
                                   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:x65923
Output report [x65923.sirna]: 
output sequence(s) [x65923.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:x65923
Output report [x65923.sirna]: 
output sequence(s) [x65923.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.

   Advanced (Unprompted) qualifiers: (none)
   Associated qualifiers:

   "-sequence" associated qualifiers
   -sbegin1            integer    Start of each sequence to be used
   -send1              integer    End of each sequence to be used
   -sreverse1          boolean    Reverse (if DNA)
   -sask1              boolean    Ask for begin/end/reverse
   -snucleotide1       boolean    Sequence is nucleotide
   -sprotein1          boolean    Sequence is protein
   -slower1            boolean    Make lower case
   -supper1            boolean    Make upper case
   -sformat1           string     Input sequence format
   -sdbname1           string     Database name
   -sid1               string     Entryname
   -ufo1               string     UFO features
   -fformat1           string     Features format
   -fopenfile1         string     Features file name

   "-outfile" associated qualifiers
   -rformat2           string     Report format
   -rname2             string     Base file name
   -rextension2        string     File name extension
   -rdirectory2        string     Output directory
   -raccshow2          boolean    Show accession number in the report
   -rdesshow2          boolean    Show description in the report
   -rscoreshow2        boolean    Show the score in the report
   -rusashow2          boolean    Show the full USA in the report
   -rmaxall2           integer    Maximum total hits to report
   -rmaxseq2           integer    Maximum hits to report for one sequence

   "-outseq" associated qualifiers
   -osformat3          string     Output seq format
   -osextension3       string     File name extension
   -osname3            string     Base file name
   -osdirectory3       string     Output directory
   -osdbname3          string     Database name to add
   -ossingle3          boolean    Separate file for each entry
   -oufo3              string     UFO features
   -offormat3          string     Features format
   -ofname3            string     Features file name
   -ofdirectory3       string     Output directory

   General qualifiers:
   -auto               boolean    Turn off prompts
   -stdout             boolean    Write standard output
   -filter             boolean    Read standard input, write standard output
   -options            boolean    Prompt for standard and additional values
   -debug              boolean    Write debug output to program.dbg
   -verbose            boolean    Report some/full command line options
   -help               boolean    Report command line options. More
                                  information on associated and general
                                  qualifiers can be found with -help -verbose
   -warning            boolean    Report warnings
   -error              boolean    Report errors
   -fatal              boolean    Report fatal errors
   -die                boolean    Report dying program messages

Input file format

   sirna reads any normal sequence USAs.

  Input files for usage example

   'tembl:x65923' is a sequence entry in the example nucleic acid
   database 'tembl'

  Database entry: tembl:x65923

ID   X65923; SV 1; linear; mRNA; STD; HUM; 518 BP.
XX
AC   X65923;
XX
DT   13-MAY-1992 (Rel. 31, Created)
DT   18-APR-2005 (Rel. 83, Last updated, Version 11)
XX
DE   H.sapiens fau mRNA
XX
KW   fau gene.
XX
OS   Homo sapiens (human)
OC   Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia
;
OC   Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae;
OC   Homo.
XX
RN   [1]
RP   1-518
RA   Michiels L.M.R.;
RT   ;
RL   Submitted (29-APR-1992) to the EMBL/GenBank/DDBJ databases.
RL   L.M.R. Michiels, University of Antwerp, Dept of Biochemistry,
RL   Universiteisplein 1, 2610 Wilrijk, BELGIUM
XX
RN   [2]
RP   1-518
RX   PUBMED; 8395683.
RA   Michiels L., Van der Rauwelaert E., Van Hasselt F., Kas K., Merregaert J.;
RT   " fau cDNA encodes a ubiquitin-like-S30 fusion protein and is expressed as
RT   an antisense sequences in the Finkel-Biskis-Reilly murine sarcoma virus";
RL   Oncogene 8(9):2537-2546(1993).
XX
DR   H-InvDB; HIT000322806.
XX
FH   Key             Location/Qualifiers
FH
FT   source          1..518
FT                   /organism="Homo sapiens"
FT                   /chromosome="11q"
FT                   /map="13"
FT                   /mol_type="mRNA"
FT                   /clone_lib="cDNA"
FT                   /clone="pUIA 631"
FT                   /tissue_type="placenta"
FT                   /db_xref="taxon:9606"
FT   misc_feature    57..278
FT                   /note="ubiquitin like part"
FT   CDS             57..458
FT                   /gene="fau"
FT                   /db_xref="GDB:135476"
FT                   /db_xref="GOA:P35544"
FT                   /db_xref="GOA:P62861"
FT                   /db_xref="HGNC:3597"
FT                   /db_xref="UniProtKB/Swiss-Prot:P35544"
FT                   /db_xref="UniProtKB/Swiss-Prot:P62861"
FT                   /protein_id="CAA46716.1"
FT                   /translation="MQLFVRAQELHTFEVTGQETVAQIKAHVASLEGIAPEDQVVLLA
G
FT                   APLEDEATLGQCGVEALTTLEVAGRMLGGKVHGSLARAGKVRGQTPKVAKQEKKKKKT
G
FT                   RAKRRMQYNRRFVNVVPTFGKKKGPNANS"
FT   misc_feature    98..102
FT                   /note="nucleolar localization signal"
FT   misc_feature    279..458
FT                   /note="S30 part"
FT   polyA_signal    484..489
FT   polyA_site      509
XX
SQ   Sequence 518 BP; 125 A; 139 C; 148 G; 106 T; 0 other;
     ttcctctttc tcgactccat cttcgcggta gctgggaccg ccgttcagtc gccaatatgc        6
0
     agctctttgt ccgcgcccag gagctacaca ccttcgaggt gaccggccag gaaacggtcg       12
0
     cccagatcaa ggctcatgta gcctcactgg agggcattgc cccggaagat caagtcgtgc       18
0
     tcctggcagg cgcgcccctg gaggatgagg ccactctggg ccagtgcggg gtggaggccc       24
0
     tgactaccct ggaagtagca ggccgcatgc ttggaggtaa agttcatggt tccctggccc       30
0
     gtgctggaaa agtgagaggt cagactccta aggtggccaa acaggagaag aagaagaaga       36
0
     agacaggtcg ggctaagcgg cggatgcagt acaaccggcg ctttgtcaac gttgtgccca       42
0
     cctttggcaa gaagaagggc cccaatgcca actcttaagt cttttgtaat tctggctttc       48
0
     tctaataaaa aagccactta gttcagtcaa aaaaaaaa                               51
8
//

