TROD (T7 RNAi Oligo Designer) is a web application that facilitates the design of DNA oligonucleotides
for the synthesis of short interfering RNAs (siRNAs) with T7 RNA polymerase. We highly recommend reading
the following references that describe and review the technique in detail (go to the References section
for more information): Donzé and Picard (2002),
Donzé et. al, (2004).
In brief: TROD takes a cDNA sequence as input and automatically generates the appropriate DNA oligos for ordering.
The output is organized so as to make it easy for the user to choose appropriate target sequences.
In detail: TROD (by default) looks for all occurrences of the sequence N2GN18C. The G and C nucleotides are required
for efficient synthesis of the RNA by T7 RNA polymerase, since it strongly favours a G at the start site. From these sequences,
the program generates both the sense and antisense strands of DNA oligos that will be used to produce the siRNAs in vitro. By definition,
the 'antisense' DNA oligo encodes the antisense siRNA strand, and vice versa.
The following (reverse complemented) T7 promoter is appended to the 3' ends of the DNA oligos: 5'-TATAGTGAGTCGTATTA-3'.
After T7 transcription, the siRNA duplexes will look like the following:
sense siRNA: 5'-GNNNNNNNNNNNNNNNNNNUUU-3'
antisense siRNA: 3'-NNCNNNNNNNNNNNNNNNNNNG-5'
Important note: TROD, by default, appends an AA dinucleotide to the 5' end of the DNA 'sense' strand, since AT-richness is preferred
at this site. The result (as shown above) is a UU dinucleotide overhang on the sense siRNA after T7 transcription. In addition, in order to destabilize
the 5' end of the antisense siRNA (so as to direct it to the RISC complex), a U replaces the complementary C, producing a GU pair.
Users have the possibility of inputing a cDNA sequence in three different ways. Clicking on the submit button will launch
the program. Details and limitations for each input are described below:
GenBank Accession Code - All GenBank accession codes are supported apart from the ones
starting with "NT_", since NT contigs contain references to clones, rather than sequences. In the future, this will
be remedied.
Sequence File - Any cDNA sequence in FASTA format is supported. Text files containing a
single sequence are also supported. In the future, many more file formats will be supported.
cDNA Sequence - A large text field is provided for pasting sequences directly.
Several options have been made available. They are described below.
siRNA length - this refers to the length of the in vitro transcribed siRNA, which ranges from 21-24 nucleotides. The default
value is 22 nt, the length that we recommend.
Sort by - the user can choose to sort the output either by GC content (default) or by the location of the target sequence
in the cDNA.
Here is a screenshot of a sample Results Page, with explanations for each column below.
ID - each pair of oligos is given a unique identification number
DNA 'antisense' + T7 - this is the DNA oligo (with appended reverse-complemented-T7 promoter) that will be used to generate the interfering
(in other words, the 'antisense') siRNA strand
DNA 'sense' + T7 -this is the DNA oligo (with appended reverse-complemented-T7 promoter) that will be used to generate the 'sense' siRNA strand
Location - the location in your cDNA sequence input of the target sequence (see "Sequence" column)
% GC - the calculated GC content of the target sequence (see "Sequence" column)
Sequence - the sequence in your cDNA that will be targeted by the siRNA duplex
Blast - provides a link to BLAST the targeted sequence (from the "Sequence" column). It is highly recommended to BLAST
your chosen sequences to ensure target specificity
Highlighted Rows
- the DNA 'antisense' oligos that produce an AT-rich 3'-dinucleotide overhang on the antisense siRNA strand
as well as an AT-rich internal dinucleotide sequence adjacent to the 5' G are highlighted, since they are the most desired
The results table has been organized to make it convenient for the user to choose ready-to-order oligos. The list of oligos is sorted by increasing GC content (by default), while those
that have an AT-rich 5'-dinucleotide on the DNA 'antisense' oligo (as well as AT-richness adjacent to the 3' C) have been highlighted (in other words, the DNA
oligos that produce the following antisense siRNA: 5'-GW2N17-20CW2-3', where W = A or T).
Although there are no well defined criteria for choosing the "right" oligos, the following guidelines and recommendations should help:
choose oligos with GC contents of 30-50% (below or higher have been known to work but the probability is lower)
choose oligos with an AT-rich 5'-dinucleotide on the 'antisense' DNA strand as well as AT-richness near the 3' C (these have been highlighted!)
try to avoid long stretches (>3) of any single nucleotide (but especially G)
remember to always BLAST the sequence to ascertain target specificity!
The in vitro transcription step requires 3 oligos: [1] DNA 'antisense' oligo, [2] DNA 'sense' oligo, [3] T7 promoter. Ordering is as simple
as copying and pasting the sequences directly from the Results Page. The sequence of the required T7 promoter oligo is shown below:
This article is the original description of the technique:
Donzé, O. and Picard, D. (2002) RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Research,
30 (10): e46 [Pubmed Link]
This article reviews the technique, and includes very detailed protocols:
Donzé, O., Dudek, P., and Picard, D. (2004) siRNA production by in vitro transcription. In: Gene Silencing by RNA Interference: Technology and Application, M. Sohail, ed. (CRC Press LLC).
Why are there so few (or no) highlighted oligos in the output?
There are a few reasons why this could occur. The most common reason is that your sequence
is too GC-rich. Since the highlighting "algorithm" favours AT-richness in certain regions of the oligo, the highlighting is biased towards sequences that have more A/T's. However, all is not lost.
One thing that may help increase the number of highlighted oligos is changing the length of the siRNA at the options section of the program page. Another thing one should keep in mind,
however, is that just because it's not highlighted, doesn't mean it won't work! The oligos don't necessarily need to meet all of the guidelines outlined in the "Interpreting Results" section. Many
"unhighlighted" oligos could still produce functional siRNAs.
Why don't the highlighted oligos print in colour?
This is due to your browser settings. By default, most browsers set the 'printing background colours' option
to off. You need to enable the printing of background colours to see the highlighted oligos in prints.
Can you please add feature/option X to the program?
If there's sufficient demand (or the change is in the best interests of the user), we may add it.
I found a bug - what should I do?
Please let us know! Email the relevant information (including how to reproduce it) to the author (dudek at cellbio.unige.ch) of the program.
