sirna.txt

emboss的linux版本的源代码

                                   sirna 



Function

   Finds siRNA duplexes in mRNA

Description

   RNA interference, or RNAi, is a phenomenon in which double stranded
   RNA (dsRNA) effects silencing of the expression of genes that are
   highly homologous to either of the RNA strands in the duplex. Gene
   silencing in RNAi results from the degradation of mRNA sequences, and
   the effect has been used to determine the function of many genes in
   Drosophilia, C. elegans, and many plant species.

   The duration of knockdown by siRNA can typically last for 7-10 days,
   and has been shown to transfer to daughter cells. Of further note,
   siRNAs are effective at quantities much lower than alternative gene
   silencing methodologies, including antisense and ribozyme based
   strategies.

   Due to various mechanisms of antiviral response to long dsRNA, RNAi at
   first proved more difficult to establish in mammalian species. Then,
   Tuschl, Elbashir, and others discovered that RNAi can be elicited very
   effectively by well-defined 21-base duplex RNAs. When these small
   interfering RNA, or siRNA, are added in duplex form with a
   transfection agent to mammalian cell cultures, the 21-base-pair RNA
   acts in concert with cellular components to silence the gene with
   sequence homology to one of the siRNA sequences. Strategies for the
   design of effective siRNA sequences have been recently documented,
   most notably by Sayda Elbashir, Thomas Tuschl, et al.

   Their studies of mammalian RNAi suggest that the most efficient
   gene-silencing effect is achieved using double-stranded siRNA having a
   19-nucleotide complementary region and a 2-nucleotide 3' overhang at
   each end. Current models of the RNAi mechanism suggest that the
   antisense siRNA strand recognizes the specific gene target.

   In gene-specific RNAi, the coding region (CDS) of the mRNA is usually
   targeted. The search for an appropriate target sequence should begin
   50-100 nucleotides downstream of the start codon. UTR-binding proteins
   and/or translation initiation complexes may interfere with the binding
   of the siRNP endonuclease complex. Tuschl, Elbashir et al. say that
   they have successfully used siRNAs targetting the 3' UTR.

   To avoid interference from mRNA regulatory proteins, sequences in the
   5' untranslated region or near the start codon should not be targeted.

   A set of rules for the design of siRNA has been suggested
   http://www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html based on the
   work of Tuschl, Elbashir et al.

   They suggest searching for 23-nt sequence motif AA(N19)TT (N, any
   nucleotide) and select hits with approx. 50% G/C-content (30% to 70%
   has also worked in for them). If no suitable sequences are found, the
   search is extended using the motif NA(N21). The sequence of the sense
   siRNA corresponds to (N19)TT or N21 (position 3 to 23 of the 23-nt
   motif), respectively. In the latter case, they convert the 3' end of
   the sense siRNA to TT.

   The rationale for this sequence conversion is to generate a symmetric
   duplex with respect to the sequence composition of the sense and
   antisense 3' overhangs. The antisense siRNA is synthesized as the
   complement to position 1 to 21 of the 23-nt motif. Because position 1
   of the 23-nt motif is not recognized sequence-specifically by the
   antisense siRNA, the 3'-most nucleotide residue of the antisense
   siRNA, can be chosen deliberately. However, the penultimate nucleotide
   of the antisense siRNA (complementary to position 2 of the 23-nt
   motif) should always be complementary to the targeted sequence. For
   simplifying chemical synthesis, they always use TT.

   More recently, they preferentially select siRNAs corresponding to the
   target motif NAR(N17)YNN, where R is purine (A, G) and Y is pyrimidine
   (C, U). The respective 21-nt sense and antisense siRNAs therefore
   begin with a purine nucleotide and can also be expressed from pol III
   expression vectors without a change in targeting site; expression of
   RNAs from pol III promoters is only efficient when the first
   transcribed nucleotide is a purine.

   They always design siRNAs with symmetric 3' TT overhangs, believing
   that symmetric 3' overhangs help to ensure that the siRNPs are formed
   with approximately equal ratios of sense and antisense target
   RNA-cleaving siRNPs Please note that the modification of the overhang
   of the sense sequence of the siRNA duplex is not expected to affect
   targeted mRNA recognition, as the antisense siRNA strand guides target
   recognition. In summary, no matter what you do to your overhangs,
   siRNAs should still function to a reasonable extent. However, using TT
   in the 3' overhang will always help your RNA synthesis company to let
   you know when you accidentally order a siRNA sequences 3' to 5' rather
   than in the recommended format of 5' to 3'.

   sirna reports both the sense and antisense siRNAs as 5' to 3'.