Output file format

   The output is a standard EMBOSS report file.

   The results can be output in one of several styles by using the
   command-line qualifier -rformat xxx, where 'xxx' is replaced by the
   name of the required format. The available format names are: embl,
   genbank, gff, pir, swiss, trace, listfile, dbmotif, diffseq, excel,
   feattable, motif, regions, seqtable, simple, srs, table, tagseq

   See: http://emboss.sf.net/docs/themes/ReportFormats.html for further
   information on report formats.

   sirna outputs a report format file. The default format is 'table'

  Output files for usage example

  File: x65923.sirna

########################################
# Program: sirna
# Rundate: Sun 15 Jul 2007 12:00:00
# Commandline: sirna
#    -sequence tembl:x65923
# Report_format: table
# Report_file: x65923.sirna
########################################

#=======================================
#
# Sequence: X65923     from: 1   to: 518
# HitCount: 85
#
# CDS region found in feature table starting at 57
#
#=======================================

  Start     End   Score    GC% Sense_siRNA Antisense_siRNA
    308     330   9.000   50.0 AAGUGAGAGGUCAGACUCCdTdT GGAGUCUGACCUCUCACUUdTdT
    309     331   9.000   50.0 AGUGAGAGGUCAGACUCCUdTdT AGGAGUCUGACCUCUCACUdTdT
    310     332   9.000   50.0 GUGAGAGGUCAGACUCCUAdTdT UAGGAGUCUGACCUCUCACdTdT
    351     373   9.000   50.0 GAAGAAGAAGACAGGUCGGdTdT CCGACCUGUCUUCUUCUUCdTdT
    166     188   8.000   55.0 GAUCAAGUCGUGCUCCUGGdTdT CCAGGAGCACGACUUGAUCdTdT
    279     301   8.000   55.0 AGUUCAUGGUUCCCUGGCCdTdT GGCCAGGGAACCAUGAACUdTdT
    330     352   8.000   55.0 GGUGGCCAAACAGGAGAAGdTdT CUUCUCCUGUUUGGCCACCdTdT
    354     376   8.000   55.0 GAAGAAGACAGGUCGGGCUdTdT AGCCCGACCUGUCUUCUUCdTdT
    357     379   8.000   55.0 GAAGACAGGUCGGGCUAAGdTdT CUUAGCCCGACCUGUCUUCdTdT
    393     415   8.000   55.0 CCGGCGCUUUGUCAACGUUdTdT AACGUUGACAAAGCGCCGGdTdT
    253     275   7.000   60.0 GUAGCAGGCCGCAUGCUUGdTdT CAAGCAUGCGGCCUGCUACdTdT
    280     302   7.000   60.0 GUUCAUGGUUCCCUGGCCCdTdT GGGCCAGGGAACCAUGAACdTdT
    339     361   7.000   40.0 ACAGGAGAAGAAGAAGAAGdTdT CUUCUUCUUCUUCUCCUGUdTdT
    340     362   7.000   40.0 CAGGAGAAGAAGAAGAAGAdTdT UCUUCUUCUUCUUCUCCUGdTdT
    348     370   7.000   40.0 GAAGAAGAAGAAGACAGGUdTdT ACCUGUCUUCUUCUUCUUCdTdT
    375     397   7.000   60.0 GCGGCGGAUGCAGUACAACdTdT GUUGUACUGCAUCCGCCGCdTdT
    408     430   7.000   60.0 CGUUGUGCCCACCUUUGGCdTdT GCCAAAGGUGGGCACAACGdTdT
    429     451   7.000   60.0 GAAGAAGGGCCCCAAUGCCdTdT GGCAUUGGGGCCCUUCUUCdTdT
    432     454   7.000   60.0 GAAGGGCCCCAAUGCCAACdTdT GUUGGCAUUGGGGCCCUUCdTdT
    435     457   7.000   60.0 GGGCCCCAAUGCCAACUCUdTdT AGAGUUGGCAUUGGGGCCCdTdT
    488     510   7.000   40.0 AAAGCCACUUAGUUCAGUCdTdT GACUGAACUAAGUGGCUUUdTdT
    489     511   7.000   40.0 AAGCCACUUAGUUCAGUCAdTdT UGACUGAACUAAGUGGCUUdTdT
    490     512   7.000   40.0 AGCCACUUAGUUCAGUCAAdTdT UUGACUGAACUAAGUGGCUdTdT
    491     513   7.000   40.0 GCCACUUAGUUCAGUCAAAdTdT UUUGACUGAACUAAGUGGCdTdT
    129     151   6.000   55.0 GGCUCAUGUAGCCUCACUGdTdT CAGUGAGGCUACAUGAGCCdTdT
    165     187   6.000   50.0 AGAUCAAGUCGUGCUCCUGdTdT CAGGAGCACGACUUGAUCUdTdT
    278     300   6.000   50.0 AAGUUCAUGGUUCCCUGGCdTdT GCCAGGGAACCAUGAACUUdTdT
    314     336   6.000   50.0 GAGGUCAGACUCCUAAGGUdTdT ACCUUAGGAGUCUGACCUCdTdT
    321     343   6.000   50.0 GACUCCUAAGGUGGCCAAAdTdT UUUGGCCACCUUAGGAGUCdTdT
    323     345   6.000   50.0 CUCCUAAGGUGGCCAAACAdTdT UGUUUGGCCACCUUAGGAGdTdT
    329     351   6.000   50.0 AGGUGGCCAAACAGGAGAAdTdT UUCUCCUGUUUGGCCACCUdTdT


  [Part of this file has been deleted for brevity]

