless absolutely necessary. GG N15 CC > G G/A N15 CC > G/A G N15 CC >>>>>>>>> A A N15 CC
8. Order oligos II) and make RNA in vitro
9. Anneal oligo A and oligo B separately to T7 primer (TAATACGACTCACTATAGG) or to primers that are complementary to oligoA and B
10. Use 2-3 ug annealed primer in a 20ul Ambion T7 Megashort script reaction Ambion Cat# 1354 (Follow Ambion's protocol Incubate 2-4 hrs)
11. Combine oligo A and B reactions

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