   Xeragon.com also suggest that choosing a region of the mRNA with a GC
   content as close as possible to 50% is a more important consideration
   than choosing a target sequence that begins with AA. They also suggest
   that a key consideration in target selection is to avoid having more
   than three guanosines in a row, since poly G sequences can hyperstack
   and form agglomerates that potentially interfere with the siRNA
   silencing mechanism.

   siRNAs appear to effectively silence genes in more than 80% of cases.
   Current data indicate that there are regions of some mRNAs where gene
   silencing does not work. To help ensure that a given target gene is
   silenced, it is advised that at least two target sequences as far
   apart on the gene as possible be chosen.

   mRNA secondary structure does not appear to have a significant effect
   on gene silencing.

  Coding region specification

   It is possible (although the evidence is not clear) that regulatory
   protein binding to regions in and near the untranslated 5' region
   might interfere with the RNAi process.

   Therefore, this program avoids choosing siRNA probes from the 5' UTR
   and from the first 50 bases of the coding region. The second 50 bases
   of the coding region has a penalty associated with it to reduce the
   reporting of possible siRNA probes in this region.

   If the input sequence has a feature table specifying a coding region,
   then this will be used, else you can specify the start of the coding
   region, where this is known by the '-sbegin' command-line qualifier
   (which is normally used to specify the start of the region of a
   sequence that should be analysed in all EMBOSS programs).

   sirna looks at the feature table of the input mRNA sequence to find
   the coding regions (CDS). It will ignore the 5' UTR and the first 50
   bases of the CDS. It will assign a penalty of 2 points to any siRNA in
   positions 51 to 100 in the CDS. If there is no CDS in the feature
   table, you can specify the CDS by using the command-line qualifier
   '-sbegin' to indicate where the CDS should start. If there is no CDS
   in the feature table and you do not use the command-line qualifier
   '-sbegin', then sirna will assume that the CDS region is not known and
   will look for siRNAs in the whole of the sequence with no penalties
   associated with the location within the sequence.

  All these confusing regions

   There are a lot of references to 23 base regions, 21 base regions, 19
   base regions, etc. in any description of siRNA.

   Perhaps an example with a sequence would be clearer?

   The 23 base region, in this case starting with an 'AA', might
   typically look like:

5' AAGUGAGAGGUCAGACUCCUATC

   The sense siRNA is made from the 19 bases of positions 3 to 21 of the
   23 base target region, so:

5'   GUGAGAGGUCAGACUCCUA

   and then typically d(TT) is added, so:

5'   GUGAGAGGUCAGACUCCUAdTdT

   The antisense siRNA sequence is made from bases 3 to 21 of the target
   region, so:

5'   GUGAGAGGUCAGACUCCUA sense
3'   CACUCUCCAGUCUGAGGAU antisense 3' -> 5'

   so the antisense sequence that should be ordered with d(TT) added is:

5'   UAGGAGUCUGACCUCUCACdTdT antisense 5' -> 3'

Algorithm

for each input sequence:

    find the start position of the CDS in the feature table
    if there is no such CDS, take the -sbegin position as the CDS start

    for each 23 base window along the sequence:

        set the score for this window = 0
        if base 2 of the window is not 'a': ignore this window
        if the window is within 50 bases of the CDS start: ignore this window
        if the window is within 100 bases of the CDS: score = -2
        measure the %GC of the 20 bases from position 2 to 21 of the window
        for the following %GC values change the score:
                %GC <= 25% (<= 5 bases): ignore this window
                %GC 30% (6 bases): score + 0
                %GC 35% (7 bases): score + 2
                %GC 40% (8 bases): score + 4
                %GC 45% (9 bases): score + 5
                %GC 50% (10 bases): score + 6
                %GC 55% (11 bases): score + 5
                %GC 60% (12 bases): score + 4
                %GC 65% (13 bases): score + 2
                %GC 70% (14 bases): score + 0
                %GC >= 75% (>= 15 bases): ignore this window
        if the window starts with a 'AA': score + 3
        if the window does not start 'AA' and it is required: ignore this windo
w
        if the window ends with a 'TT': score + 1
        if the window does not end 'TT' and it is required: ignore this window
        if 4 G's in a row are found: ignore this window
        if any 4 bases in a row are present and not required: ignore this windo
w
        if PolIII probes are required and the window is not NARN(17)YNN: ignore
 this window
        if the score is > 0: store this window for output