    353     375   5.000   55.0 AGAAGAAGACAGGUCGGGCdTdT GCCCGACCUGUCUUCUUCUdTdT
    360     382   5.000   65.0 GACAGGUCGGGCUAAGCGGdTdT CCGCUUAGCCCGACCUGUCdTdT
    374     396   5.000   55.0 AGCGGCGGAUGCAGUACAAdTdT UUGUACUGCAUCCGCCGCUdTdT
    383     405   5.000   55.0 UGCAGUACAACCGGCGCUUdTdT AAGCGCCGGUUGUACUGCAdTdT
    387     409   5.000   55.0 GUACAACCGGCGCUUUGUCdTdT GACAAAGCGCCGGUUGUACdTdT
    390     412   5.000   55.0 CAACCGGCGCUUUGUCAACdTdT GUUGACAAAGCGCCGGUUGdTdT
    392     414   5.000   55.0 ACCGGCGCUUUGUCAACGUdTdT ACGUUGACAAAGCGCCGGUdTdT
    407     429   5.000   55.0 ACGUUGUGCCCACCUUUGGdTdT CCAAAGGUGGGCACAACGUdTdT
    428     450   5.000   55.0 AGAAGAAGGGCCCCAAUGCdTdT GCAUUGGGGCCCUUCUUCUdTdT
    431     453   5.000   55.0 AGAAGGGCCCCAAUGCCAAdTdT UUGGCAUUGGGGCCCUUCUdTdT
    434     456   5.000   60.0 AGGGCCCCAAUGCCAACUCdTdT GAGUUGGCAUUGGGGCCCUdTdT
    444     466   5.000   35.0 UGCCAACUCUUAAGUCUUUdTdT AAAGACUUAAGAGUUGGCAdTdT
    487     509   5.000   35.0 AAAAGCCACUUAGUUCAGUdTdT ACUGAACUAAGUGGCUUUUdTdT
    123     145   4.000   50.0 GAUCAAGGCUCAUGUAGCCdTdT GGCUACAUGAGCCUUGAUCdTdT
    125     147   4.000   50.0 UCAAGGCUCAUGUAGCCUCdTdT GAGGCUACAUGAGCCUUGAdTdT
    128     150   4.000   50.0 AGGCUCAUGUAGCCUCACUdTdT AGUGAGGCUACAUGAGCCUdTdT
    155     177   4.000   50.0 UUGCCCCGGAAGAUCAAGUdTdT ACUUGAUCUUCCGGGGCAAdTdT
    234     256   4.000   60.0 GGCCCUGACUACCCUGGAAdTdT UUCCAGGGUAGUCAGGGCCdTdT
    259     281   4.000   60.0 GGCCGCAUGCUUGGAGGUAdTdT UACCUCCAAGCAUGCGGCCdTdT
    266     288   4.000   40.0 UGCUUGGAGGUAAAGUUCAdTdT UGAACUUUACCUCCAAGCAdTdT
    342     364   4.000   40.0 GGAGAAGAAGAAGAAGAAGdTdT CUUCUUCUUCUUCUUCUCCdTdT
    347     369   4.000   40.0 AGAAGAAGAAGAAGACAGGdTdT CCUGUCUUCUUCUUCUUCUdTdT
    359     381   4.000   60.0 AGACAGGUCGGGCUAAGCGdTdT CGCUUAGCCCGACCUGUCUdTdT
    111     133   3.000   55.0 AACGGUCGCCCAGAUCAAGdTdT CUUGAUCUGGGCGACCGUUdTdT
    113     135   3.000   65.0 CGGUCGCCCAGAUCAAGGCdTdT GCCUUGAUCUGGGCGACCGdTdT
    172     194   3.000   70.0 GUCGUGCUCCUGGCAGGCGdTdT CGCCUGCCAGGAGCACGACdTdT
    443     465   3.000   35.0 AUGCCAACUCUUAAGUCUUdTdT AAGACUUAAGAGUUGGCAUdTdT
    456     478   3.000   35.0 AGUCUUUUGUAAUUCUGGCdTdT GCCAGAAUUACAAAAGACUdTdT
    468     490   3.000   30.0 UUCUGGCUUUCUCUAAUAAdTdT UUAUUAGAGAAAGCCAGAAdTdT
    484     506   3.000   30.0 UAAAAAAGCCACUUAGUUCdTdT GAACUAAGUGGCUUUUUUAdTdT
    108     130   2.000   60.0 GGAAACGGUCGCCCAGAUCdTdT GAUCUGGGCGACCGUUUCCdTdT
    135     157   2.000   60.0 UGUAGCCUCACUGGAGGGCdTdT GCCCUCCAGUGAGGCUACAdTdT
    139     161   2.000   60.0 GCCUCACUGGAGGGCAUUGdTdT CAAUGCCCUCCAGUGAGGCdTdT
    150     172   2.000   60.0 GGGCAUUGCCCCGGAAGAUdTdT AUCUUCCGGGGCAAUGCCCdTdT
    171     193   2.000   65.0 AGUCGUGCUCCUGGCAGGCdTdT GCCUGCCAGGAGCACGACUdTdT
    201     223   2.000   65.0 GGAUGAGGCCACUCUGGGCdTdT GCCCAGAGUGGCCUCAUCCdTdT
    204     226   2.000   65.0 UGAGGCCACUCUGGGCCAGdTdT CUGGCCCAGAGUGGCCUCAdTdT
    245     267   2.000   65.0 CCCUGGAAGUAGCAGGCCGdTdT CGGCCUGCUACUUCCAGGGdTdT
    256     278   2.000   65.0 GCAGGCCGCAUGCUUGGAGdTdT CUCCAAGCAUGCGGCCUGCdTdT
    285     307   2.000   65.0 UGGUUCCCUGGCCCGUGCUdTdT AGCACGGGCCAGGGAACCAdTdT
    338     360   2.000   35.0 AACAGGAGAAGAAGAAGAAdTdT UUCUUCUUCUUCUCCUGUUdTdT
    345     367   2.000   35.0 GAAGAAGAAGAAGAAGACAdTdT UGUCUUCUUCUUCUUCUUCdTdT
    486     508   2.000   35.0 AAAAAGCCACUUAGUUCAGdTdT CUGAACUAAGUGGCUUUUUdTdT

#---------------------------------------
#---------------------------------------

#---------------------------------------
# Total_sequences: 1
# Total_hitcount: 85
#---------------------------------------

  File: x65923.fasta

>X65923_308 %GC 50.0 Score 9 H.sapiens fau mRNA
aaaagtgagaggtcagactccta
>X65923_309 %GC 50.0 Score 9 H.sapiens fau mRNA
aaagtgagaggtcagactcctaa
>X65923_310 %GC 50.0 Score 9 H.sapiens fau mRNA
aagtgagaggtcagactcctaag
>X65923_351 %GC 50.0 Score 9 H.sapiens fau mRNA
aagaagaagaagacaggtcgggc
>X65923_166 %GC 55.0 Score 8 H.sapiens fau mRNA
aagatcaagtcgtgctcctggca
>X65923_279 %GC 55.0 Score 8 H.sapiens fau mRNA
aaagttcatggttccctggcccg
>X65923_330 %GC 55.0 Score 8 H.sapiens fau mRNA
aaggtggccaaacaggagaagaa
>X65923_354 %GC 55.0 Score 8 H.sapiens fau mRNA
aagaagaagacaggtcgggctaa
>X65923_357 %GC 55.0 Score 8 H.sapiens fau mRNA
aagaagacaggtcgggctaagcg
>X65923_393 %GC 55.0 Score 8 H.sapiens fau mRNA
aaccggcgctttgtcaacgttgt
>X65923_253 %GC 60.0 Score 7 H.sapiens fau mRNA
aagtagcaggccgcatgcttgga
>X65923_280 %GC 60.0 Score 7 H.sapiens fau mRNA
aagttcatggttccctggcccgt
>X65923_339 %GC 40.0 Score 7 H.sapiens fau mRNA
aaacaggagaagaagaagaagaa
>X65923_340 %GC 40.0 Score 7 H.sapiens fau mRNA
aacaggagaagaagaagaagaag
>X65923_348 %GC 40.0 Score 7 H.sapiens fau mRNA
aagaagaagaagaagacaggtcg
>X65923_375 %GC 60.0 Score 7 H.sapiens fau mRNA
aagcggcggatgcagtacaaccg
>X65923_408 %GC 60.0 Score 7 H.sapiens fau mRNA
aacgttgtgcccacctttggcaa
>X65923_429 %GC 60.0 Score 7 H.sapiens fau mRNA
aagaagaagggccccaatgccaa
>X65923_432 %GC 60.0 Score 7 H.sapiens fau mRNA
aagaagggccccaatgccaactc
>X65923_435 %GC 60.0 Score 7 H.sapiens fau mRNA
aagggccccaatgccaactctta
>X65923_488 %GC 40.0 Score 7 H.sapiens fau mRNA
aaaaagccacttagttcagtcaa
>X65923_489 %GC 40.0 Score 7 H.sapiens fau mRNA
aaaagccacttagttcagtcaaa
>X65923_490 %GC 40.0 Score 7 H.sapiens fau mRNA
aaagccacttagttcagtcaaaa
>X65923_491 %GC 40.0 Score 7 H.sapiens fau mRNA
aagccacttagttcagtcaaaaa
>X65923_129 %GC 55.0 Score 6 H.sapiens fau mRNA
aaggctcatgtagcctcactgga


  [Part of this file has been deleted for brevity]

gaggccctgactaccctggaagt
>X65923_259 %GC 60.0 Score 4 H.sapiens fau mRNA
caggccgcatgcttggaggtaaa
>X65923_266 %GC 40.0 Score 4 H.sapiens fau mRNA
catgcttggaggtaaagttcatg
>X65923_342 %GC 40.0 Score 4 H.sapiens fau mRNA
caggagaagaagaagaagaagac
>X65923_347 %GC 40.0 Score 4 H.sapiens fau mRNA
gaagaagaagaagaagacaggtc
>X65923_359 %GC 60.0 Score 4 H.sapiens fau mRNA
gaagacaggtcgggctaagcggc
>X65923_111 %GC 55.0 Score 3 H.sapiens fau mRNA
gaaacggtcgcccagatcaaggc
>X65923_113 %GC 65.0 Score 3 H.sapiens fau mRNA
aacggtcgcccagatcaaggctc
>X65923_172 %GC 70.0 Score 3 H.sapiens fau mRNA
aagtcgtgctcctggcaggcgcg
>X65923_443 %GC 35.0 Score 3 H.sapiens fau mRNA
caatgccaactcttaagtctttt
>X65923_456 %GC 35.0 Score 3 H.sapiens fau mRNA
taagtcttttgtaattctggctt
>X65923_468 %GC 30.0 Score 3 H.sapiens fau mRNA
aattctggctttctctaataaaa
>X65923_484 %GC 30.0 Score 3 H.sapiens fau mRNA
aataaaaaagccacttagttcag
>X65923_108 %GC 60.0 Score 2 H.sapiens fau mRNA
caggaaacggtcgcccagatcaa
>X65923_135 %GC 60.0 Score 2 H.sapiens fau mRNA
catgtagcctcactggagggcat
>X65923_139 %GC 60.0 Score 2 H.sapiens fau mRNA
tagcctcactggagggcattgcc
>X65923_150 %GC 60.0 Score 2 H.sapiens fau mRNA
gagggcattgccccggaagatca
>X65923_171 %GC 65.0 Score 2 H.sapiens fau mRNA
caagtcgtgctcctggcaggcgc
>X65923_201 %GC 65.0 Score 2 H.sapiens fau mRNA
gaggatgaggccactctgggcca
>X65923_204 %GC 65.0 Score 2 H.sapiens fau mRNA
gatgaggccactctgggccagtg
>X65923_245 %GC 65.0 Score 2 H.sapiens fau mRNA
taccctggaagtagcaggccgca
>X65923_256 %GC 65.0 Score 2 H.sapiens fau mRNA
tagcaggccgcatgcttggaggt
>X65923_285 %GC 65.0 Score 2 H.sapiens fau mRNA
catggttccctggcccgtgctgg
>X65923_338 %GC 35.0 Score 2 H.sapiens fau mRNA
caaacaggagaagaagaagaaga
>X65923_345 %GC 35.0 Score 2 H.sapiens fau mRNA
gagaagaagaagaagaagacagg
>X65923_486 %GC 35.0 Score 2 H.sapiens fau mRNA
taaaaaagccacttagttcagtc