    sort the windows found by their score
    output the 23-base windows to the sequence file
    if the 'context' qualifier is specified, output window bases 1 and 2 in bra
ckets to the report file
    take the window bases 3 to 21, add 'dTdT' output to the report file
    take the window bases 3 to 21, reverse complement, add 'dTdT' output to the
 report file

Usage

   Here is a sample session with sirna


% sirna 
Finds siRNA duplexes in mRNA
Input nucleotide sequence(s): tembl:hsfau
Output report [hsfau.sirna]: 
output sequence(s) [hsfau.fasta]: 

   Go to the input files for this example
   Go to the output files for this example

   Example 2

   Show the first two bases of the 23 base target region in brackets.
   These do not form part of the sequence to be ordered, but it is useful
   to see if the 23 base region starts with an 'AA'.


% sirna -context 
Finds siRNA duplexes in mRNA
Input nucleotide sequence(s): tembl:hsfau
Output report [hsfau.sirna]: 
output sequence(s) [hsfau.fasta]: 

   Go to the output files for this example

Command line arguments

   Standard (Mandatory) qualifiers:
  [-sequence]          seqall     Nucleotide sequence(s) filename and optional
                                  format, or reference (input USA)
  [-outfile]           report     [*.sirna] The output is a table of the
                                  forward and reverse parts of the 21 base
                                  siRNA duplex. Both the forward and reverse
                                  sequences are written 5' to 3', ready to be
                                  ordered. The last two bases have been
                                  replaced by 'dTdT'. The starting position of
                                  the 23 base region and the %GC content is
                                  also given. If you wish to see the complete
                                  23 base sequence, then either look at the
                                  sequence in the other output file, or use
                                  the qualifier '-context' which will display
                                  the 23 bases of the forward sequence in this
                                  report withthe first two bases in brackets.
                                  These first two bases do not form part of
                                  the siRNA probe to be ordered.
  [-outseq]            seqoutall  [.] This is a file of the
                                  sequences of the 23 base regions that the
                                  siRNAs are selected from. You may use it to
                                  do searches of mRNA databases (e.g. REFSEQ)
                                  to confirm that the probes are unique to the
                                  gene you wish to use it on.

   Additional (Optional) qualifiers:
   -poliii             boolean    [N] This option allows you to select only
                                  the 21 base probes that start with a purine
                                  and so can be expressed from Pol III
                                  expression vectors. This is the NARN(17)YNN
                                  pattern that has been suggested by Tuschl et
                                  al.
   -aa                 boolean    [N] This option allows you to select only
                                  those 23 base regions that start with AA. If
                                  this option is not selected then regions
                                  that start with AA will be favoured by
                                  giving them a higher score, but regions that
                                  do not start with AA will also be reported.
   -tt                 boolean    [N] This option allows you to select only
                                  those 23 base regions that end with TT. If
                                  this option is not selected then regions
                                  that end with TT will be favoured by giving
                                  them a higher score, but regions that do not
                                  end with TT will also be reported.
   -[no]polybase       boolean    [Y] If this option is FALSE then only those
                                  23 base regions that have no repeat of 4 or
                                  more of any bases in a row will be reported.
                                  No regions will ever be reported that have
                                  4 or more G's in a row.
   -context            boolean    [N] The output report file gives the
                                  sequences of the 21 base siRNA regions ready
                                  to be ordered. This does not give you an
                                  indication of the 2 bases before the 21
                                  bases. It is often interesting to see which
                                  of the suggested possible probe regions have
                                  an 'AA' in front of them (i.e. it is useful
                                  to see which of the 23 base regions start
                                  with an 'AA'). This option displays the
                                  whole 23 bases of the region with the first
                                  two bases in brackets, e.g. '(AA)' to give
                                  you some context for the probe region. YOU
                                  SHOULD NOT INCLUDE THE TWO BASES IN BRACKETS
                                  WHEN YOU PLACE AN ORDER FOR THE PROBES.