  Output files for usage example 2

  File: x65923.sirna

########################################
# Program: sirna
# Rundate: Sun 15 Jul 2007 12:00:00
# Commandline: sirna
#    -context
#    -sequence tembl:x65923
# Report_format: table
# Report_file: x65923.sirna
########################################

#=======================================
#
# Sequence: X65923     from: 1   to: 518
# HitCount: 85
#
# The forward sense sequence shows the first 2 bases of
# the 23 base region in brackets, this should be ignored
# when ordering siRNA probes.
# CDS region found in feature table starting at 57
#
#=======================================

  Start     End   Score    GC% Sense_siRNA Antisense_siRNA
    308     330   9.000   50.0 (AA)AAGUGAGAGGUCAGACUCCdTdT GGAGUCUGACCUCUCACUUd
TdT
    309     331   9.000   50.0 (AA)AGUGAGAGGUCAGACUCCUdTdT AGGAGUCUGACCUCUCACUd
TdT
    310     332   9.000   50.0 (AA)GUGAGAGGUCAGACUCCUAdTdT UAGGAGUCUGACCUCUCACd
TdT
    351     373   9.000   50.0 (AA)GAAGAAGAAGACAGGUCGGdTdT CCGACCUGUCUUCUUCUUCd
TdT
    166     188   8.000   55.0 (AA)GAUCAAGUCGUGCUCCUGGdTdT CCAGGAGCACGACUUGAUCd
TdT
    279     301   8.000   55.0 (AA)AGUUCAUGGUUCCCUGGCCdTdT GGCCAGGGAACCAUGAACUd
TdT
    330     352   8.000   55.0 (AA)GGUGGCCAAACAGGAGAAGdTdT CUUCUCCUGUUUGGCCACCd
TdT
    354     376   8.000   55.0 (AA)GAAGAAGACAGGUCGGGCUdTdT AGCCCGACCUGUCUUCUUCd
TdT
    357     379   8.000   55.0 (AA)GAAGACAGGUCGGGCUAAGdTdT CUUAGCCCGACCUGUCUUCd
TdT
    393     415   8.000   55.0 (AA)CCGGCGCUUUGUCAACGUUdTdT AACGUUGACAAAGCGCCGGd
TdT
    253     275   7.000   60.0 (AA)GUAGCAGGCCGCAUGCUUGdTdT CAAGCAUGCGGCCUGCUACd
TdT
    280     302   7.000   60.0 (AA)GUUCAUGGUUCCCUGGCCCdTdT GGGCCAGGGAACCAUGAACd
TdT
    339     361   7.000   40.0 (AA)ACAGGAGAAGAAGAAGAAGdTdT CUUCUUCUUCUUCUCCUGUd
TdT
    340     362   7.000   40.0 (AA)CAGGAGAAGAAGAAGAAGAdTdT UCUUCUUCUUCUUCUCCUGd
TdT
    348     370   7.000   40.0 (AA)GAAGAAGAAGAAGACAGGUdTdT ACCUGUCUUCUUCUUCUUCd
TdT
    375     397   7.000   60.0 (AA)GCGGCGGAUGCAGUACAACdTdT GUUGUACUGCAUCCGCCGCd
TdT
    408     430   7.000   60.0 (AA)CGUUGUGCCCACCUUUGGCdTdT GCCAAAGGUGGGCACAACGd
TdT
    429     451   7.000   60.0 (AA)GAAGAAGGGCCCCAAUGCCdTdT GGCAUUGGGGCCCUUCUUCd
TdT
    432     454   7.000   60.0 (AA)GAAGGGCCCCAAUGCCAACdTdT GUUGGCAUUGGGGCCCUUCd
TdT
    435     457   7.000   60.0 (AA)GGGCCCCAAUGCCAACUCUdTdT AGAGUUGGCAUUGGGGCCCd
TdT
    488     510   7.000   40.0 (AA)AAAGCCACUUAGUUCAGUCdTdT GACUGAACUAAGUGGCUUUd
TdT
    489     511   7.000   40.0 (AA)AAGCCACUUAGUUCAGUCAdTdT UGACUGAACUAAGUGGCUUd
TdT
    490     512   7.000   40.0 (AA)AGCCACUUAGUUCAGUCAAdTdT UUGACUGAACUAAGUGGCUd
TdT
    491     513   7.000   40.0 (AA)GCCACUUAGUUCAGUCAAAdTdT UUUGACUGAACUAAGUGGCd
TdT
    129     151   6.000   55.0 (AA)GGCUCAUGUAGCCUCACUGdTdT CAGUGAGGCUACAUGAGCCd
TdT
    165     187   6.000   50.0 (GA)AGAUCAAGUCGUGCUCCUGdTdT CAGGAGCACGACUUGAUCUd
TdT
    278     300   6.000   50.0 (UA)AAGUUCAUGGUUCCCUGGCdTdT GCCAGGGAACCAUGAACUUd
TdT


  [Part of this file has been deleted for brevity]

    353     375   5.000   55.0 (GA)AGAAGAAGACAGGUCGGGCdTdT GCCCGACCUGUCUUCUUCUd
TdT
    360     382   5.000   65.0 (AA)GACAGGUCGGGCUAAGCGGdTdT CCGCUUAGCCCGACCUGUCd
TdT
    374     396   5.000   55.0 (UA)AGCGGCGGAUGCAGUACAAdTdT UUGUACUGCAUCCGCCGCUd
TdT
    383     405   5.000   55.0 (GA)UGCAGUACAACCGGCGCUUdTdT AAGCGCCGGUUGUACUGCAd
TdT
    387     409   5.000   55.0 (CA)GUACAACCGGCGCUUUGUCdTdT GACAAAGCGCCGGUUGUACd
TdT
    390     412   5.000   55.0 (UA)CAACCGGCGCUUUGUCAACdTdT GUUGACAAAGCGCCGGUUGd
TdT
    392     414   5.000   55.0 (CA)ACCGGCGCUUUGUCAACGUdTdT ACGUUGACAAAGCGCCGGUd
TdT
    407     429   5.000   55.0 (CA)ACGUUGUGCCCACCUUUGGdTdT CCAAAGGUGGGCACAACGUd
TdT
    428     450   5.000   55.0 (CA)AGAAGAAGGGCCCCAAUGCdTdT GCAUUGGGGCCCUUCUUCUd
TdT
    431     453   5.000   55.0 (GA)AGAAGGGCCCCAAUGCCAAdTdT UUGGCAUUGGGGCCCUUCUd
TdT
    434     456   5.000   60.0 (GA)AGGGCCCCAAUGCCAACUCdTdT GAGUUGGCAUUGGGGCCCUd
TdT
    444     466   5.000   35.0 (AA)UGCCAACUCUUAAGUCUUUdTdT AAAGACUUAAGAGUUGGCAd
TdT
    487     509   5.000   35.0 (AA)AAAAGCCACUUAGUUCAGUdTdT ACUGAACUAAGUGGCUUUUd
TdT
    123     145   4.000   50.0 (CA)GAUCAAGGCUCAUGUAGCCdTdT GGCUACAUGAGCCUUGAUCd
TdT
    125     147   4.000   50.0 (GA)UCAAGGCUCAUGUAGCCUCdTdT GAGGCUACAUGAGCCUUGAd
TdT
    128     150   4.000   50.0 (CA)AGGCUCAUGUAGCCUCACUdTdT AGUGAGGCUACAUGAGCCUd
TdT
    155     177   4.000   50.0 (CA)UUGCCCCGGAAGAUCAAGUdTdT ACUUGAUCUUCCGGGGCAAd
TdT
    234     256   4.000   60.0 (GA)GGCCCUGACUACCCUGGAAdTdT UUCCAGGGUAGUCAGGGCCd
TdT
    259     281   4.000   60.0 (CA)GGCCGCAUGCUUGGAGGUAdTdT UACCUCCAAGCAUGCGGCCd
TdT
    266     288   4.000   40.0 (CA)UGCUUGGAGGUAAAGUUCAdTdT UGAACUUUACCUCCAAGCAd
TdT
    342     364   4.000   40.0 (CA)GGAGAAGAAGAAGAAGAAGdTdT CUUCUUCUUCUUCUUCUCCd
TdT
    347     369   4.000   40.0 (GA)AGAAGAAGAAGAAGACAGGdTdT CCUGUCUUCUUCUUCUUCUd
TdT
    359     381   4.000   60.0 (GA)AGACAGGUCGGGCUAAGCGdTdT CGCUUAGCCCGACCUGUCUd
TdT
    111     133   3.000   55.0 (GA)AACGGUCGCCCAGAUCAAGdTdT CUUGAUCUGGGCGACCGUUd
TdT
    113     135   3.000   65.0 (AA)CGGUCGCCCAGAUCAAGGCdTdT GCCUUGAUCUGGGCGACCGd
TdT
    172     194   3.000   70.0 (AA)GUCGUGCUCCUGGCAGGCGdTdT CGCCUGCCAGGAGCACGACd
TdT
    443     465   3.000   35.0 (CA)AUGCCAACUCUUAAGUCUUdTdT AAGACUUAAGAGUUGGCAUd
TdT
    456     478   3.000   35.0 (UA)AGUCUUUUGUAAUUCUGGCdTdT GCCAGAAUUACAAAAGACUd
TdT
    468     490   3.000   30.0 (AA)UUCUGGCUUUCUCUAAUAAdTdT UUAUUAGAGAAAGCCAGAAd
TdT
    484     506   3.000   30.0 (AA)UAAAAAAGCCACUUAGUUCdTdT GAACUAAGUGGCUUUUUUAd
TdT
    108     130   2.000   60.0 (CA)GGAAACGGUCGCCCAGAUCdTdT GAUCUGGGCGACCGUUUCCd
TdT
    135     157   2.000   60.0 (CA)UGUAGCCUCACUGGAGGGCdTdT GCCCUCCAGUGAGGCUACAd
TdT
    139     161   2.000   60.0 (UA)GCCUCACUGGAGGGCAUUGdTdT CAAUGCCCUCCAGUGAGGCd
TdT
    150     172   2.000   60.0 (GA)GGGCAUUGCCCCGGAAGAUdTdT AUCUUCCGGGGCAAUGCCCd
TdT
    171     193   2.000   65.0 (CA)AGUCGUGCUCCUGGCAGGCdTdT GCCUGCCAGGAGCACGACUd
TdT
    201     223   2.000   65.0 (GA)GGAUGAGGCCACUCUGGGCdTdT GCCCAGAGUGGCCUCAUCCd
TdT
    204     226   2.000   65.0 (GA)UGAGGCCACUCUGGGCCAGdTdT CUGGCCCAGAGUGGCCUCAd
TdT
    245     267   2.000   65.0 (UA)CCCUGGAAGUAGCAGGCCGdTdT CGGCCUGCUACUUCCAGGGd
TdT
    256     278   2.000   65.0 (UA)GCAGGCCGCAUGCUUGGAGdTdT CUCCAAGCAUGCGGCCUGCd
TdT
    285     307   2.000   65.0 (CA)UGGUUCCCUGGCCCGUGCUdTdT AGCACGGGCCAGGGAACCAd
TdT
    338     360   2.000   35.0 (CA)AACAGGAGAAGAAGAAGAAdTdT UUCUUCUUCUUCUCCUGUUd
TdT
    345     367   2.000   35.0 (GA)GAAGAAGAAGAAGAAGACAdTdT UGUCUUCUUCUUCUUCUUCd
TdT
    486     508   2.000   35.0 (UA)AAAAAGCCACUUAGUUCAGdTdT CUGAACUAAGUGGCUUUUUd
TdT

#---------------------------------------
#---------------------------------------

#---------------------------------------
# Total_sequences: 1
# Total_hitcount: 85
#---------------------------------------

   The siRNAs are reported in order of best score first.

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

Data files

   None.

Notes

   None.

References

    1. Elbashir, S. M., et al. (2001a). Duplexes of 21-nucleotide RNAs
       mediate RNA interference in mammalian cell culture. Nature 411:
       494-498.
    2. Elbashir, S. M., W. Lendeckel and T. Tuschl (2001b). RNA
       interference is mediated by 21 and 22 nt RNAs. Genes & Dev. 15:
       188-200.

Warnings

   It is assumed that the input sequence is mRNA.

Diagnostic Error Messages

   None.

Exit status

   It always exits with status 0.

Known bugs

   None.

See also

   Program name                        Description
   banana       Bending and curvature plot in B-DNA
   btwisted     Calculates the twisting in a B-DNA sequence
   chaos        Create a chaos game representation plot for a sequence
   compseq      Count composition of dimer/trimer/etc words in a sequence
   dan          Calculates DNA RNA/DNA melting temperature
   freak        Residue/base frequency table or plot
   isochore     Plots isochores in large DNA sequences
   wordcount    Counts words of a specified size in a DNA sequence

Author(s)

   Gary Williams (gwilliam © rfcgr.mrc.ac.uk)
   MRC Rosalind Franklin Centre for Genomics Research Wellcome Trust
   Genome Campus, Hinxton, Cambridge, CB10 1SB, UK

History

   Written (November 2002) - Gary Williams.

Target users

   This program is intended to be used by everyone and everything, from
   naive users to embedded scripts.

Comments

   None
